1. health and fitness
2. disease

THE EFFECT OF PAX6 ON ANTERIOR SEGMENT ANATOMY, CORNEAL WOUND
HEALING, LIMBAL PROGENITOR CELLS, AND CORNEAL VASCULARIZATION.
PJ Accola, PA Moore, KP Carmichael, JD Lauderdale
The purpose of this study was to establish a corneal wounding model and characterize corneal
wound healing and anterior segment pathology in Small eye (Sey) (Pax6+/-) mice.
The anterior segment of Wild Type (WT) (n=79) and Sey (n=40) mice (age 2-3 months) were
examined with slit lamp biomicroscopy. Corneal wounding with n-heptanol was performed and
healing evaluated on days 1, 2, 3 (if fluorescein positive on day 2), 4, 7, 14, and 28 post wounding.
Each day, 3-5 mice were euthanized. Anterior segment histopathology was characterized.
Immunohistochemistry (p63, sVEGFR-1) was performed. Days to negative fluorescein staining
were compared using the Chi-Square test. The number of p63 staining basal cells was recorded
and statistically evaluated by ANOVA. Staining for sVEGFR1 in non-wounded eyes was recorded
as positive or negative and anatomic location reported.
Ophthalmic examination and histopathologic findings in Sey mice included corneal opacity,
corneal vascularization, iris hypoplasia, and cataract. The wounding protocol resulted in precise
and accurate removal of corneal epithelium. There was no significant difference in the amount of
cornea wounded in WT and Sey mice (p>0.05). There was a statistically significant delay in
corneal wound healing in Sey mice at days 2 and 3 when compared to WT mice (p<0.05). There
was no significant difference in p63 staining (p>0.05). All mice exhibited comparable sVEGFR1
staining.
Histopathologic findings are consistent with impaired anterior segment development. The
delayed corneal healing is unlikely to be due to a deficiency in p63 cellular expression. The
comparable expression of sVEGFR1 suggests that it alone is likely not responsible for the corneal
vascularization present in Sey mice.
A RETROSPECTIVE INVESTIGATION OF CLINICAL CASES OF CANINE
SALMONELLOSIS REPORTED IN GEORGIA
Laura Adams1, Koren Moore1, Susan Sanchez2, Steve Valeika3, Ying Cheng2 and John J. Maurer2.
Departments of Population Health1, and Infectious Diseases2, College of Veterinary Medicine, and
Department of Biostatistics and Epidemiology3, College of Public Health, the University of
Georgia, Athens, GA
Salmonella causes a variety of diseases in many animal species. Most of the research regarding
canine salmonellosis has focused on outbreaks where many animals were infected and a common
source such as dog food was linked to the outbreak. However, there is a paucity of information
regarding sporadic cases of salmonellosis in dogs. To investigate this issue, we did a retrospective
study of clinical cases of canine salmonellosis reported to the Athens Veterinary Diagnostic
Laboratory from September 1999-December 2008. Twenty-one Salmonella isolates were typed
serologically and by pulse field gel electrophoresis (PFGE). Clinical and epidemiological
information was also collected for each case including: symptoms, age, breed, sex, geographical
location, site of sample taken, predisposing factors, and date of isolation. The information was
then reviewed to investigate potential epidemiologic links between the cases. For the twenty-one
canine Salmonella isolates examined, not a single Salmonella serovar or strain was common to
these reported cases of canine salmonellosis. Many of the Salmonella enterica serovars identified
were many of the same serovars that commonly infect humans. Salmonella enterica subspecies
arizoneae was isolated from a dog, uncommon in its isolation, especially in humans where most
cases are generally associated with an underlying predisposing factor (ex. cancer). In reviewing
the clinical case file, this patient was also diagnosed as having cancer, which might explain the
isolation of this unusual Salmonella serovar from this dog. The finding, of such a diverse array of
S. enterica serovars and strains, in animals within this state across time lends support to the
sporadic nature of this illness in dogs. We are presently investigating whether these canine
Salmonella isolates match with humans isolates reported to the Centers for Disease Control and
Prevention in Atlanta, GA. The potential for zoonotic transmission of Salmonella exist between
man and his “best friend” due to the close relationship between humans and dogs, and the biology
and behavior of canines.
THE PRESENCE OF RESIDUAL DISC AND ITS INFLUENCE ON RECOVERY AND RECURRENCE
IN 43 CHONDRODYSTROPHIC DOGS WITH ACUTE THORACOLUMBAR INTERVERTEBRAL
DISC DISEASE. ROACH W, THOMAS M, WEH J, PLATT S, KENT M, BLEEDORN J, WELLS K.
The purpose of this study was to report the amount of residual disc material following
hemilaminectomy, to determine if certain hemilaminectomy techniques are associated with
less residual disc, and to determine if the amount of residual disc effects patient functional
outcome in chondrodystrophic dogs with acute intervertebral disc disease (IVDD).
Computed tomography (CT) was performed immediately pre-operatively and postoperatively on 43 dogs undergoing hemilaminectomy to determine the amount of residual
disc. Patient recovery and long-term functional outcome was recorded while in-hospital and
with two telephone interviews with the owners.
Residual disc was present in all of the dogs in this study, which has not been previously
reported. More ventral hemilaminectomies in this study were associated with more residual
disc. There was a positive correlation between maximum residual effacement percentage
and days until ambulation.
There was a significant effect of residual disc percentage on patient functional outcome with
respect to residual neurologic deficits at long-term follow-up.
The findings of this study reveal the presence of residual disc in all dogs, and suggest that
the amount residual disc is an important factor in patient functional recovery.
Multiple-anthelmintic resistance on a llama farm in the southeastern
United States
Daniel Zarate1, Bob Storey1, Lisa Williamson2, and Ray M. Kaplan1
University of Georgia, College of Veterinary Medicine, Department of Infectious Diseases1, and
Department of Large Animal Clinical Sciences2, Athens, Georgia USA
Documentation of anthelmintic resistance on goat and sheep farms in the southeastern United
States is substantial; however, little is known about its prevalence on llama farms. A study was
conducted on a llama farm in Florida to test for the presence of resistance to fenbendazole,
levamisole, ivermectin, and moxidectin using both fecal egg count reduction test (FECRT) and
larval development assay (LDA, DrenchRite®). Seventy-two llamas were allocated randomly
into six treatment groups (n=12/group): fenbendazole oral(FBZ 20 mg/kg), levamisole oral (LEV
12 mg/kg), ivermectin injectable (IVM 0.3 mg/kg), moxidectin oral (MOX 0.3 mg/kg), moxidectin
injectable (MOX 0.3 mg/kg), and untreated control. For the LDA, nematode eggs were isolated
from a pooled fecal sample collected at the time of treatment. FEC reductions were 0%, 96%,
0%, 90%, and 58% for FBZ, LEV, IVM, MOX PO, and MOX SC, respectively. Based on
WAAVP guidelines, these results indicate resistance to all drugs tested except levamisole. In
contrast, the LDA data indicated resistance for BZ and IVM, and sensitivity to LEV and MOX.
Based on coprocultures, the most common nematode was Haemonchus contortus, followed by
Nematodirus spp. and Trichostrongylus spp. These findings confirm the presence of multipleanthelmintic resistance on a llama farm in the southeastern US. Furthermore, the incongruent
results for MOX in the LDA and FECRT suggest that the dose applied may not be adequate,
and that optimal route of administration requires further investigation. . Further research is
required to assess the prevalence of anthelmintic resistance on camelid farms in US.
JOINT CONTRACTURES IN GOLDEN RETRIEVER MUSCULAR DYSTROPHY: A
MODEL FOR DUCHENNE MUSCULAR DYSTROPHY.
Nghiem PP1,2, Schatzberg SJ1, Hoffman EP2, Wang Z2, Ghimbovschi S2, Rayavarapu S2,
Knoblach S2, Nagaraju K2, and Kornegay JN3. 1University of Georgia, College of Veterinary
Medicine, Athens, GA, 2Children’s National Medical Center, Center for Genetic Medicine
Research, Washington, D.C., 3University of North Carolina, School of Medicine, Chapel Hill, NC.
In both Duchenne muscular dystrophy (DMD) patients and Golden retriever muscular dystrophy
(GRMD) dogs, joint contractures are the result of the dystrophic process and are extremely
debilitating. In DMD, both muscle weakness and joint contractures are thought to contribute to
postural instability and ultimately the loss of ambulation. Conventional thought is that joint
contractures are driven by progressive skeletal muscle wasting and weakness. However, in
GRMD, our functional phenotypic data suggest differently, as joint contractures seem to be driven
by an imbalance of skeletal muscle extensor and flexor forces and skeletal muscle hypertrophy. In
an attempt to correlate functional abnormalities (i.e. increased Cranial sartorius (CS) m.
circumference, increased tetanic flexor force, decreased tetanic extensor force, and decreased
tibiotarsal joint angle) with skeletal muscle molecular pathways, we have performed genome-wide
mRNA expression profiling on archived biopsy samples from three muscles (CS, Long digital
extensor, and Vastus lateralis) that were collected at 6 months of age from 8 GRMD and 4 normal
Golden retriever dogs. We have identified a major cytokine, osteopontin (OPN) that may be
associated with the inflammatory and hypertrophic changes in the CS muscle. We also have
identified three genes that are over-expressed in the hypertrophied CS muscle including DAG1,
LARGE, and SNTA1, that code for α- and β-dystroglycan, like-glycosyltransferase, and
syntrophin alpha 1, respectively. To validate the expression profiling results, quantitative PCR
and immunohistochemical studies of these gene transcripts and proteins, respectively, are
underway in variably affected GRMD and control dogs. OPN, DAG1, LARGE, and SNTA1
ultimately may prove to be important therapeutic targets for GRMD dogs and DMD patients.
DEVELOPMENT OF AN IN VITRO BIOASSAY TO DETECT THE PRESENCE OF
ANTHELMINTIC RESISTANCE IN DIROFILARIA IMMITIS
A. R. Moorhead, M.T. Dzimianski, P. Supakorndej and R. M. Kaplan
Heartworm disease is a significant threat to canine health. Over the past few decades,
the prevalence of heartworm infection in pet dogs has been greatly reduced by monthly
prophylaxis using anthelmintics of the avermectin/milbemycin class. However, recent
reports of heartworm infections in dogs receiving documented monthly prophylaxis are
cause for concern. Two possible explanations for this observation are failure of owner
compliance in ensuring proper administration of prophylaxis, and the development of
anthelmintic resistance. However, it is currently not possible to distinguish these two
scenarios because compliance is not possible to verify, and there are no validated assays
for detecting resistance in D. immitis. In order to address this problem, we developed a
larval migration inhibition bioassay (LMIA) for D. immitis, using a 96-well plate format.
Dirofilaria immitis L3 obtained from mosquitoes fed on microfilaremic dog blood were
incubated for 3 h in the presence of increasing concentrations of ivermectin. Following
incubation, L3 were transferred to wells containing a 20-micron mesh filter, and numbers
of larvae migrating through the mesh were measured after 12 h. Preliminary results
indicate that the LMIA produces a sigmoidal dose response with D. immitis L3. We are
currently in the process of further optimizing this assay and testing the dose response
characteristics of a variety of avermectin/milbemycin drugs. Our goal is to validate this
assay so it can be used to screen D. immitis L3 produced from microfilaremic blood
samples obtained from dogs for which a failure of anthelmintic prophylaxis has been
reported.
ESTABLISHING A REPRODUCIBLE METHOD FOR THE CULTURE OF PRIMARY
EQUINE CORNEAL CELLS (RL Mathes 1, UM Dietrich 1, TM Krunkosky 2, DJ Hurley
3, AJ Reber 3) 1 Department of Small Animal Medicine and Surgery, College of
Veterinary Medicine, University of Georgia; 2 Department of Anatomy and Radiology,
College of Veterinary Medicine, University of Georgia; 3 Department of Population
Health, College of Veterinary Medicine, University of Georgia
Purpose. To establish a reproducible method for the culture of primary equine corneal
epithelial cells, keratocytes and endothelial cells and to describe each cell’s
morphologic characteristics, immunocytochemical staining properties and conditions
required for cryopreservation.
Methods. Corneas from eight horses recently euthanized for reasons unrelated to this
study were collected aseptically and enzymatically separated into three individual layers
for cell isolation. The cells were plated, grown in culture and continued for several
passages. Each cell type was characterized by morphology and immunocytochemical
staining.
Results. All three equine corneal cell types were successfully grown in culture.
Cultured corneal endothelial cells were large, hexagonal cells with a moderate growth
rate. Keratocytes were small, spindloid cells that grew rapidly. Epithelial cells had
heterogenous morphology and grew slowly. The endothelial cells and keratocytes
stained positive for vimentin and were morphologically distinguishable from one
another. The epithelial cells stained positive for cytokeratin. Keratocytes and
endothelial cells were able to be cryopreserved and recovered. The cryopreserved cells
maintained their morphological and immunocytochemical features after
cryopreservation and recovery.
Conclusion. This work establishes reproducible methods for isolation and culture of all
three equine corneal cells. Cell morphology and cytoskeletal element expression for
each cell type is also described. This has not previously been reported for equine
corneal cells. This report also demonstrates the ability to preserve equine keratocytes
and endothelial cells for extended periods of time and utilize them long after primary cell
collection, a feature that has not been reported for veterinary corneal cell culture.
University of Georgia Veterinary Ophthalmology Research Fund (VORF). None.
RISK
FACTORS
FOR
SEPTIC
PERITONITIS
AND
SURVIVAL
AFTER
GASTROINTESTINAL SURGERY IN DOGS: 225 CASES. JA Grimes, CW Schmiedt, K
Cornell, M Radlinsky. University of Georgia College of Veterinary Medicine, Athens, GA.
Gastrointestinal surgery is commonly performed in small animal surgical practice. The purpose
of this retrospective study is to identify risk factors for survival and development of septic
peritonitis (SP) following a full thickness gastrointestinal incision in different populations of dogs.
Our hypothesis is that hypoalbuminemia and hypoproteinemia are risk factors for both postoperative SP and mortality and that risk of mortality is affected by the presence of pre-operative
SP.
Medical records of dogs undergoing full-thickness intestinal incisions between 1998 and 2007
were reviewed. Data was collected on pre-, intra-, and post-operative variables and significance as
it related to survival and development of SP was evaluated using logistic regression analysis.
Two hundred and twenty five cases were identified; 28 (12.4%) cases developed post-operative
SP and 29 (12.9%) cases did not survive to discharge. Forty four (19.5%) cases had pre-operative
SP, of those 16 (36.4%) had post-operative SP and 14 (31.8%) died. In cases without pre-operative
SP, 11 (6.0%) developed post-operative SP and 20 (11.0%) died. When all cases are considered,
common significant risk factors include presence of pre-operative SP, pre-operative and postoperative albumin and protein concentrations, and intra-operative hypotension. The presence of a
foreign body was frequently a protective factor.
Multiple factors are involved in survival rates and the development of SP post-operatively.
Dogs with decreased plasma protein and albumin concentrations are at risk for decreased survival
and development of SP. Aggressive peri-operative attempts to increase protein concentrations and
intra-operative surgical strategies to minimize morbidity and mortality may be indicated in
patients with risk factors identified here.
5-LIPOXYGENASE EXPRESSION IN HYPERPLASTIC, INFLAMED, AND NEOPLASTIC
CANINE PROSTATE TISSUES
Laura Goodman, Carla Jarrett, Thomas Krunkosky, Steve Budsberg, Nicole Northrup, Corey
Saba, Bruce LeRoy
5-lipoxygenase (5-LO) metabolizes arachidonic acid to produce lipid mediators of inflammation.
5-lipoxygenase is overexpressed in human prostate carcinomas and 5-LO inhibition induces
apoptosis in prostate cancer cell lines. We hypothesized that as in human prostate carcinoma, 5LO would be overexpressed in canine prostate carcinoma and may be important in the
pathogenesis of the disease. We evaluated 5-LO expression in hyperplastic, inflamed, and
neoplastic canine prostate tissues.
Western blots were performed to validate the antibody in canine tissue and to evaluate 5-LO
expression in prostate carcinomas compared to benign prostatic hyperplasia (BPH) tissues.
Subsequently, immunohistochemistry was performed to evaluate 5-LO expression in BPH,
suppurative prostatitis, prostate carcinoma, and metastatic tissues. Stain distribution and intensity
were determined for the epithelial and stromal components of all samples.
Western blot analysis validated the antibody and demonstrated 5-LO expression in both prostate
carcinoma and BPH tissue. 5-lipoxygenase staining was not significantly different between
epithelial and stromal cells in BPH, prostatitis, and prostate carcinoma. However, 5-LO
immunoreactivity was markedly decreased in intensity and distribution within the epithelial and
stromal components of metastatic lesions compared to primary prostate carcinomas (p<0.015).
5-lipoxygenase is expressed in hyperplastic, inflammatory, and neoplastic lesions of the canine
prostate. The similarities in 5-LO expression between BPH, prostatitis, and prostate carcinoma
suggest that there is not differential expression of the enzyme in these conditions. Decreased
expression of 5-LO in metastatic lesions may indicate down-regulation or altered expression of the
enzyme with progression of canine prostate carcinoma to a metastatic phenotype.
PHARMACOKINETICS OF A NOVEL INTRANASAL MIDAZOLAM GEL IN DOGS.
JS Eagleson,1 M Kent,1 AC Freeman,1 AC Haley,1 P Nghiem,1 AC Durden,1 D Strong,2 S White,2
SJ Schatzberg,1 SR Platt.1 University of Georgia Colleges of Veterinary Medicine1 and Pharmacy,2
Athens, GA.
The pharmacokinetics of a novel bioadhesive gel formulation of midazolam administered
intranasally (IN) were compared to those of parenteral midazolam solution administered IN,
intravenously (IV) and rectally.
Ten healthy beagles were randomly assigned to treatment groups in a crossover design.
Parenteral midazolam solution was administered IV to group 1, rectally to group 2, and IN to
group 3 (0.2 mg/kg). A 0.2 % carbopol midazolam gel formulation was administered IN to group
4 (0.2 mg/kg). Blood was collected from the jugular vein of each dog into tubes containing
lithium heparin before and 3, 6, 9, 12, 15, 20, 30, 60, 120, 240, and 480 minutes following
midazolam administration. The protocol was repeated 4 times with a 7-day wash out period
between administrations. Each dog received all 4 forms of midazolam administration. Plasma
concentration of midazolam was determined using high performance liquid chromatography.
Mean (+/- SD) peak plasma concentrations (Cmax) of midazolam were 1.82 (+/- 0.278) μg/mL
(IV), 0.45 (+/- 0.09) μg/L (IN gel), 0.21 (+/- 0.02) μg/mL (IN solution) and 0.15 (+/− 0.01) μg/mL
(rectal). The Cmax following IN gel administration was significantly higher than IN solution and
rectal administration (p<0.0001). Mean plasma concentrations of midazolam following IV
administration were significantly higher at 3, 6 , 9 and 12 minutes post-delivery when compared to
all other methods of delivery (p<0.0001). Mean plasma concentrations of midazolam following
IN gel administration were significantly higher at 6, 9 and 12 minutes post-delivery when
compared to IN solution and rectal administration (p<0.0001). Time (+/- SD) to peak
concentration (Tmax) was <3 minutes (IV), 11.70 (+/- 2.63) minutes (IN gel), 17.50 (+/- 2.64)
minutes (IN solution) and 39 (+/- 14.49) minutes (rectal). The Tmax following rectal
administration was significantly longer than both IN gel and IN solution administration
(p<0.0001). Mean bioavailability of midazolam was 70.4% (IN gel), 52.0% (IN solution) and
49.0% (rectal). Bioavailability following IN gel administration was significantly higher than IN
solution and rectal administration (p<0.0001). The half lives (+/- SD) were 121 (+/- 25.74)
minutes (IV), 119.61 (+/- 22.83) minutes (IN gel), 118.66 (+/- 18.84) minutes (IN solution) and
115.83 (+/- 15.56) minutes (rectal).
Intranasal midazolam gel was superior to IN solution administration and rectal administration
with respect to peak plasma concentration and bioavailability. Although IV administration of
benzodiazepines is still considered the treatment of choice for short acting anti-convulsant therapy,
intranasal administration of this novel midazolam gel may be useful for treatment of seizures in
dogs by owners or when intravenous access is not readily available.
Combination of molecular and bioinformatics tools for detection of antigenic variant
strains of Infectious Bursal Disease Virus
Vijay Durairaj & Egbert Mundt
Poultry Diagnostic and Research Center,
College of Veterinary Medicine,
University of Georgia, Athens.
Infectious bursal disease (IBD) is a highly contagious and immunosuppressive disease in
chickens and is a worldwide problem. It is caused by Infectious Bursal Disease Virus (IBDV)
belonging to family Birnaviridae. Antigenic different strains of IBDV are isolated in poultry
flocks. This is likely caused by antigenic drift due to existing vaccination programs. These new
antigenic variant strains can break through current vaccination programs and cause devastating
losses to the poultry industry. The aim of our research is to detect, identify and characterize new
antigenic variant strains of IBDV. We apply reverse genetics as a diagnostic tool in identifying
these strains and characterize them based on their reaction with panel of monoclonal antibodies.
By applying this technique, we were able to identify strains of IBDV with an unusual reaction
pattern based on their reactivity with the panel of monoclonal antibodies. Our studies indicate
that IBDV strains are circulating in poultry flocks which are antigenic different from known
IBDV isolates. These new isolates might become candidates for future IBDV vaccines to induce
protection of young chickens against this important disease in poultry.
POTENTIAL ROLE OF HEME OXYGENASE-1 IN THE ENHANCEMENT
INFLAMMATION AND ENSUING NEUROTOXICITY CAUSED BY MANGANESE
OF
CA Dodd, II Georgieva, and NM Filipov. Physiology and Pharmacology, University of Georgia,
Athens, GA.
In humans, excess exposure to manganese (Mn) is associated with a Parkinsonian type
neurological disorder. Several studies provide evidence for the role of glial-derived (microglia and
astrocytes) inflammatory products in the development of Mn neurotoxicity. In fact, Mn was found
to potentiate the production of inflammatory cytokines induced by the inflammagen
lipopolysaccharide (LPS) in microglia cells. Inducible heme oxygenase (HO-1), an enzyme
responsible for the cleavage of the oxidant heme into biliverdin, carbon monoxide, and iron (Fe),
is increased in response to oxidative stress caused by various toxicants, including metals, and has
been reported to play a role in the regulation of inflammation. While the initial function of HO-1
appears to be anti-inflammatory, excessive induction of HO-1 can deregulate Fe homeostasis and
contribute to the increased production of free radicals and potentially inflammatory mediators.
Expression of HO-1 is increased in response to LPS; however, at present the effect of Mn on HO1 has yet to be determined. This study was designed to examine the effect of Mn on HO-1
induction and on LPS induced HO-1 in microglia (N9) and dopaminergic neuronal (N27) cells. N9
microglia and N27 neuronal cells were exposed to either LPS (100 ng/ml), Mn (100 µM), or
combined Mn+LPS for 24 hours. In microglia, while Mn had minimal effects on its own,
induction of HO-1 (protein and mRNA) by LPS was potentiated by Mn. The increase in HO-1
was not due to increased intracellular Mn, but was accompanied by a small but significant increase
in Fe concentration and an increase in mRNA for iNOS, and the inflammatory cytokines TNF-α
and IL-6. In contrast, neither LPS nor Mn had any effect on HO-1 in the N27 neuronal cells.
Moreover, combined Mn+LPS treatment caused a small, but significant decrease in HO-1. These
results indicate that Mn potentiates the induction of HO-1 by LPS in microglia, but not in neuronal
cells. Because the microglial increase in HO-1 is accompanied by an increase in inflammatory
mediators as well as Fe, there is a possibility that in the presence of Mn, HO-1 acts as a prooxidant and is involved in the enhanced cytokine production by Mn-exposed activated microglia
that has been reported previously. Future studies incorporating inhibition of HO-1 will enable us
to uncover whether induction of HO-1 is important for the increase in inflammatory cytokines
observed with Mn+LPS treatment in microglia cells.
INFLUENCE OF SALINE, ACEPROMAZINE OR MIDAZOLAM AS PREMEDICATION ON
THE QUALITY OF ANESTHESIA IN ISOFLURANE ANESTHETIZED NEW ZEALAND
WHITE RABBITS Cremer J1, Allworth L2, Harvey S2, Hofmeister E3.
1
Department of Large Anima Medicine, 2University Research Animal Resources and Department
of Population Health, 3Department of Small Animal Medicine, University of Georgia College of
Veterinary Medicine, Athens, Georgia
Certain procedures in laboratory animals (i.e. blood sampling), require short term anesthesia or
sedation. Acepromazine and midazolam are commonly used sedatives in small animals, however,
their sedative effects have not been evaluated in rabbits. The purpose of this study was to
investigate the effects of premedication with acepromazine, midazolam, or saline on sedation,
induction, and recovery in normal New Zealand White (NZW) rabbits.
Twelve adult female NZW rabbits were randomly assigned to one of three groups in a crossover
design: sterile saline 0.2 ml/kg, acepromazine 1mg/kg, and midazolam 0.5 mg/kg, all given IM.
Thirty minutes after premedication the rabbits were anesthetized by mask with 5% isoflurane in
2L/min oxygen. Induction was considered completed when the animal could be placed in lateral
recumbency. Quality of sedation, induction, recovery, and anesthesia were evaluated using a VAS,
and data was analyzed with a one-way ANOVA.
No significant difference was seen in quality of sedation (mean VAS scores: saline: 2.7+/- 3.0,
midazolam: 3.1+/- 3.8, acepromazine: 5.8+/- 3.0) and quality of induction (saline: 4.6+/- 3.9,
midazolam: 5.2+/- 3.0, acepromazine: 4.2+/- 2.4). Induction time was similar for all three
treatments (saline: 280s+/- 98, midazolam: 230s+/- 232, acepromazine: 264s+/- 215). No
difference was seen in quality of recovery (saline: 8.0+/- 1.7, midazolam: 7.0+/- 2.7,
acepromazine: 8.1+/- 1.2) or overall quality of anesthesia (saline: 6.8+/- 2.5, midazolam: 7.4+/2.5, acepromazine: 7.5+/- 1.1).
There was no effect of premedication with acepromazine or midazolam on sedation, induction
time or quality, or recovery time or quality in laboratory-used NZW rabbits.
EFFECT OF ALDOSTERONE BLOCKADE ON CHRONIC ALLOGRAFT NEPHROPATHY IN RATS
COGAR SM; SCHMIEDT CW; CHESSMAN CH; BROWN CA; and HURLEY DJ
College of Veterinary Medicine, University of Georgia
The long-term efficacy of human and feline renal transplantation is adversely impacted by the occurrence of
chronic allograft nephropathy (CAN), a common condition which ultimately results in deterioration of graft function
and resurgence of systemic signs of kidney disease. Recent studies suggest aldosterone potentiates renal
parenchymal damage in patients with kidney disease; however its role in modulating CAN is unknown. The purpose
of this study was to evaluate the effect of an aldosterone receptor blockade on the severity of lesions associated with
CAN in a F344 to Lewis rat renal transplantation model. We hypothesized that the inhibition of aldosterone will
result in a reduction in renal allograft damage evaluated by allograft function, characteristic histopathologic lesions,
and activation of proinflammatory and profibrotic genes.
Lewis rats were divided into 4 groups. Two groups underwent heterotropic kidney transplantation followed by a
bilateral native nephrectomy during 2 staged procedures approximately 10 days apart. Donors for all transplanted
kidneys were male F344 rats. Kidney transplantation was performed in an end-to-side anastomosis. The cold
ischemia and anastomosis time was also recorded for each rat. Following renal transplantation, 1 group received
spironolactone (Group 1, 10 mg/kg/day) and the other received water (Group 2, 0.25 ml/day). Groups 3 and 4 were
right sided nephrectomy control groups to control for reduction in nephron number. Of these 2 groups, 1 received
spironolactone at the same dose and the other received water. Each group was medicated daily for 112 days. All
rats received cyclosporine daily for 10 days following surgery. All medication was administered via gavage. Serum
creatinine and urine protein : urine creatinine (UP:UC) was measured at set intervals and at the conclusion of the
study. Kidneys were also obtained for histopathologic scoring for the lesions associated with CAN based on the
Baniff 97’ scoring system. The remained of the kidney was stored in a -80°C freezer. These samples were thawed
at a later date and underwent RT-qPCR to quantitate the up regulation of pro-inflammatory and pro-fibrotic genes.
Statistical analysis was performed to determine significance of measured parameters between groups.
Cold ischemia time (p=0.33) and anastomosis time (p=0.34) were not statistically different between transplant
groups. In general there was an increase in UP:UC values following transplantation in both groups. When the 16
weeks end-point UP:UC levels were compared to the baseline there was a significant increase in both transplant
groups, but a difference between groups was not observed at any time point. Regarding serum creatinine
concentration there were significant differences between weeks (p<0.001) and groups (p<0.001). Notably, serum
creatinine was significantly different between the nephrectomy and the transplant groups 1 day after transplantation,
but was not significantly different between the two transplant groups at any time points. In general the
transplantation groups had an increase in serum creatinine compared to baseline following transplantation at
multiple points during the 16 week study when compared to baseline. There were no differences in the nephrectomy
groups at any time. Histological and gene activation data analysis between groups are pending.
In conclusion, the data analyzed from the urine protein : urine creatinine and serum creatinine suggests that
aldosterone inhibition with spironolactone does not result in improved allograft function over 16 weeks when
compared to either nephrectomy and transplant control rats. However, analysis of histopathologic and RT-qPCR
data is still pending.
EVALUATION OF THE POSITIVE PREDICTIVE VALUE OF SERUM PROTEIN
ELECTROPHORESIS BETA-GAMMA BRIDGING FOR HEPATIC DISEASE IN THREE
DOMESTIC ANIMAL SPECIES.
MS Camus, PM Krimer, FS Almy, BE LeRoy.
College of Veterinary Medicine, University of Georgia, Athens, GA
Based on human studies conducted in the 1950’s, beta-gamma bridging (β-γ bridging) on protein
electrophoresis is presented in the human and veterinary literature as virtually pathognomonic for
hepatic disease. However, the criteria for β-γ bridging are not defined and very few veterinary
publications exist to support a relationship between β-γ bridging and liver disease. The goal of
this retrospective study was to confirm or repudiate an association between the two by measuring
the positive predictive value of β-γ bridging for liver disease. All electrophoretograms generated
at the University of Georgia between 1994 and 2008 were evaluated for the presence of β-γ
bridging. β-γ bridging was identified if there were 1) an albumin to globulin ratio below the
established reference interval; 2) indistinct separation/demarcation between all β and γ globulin
fractions or between the β2 and γ fractions, with a negative shoulder slope of <5%; and 3)
predominance of γ proteins. There were 237 electrophoretograms examined, of which 25 cases
met the inclusion criteria for a β-γ bridge. Of these cases, 8/25 (32%) had hepatic disease based on
biochemistry, cytology, histopathology, and/or necropsy findings, while 9/25 (36%) had infectious
diseases. The positive predictive value of β-γ bridging for hepatic disease was determined to be
32.0% with a 95% confidence interval of 15.0-53.5% (p<0.001). The positive predictive value of
this phenomenon for infectious disease was determined to be 36.0% with a 95% confidence
interval of 18.0-57.5% (p<0.001). Though β-γ bridging can be found in some cases of hepatic
pathology, it is not “pathognomonic” for liver diseases and is as frequently found with infectious
diseases.
THE EFFECT OF PAX6 ON ANTERIOR SEGMENT ANATOMY, CORNEAL WOUND
HEALING, LIMBAL PROGENITOR CELLS, AND CORNEAL VASCULARIZATION.
PJ Accola, PA Moore, KP Carmichael, JD Lauderdale
The purpose of this study was to establish a corneal wounding model and characterize corneal
wound healing and anterior segment pathology in Small eye (Sey) (Pax6+/-) mice.
The anterior segment of Wild Type (WT) (n=79) and Sey (n=40) mice (age 2-3 months) were
examined with slit lamp biomicroscopy. Corneal wounding with n-heptanol was performed and
healing evaluated on days 1, 2, 3 (if fluorescein positive on day 2), 4, 7, 14, and 28 post wounding.
Each day, 3-5 mice were euthanized. Anterior segment histopathology was characterized.
Immunohistochemistry (p63, sVEGFR-1) was performed. Days to negative fluorescein staining
were compared using the Chi-Square test. The number of p63 staining basal cells was recorded
and statistically evaluated by ANOVA. Staining for sVEGFR1 in non-wounded eyes was recorded
as positive or negative and anatomic location reported.
Ophthalmic examination and histopathologic findings in Sey mice included corneal opacity,
corneal vascularization, iris hypoplasia, and cataract. The wounding protocol resulted in precise
and accurate removal of corneal epithelium. There was no significant difference in the amount of
cornea wounded in WT and Sey mice (p>0.05). There was a statistically significant delay in
corneal wound healing in Sey mice at days 2 and 3 when compared to WT mice (p<0.05). There
was no significant difference in p63 staining (p>0.05). All mice exhibited comparable sVEGFR1
staining.
Histopathologic findings are consistent with impaired anterior segment development. The
delayed corneal healing is unlikely to be due to a deficiency in p63 cellular expression. The
comparable expression of sVEGFR1 suggests that it alone is likely not responsible for the corneal
vascularization present in Sey mice.
NEMATODE ION CHANNELS AS TARGETS FOR ANTHELMINTIC COMPOUNDS
SM Williamson, HM Bennett, S McCavera, AJ Wolstenholme
Dept. Infectious Diseases, College of Veterinary Medicine, University of Georgia and Dept of
Biology & Biochemistry, University of Bath, Bath, U.K.
Nematode parasites have a huge impact on the health of both companion animals and livestock.
Antiparasitics are the largest-selling class of veterinary drugs. Many of these drugs act by binding
to the ligand-gated ion channels of the nematode, causing paralysis and cessation of feeding and
egg production by the parasite. Despite decades of routine administration of these compounds, the
molecular basis underlying the exact mode of action of these drugs is still poorly understood. The
aim of our work is to gain a more complete understanding of the drug targets and understand how
nematode parasites become resistant to dewormers. We use molecular biology techniques to study
the nematode ion channels targeted by ivermectin and the cholinergic anthelmintics such as
pyrantel and levamisole. We have studied the glutamate-gated chloride channels of Haemonchus
contortus, a gastrointestinal parasite of small ruminants, to determine the molecular basis of
ivermectin sensitivity; we have also studied the nicotinic acetylcholine receptors of Ascaris suum,
the large roundworm of pigs, to investigate the molecular basis of parasite sensitivity to
levamisole, pyrantel and oxantel. We have amplified and cloned the ion-channel subunit
sequences from the parasites, then used a heterologous expression system to produce recombinant
parasite ion channels which can be used for in vitro pharmacological studies. The anthelmintics
open the channels, resulting in uncontrolled ion fluxes that, in vivo, cause the paralysis and
expulsion or death of the worm. These studies have identified which ion channel subunits in these
parasites are targeted by the anthelmintic drugs, and have also demonstrated that heterologous
expression of parasite ion channels is an extremely useful method for both investigating the effects
of potential resistance mutations on drug sensitivity in vitro, and also as a tool for screening novel
compounds for potential anthelmintic activity.
RETROSPECTIVE STUDY ON FINDINGS AND PROGNOSIS IN MIDDLE CARPAL
JOINT DIAGNOSTIC ARTHROSCOPIES: 68 CASES
Fernando Canonici1, Valeria Albanese1,2
1:Equine Practice equine hospital, Rome, Italy. 2:University of Georgia VTH, Athens GA
Objectives. This retrospective study is aimed to show findings and correlated prognosis in
arthroscopies performed on middle carpal joints of racehorses for diagnostic purpose in a
private practice over 4 years.
Material and Methods. The study was carried on surgery records from 68 horses elected for
diagnostic arthroscopy of the middle carpal joint(s). Horses presented for lameness or poor
performance, localized to the middle carpal joint by intraarticular anesthesia, and refractory to
conservative treatment. Condition to be included in the study was to to show little or no
radiographic findings on a full set of radiographs of the carpus, including skyline views.
The follow-up information was obtained from official race records. Both return to racing and
performance were recorded. To evaluate the outcome, performance records after and before
surgery have been compared, where not available, racing class was used. For unraced horses, a
comparison was made with matched siblings. The outcome was considered positive when at
least one of the following criteria was met: same or better performance or racing class after
surgery, same or better performance or racing class than matched siblings.
Pathological findings were classified retrospectively based on surgery reports and
intraoperatory pictures, depending on their location and type. Osteochondral lesions were
graded as I (articular cartilage only), II (articular cartilage and subchondral bone) and
III(fragment). Soft tissue lesions, namely medial palmar intercarpal ligament tears, were
graded as I (less than 50% of the ligament torn), II(over 50%) and III(100%)
Results. 67/68 horses resumed racing after surgery, 49 of which had raced before. 33/49 had
same or superior performance than before, whereas 16/49 had lower. Of 18 that had not raced
before surgery, 9 had same or superior performance than siblings, 5 had lower and 4 could not
be evaluated.
116 lesions were identified in the middle carpal joints examined: 48.2% of them were
localized on the radial facet of the third carpal bone, 23.3% were MPIL tears and 22.4% were
located on the distal radial carpal bone.
Osteochondral lesions appear to be associated with a fair to favourable prognosis, whilst
horses with MPIL tears have only 50% chance to go back to race at the same level they had
before.
Conclusions. Our study gives an overview of the diverse conditions that can affect the middle
carpal joint yielding little or no radiographic changes, and provides informations regarding
their prognoses.
BROADLY REACTIVE METHODOLOGIES FOR PATHOGEN DETECTION IN CANINE
GRANULOMATOUS AND NECROTIZING MENINGOENCEPHALITIS. RM Barber1, SJ
Schatzberg1, Q Li1, K Greer2, B Porter3, PPVP Diniz4, A Allison1, M May5, JM Levine3, GJ
Levine3, EB Breitschwerdt4, AJ Birkenheuer4, S Roune6, SR Platt1, M Kent1, LJ Anderson6, D
Brown5, S Tong6. 1. University of Georgia, College of Veterinary Medicine (CVM), Athens, GA.
2. Indiana University East, School of Natural Sciences and Mathematics, Richmond, IN. 3. Texas
A&M University, CVM, College Station, TX. 4. North Carolina State University, CVM, Raleigh,
NC. 5. University of Florida, CVM, Gainesville, FL. 6. Centers for Disease Control and
Prevention, Division of Viral Diseases, Atlanta, GA.
Granulomatous and necrotizing meningoencephalitis, GME and NME respectively, are clinically
important idiopathic inflammatory disorders of the canine central nervous system. These
disorders share histopathological similarities with several infectious meningoencephalitides of
dogs and people, and identification of infectious etiologies could lead to directed therapy and
improved clinical outcomes. This investigation evaluated brain tissue from histopathologically
confirmed cases of GME and NME with broadly reactive pathogen identification methodologies
to determine if viral or bacterial pathogens are associated with these disorders.
Brain tissues from 6 cases of GME and 25 cases of NME were evaluated. Total nucleic acids
were extracted for polymerase chain reaction (PCR) and reverse transcriptase-PCR. Nucleic acid
integrity was confirmed by amplification of canine housekeeping genes, and appropriate positive
and negative controls were included in all reactions. Samples were evaluated by broadly reactive
consensus degenerate hybrid oligonucleotide PCR assays to detect adeno-, herpes-, alpha-, flavi-,
bunya-, corona-, borna-, rhabdo-, paramyxo-, polyoma- and picornaviruses and broadly reactive
degenerate PCR assays for vector-borne microorganisms in the genera Ehrlichia, Anaplasma,
Spotted Fever Group Rickettsia, Bartonella and Borrelia. Additionally, homogenized brain tissue
from each case was inoculated onto XC cell cultures and supernatants from cultures with a
cytopathic effect were pooled for sequence-independent single primer amplification (SISPA); the
presence of SISPA-identified bacterial species was confirmed by culture and 16S PCR.
Comparison of PCR amplicon sequences with those in GenBank was used to identify organisms.
Although the results of this investigation were predominantly negative, Bartonella vinsonii
subsp. berkhoffii was identified in brain tissue from one dog with GME and Mycoplasma canis
was identified in 6/31 cases by 16S PCR and 8/10 cases by culture.
The primarily negative results suggest the pathogens evaluated in this investigation are not
commonly detected in brain tissue from dogs with GME and NME. Further investigation is
warranted to determine the importance of identified Bartonella and Mycoplasma organisms, and
follow-up studies utilizing immunohistochemistry, fluorescent in situ hybridization and electron
microscopy are ongoing.
GASTRO-INTESTINAL TRANSIT TIME IN THE RHINOCEROS
Kruger M, Betts E, Virgo J, Ball BA, Pitts N, and Fayrer-Hosken RA
Veterinary Wildlife Services, Kruger National Park, Skukuza, RSA, Department of Population
Health and Reproduction, University of California, Davis, Davis, CA, Department of Large
Animal Medicine, College Veterinary Medicine, University of Georgia, Athens GA.
Kruger National Park (KNP) has the majority of the world’s white rhinoceroses. KNP
captures and relocates between eighty to one hundred rhinoceroses annually. Of these
rhinoceroses, forty to fifty are boma-confined or boma-trained. The relocation of white
rhinoceros is essential to re-establish rhinoceros populations and to increase their numbers in
the world. The relocation is however stressful to the animals and this stress may be
detrimental to their subsequent adaptation and reproduction. The aim of this research was to
measure gastrointestinal transit times in rhinoceroses that have been anesthetized. These
studies were extrapolated from preliminary studies in the horse. Knowing the intestinal transit
times will allow evaluation of fecal corticoid profiles. The profile will quantitate stress in
captured white rhinoceroses. Glitter and Kayro syrup were administered to two bomahabituated rhinoceroses (Klokkies and Rosa) before reversal of their anesthesia. Klokkies
(Figure 1) and Rosa (Figure 2) were administered saline (control) or ACTH (Synacthen®, 0.5
(Rosa and Klokkies) or 1.5 (Klokkies) IU/kg) in experimental trials.
The intestinal transit times appeared to be delayed at 0.5 IU/kg Synacthen® administration,
but this was not supported at 1.5 IU/kg (Klokkies). The treatments were not statistically
(p<0.05) affected by time of day, weather for the day, volume of injection, type and amount of
anesthesia/sedative. The conclusion is that the rhinoceroses intestinal transit times are
variable, but that colored glitter provides a comprehensive profile. This will allow
interpretation of fecal corticoid profiles.
EFFICACY OF SELECT DISINFECTANTS AT INACTIVATING RANAVIRUS. LK Bryan,
CA Baldwin, MJ Gray, DL Miller. University of Georgia, College of Veterinary Medicine,
Athens, GA. University of Georgia, Veterinary Diagnostic and Investigational Laboratory, Tifton,
GA. University of Tennessee, Center for Wildlife Health, Knoxville, TN.
Ranavirus is an emerging viral disease affecting amphibians and reptiles. Because survival time
of the virus outside a host remains unknown, disinfection of field equipment is essential to prevent
the spread of Ranavirus from infected to naïve populations. A 0.75% solution of chlorhexidine
diacetate (Nolvasan®) and a 1% solution of sodium hypochlorite (bleach) at 10 min contact time
are currently recommended for direct use with amphibians. Long contact times and high
disinfectant concentrations increase the risk of toxicity to wildlife. This study was instigated to
determine if current disinfection protocols used against Ranavirus are effective and to determine
the most efficacious concentration and contact time for several widely used disinfectants: bleach,
Nolvasan®, potassium peroxymonosulfate (Virkon S®) and potassium permanganate.
The efficacy of Nolvasan® (0.25, 0.75 and 2.0%), bleach (0.2, 1.0, 3.0 and 5.0%), and
Virkon S® (1.0%) solutions at inactivating Ranavirus at 1 and 5 min contact durations was
determined by calculating the reduction in viral titer, from a known starting concentration, via
plaque assay for each disinfectant at each concentration and contact time. Potassium permanganate
(2.0 and 5.0 ppm) was also tested, but with a 60 min contact time. Equal parts of a wild-type
Ranavirus and a disinfectant were allowed to react for 1, 5 or 60 min at room temperature and
were then processed through gel column centrifugation to remove residual disinfectant. The
mixtures were then applied in ten-fold dilutions (10-2 to 10-8) to 6-well plates seeded with fat-head
minnow cell cultures and were incubated for 6 d at room temperature. Viral titer was determined
after 6 d by staining the plates with crystal violet and counting the plaques present in each well. A
minimum 3 log10 reduction (99.9% inactivated) in titer was necessary for a disinfectant to be
considered effective.
Nolvasan® at 0.75 and 2.0% and bleach at 3.0 and 5.0% concentration were effective for 1 and 5
min. Virkon S® was effective for both contact durations, but KMnO4 was not effective at either
concentration tested at 60 min contact time.
These results support the use of 0.75% Nolvasan® as an effective disinfectant rinse for
amphibians, even with 1 minute contact time. Bleach is only effective at short contact times at a
concentration of at least 3%, an unsafe concentration for direct use with amphibians. Virkon S®
and higher concentrations of bleach and Nolvasan® can be used in non-contact situations, but
toxicity to wildlife should be considered before use. Potassium permanganate was not effective
and should not be used as a disinfectant for Ranavirus.
EVALUATION OF THE POSITIVE PREDICTIVE VALUE OF SERUM PROTEIN
ELECTROPHORESIS BETA-GAMMA BRIDGING FOR HEPATIC DISEASE IN THREE
DOMESTIC ANIMAL SPECIES.
MS Camus, PM Krimer, FS Almy, BE LeRoy.
College of Veterinary Medicine, University of Georgia, Athens, GA
Based on human studies conducted in the 1950’s, beta-gamma bridging (β-γ bridging) on protein
electrophoresis is presented in the human and veterinary literature as virtually pathognomonic for
hepatic disease. However, the criteria for β-γ bridging are not defined and very few veterinary
publications exist to support a relationship between β-γ bridging and liver disease. The goal of
this retrospective study was to confirm or repudiate an association between the two by measuring
the positive predictive value of β-γ bridging for liver disease. All electrophoretograms generated
at the University of Georgia between 1994 and 2008 were evaluated for the presence of β-γ
bridging. β-γ bridging was identified if there were 1) an albumin to globulin ratio below the
established reference interval; 2) indistinct separation/demarcation between all β and γ globulin
fractions or between the β2 and γ fractions, with a negative shoulder slope of <5%; and 3)
predominance of γ proteins. There were 237 electrophoretograms examined, of which 25 cases
met the inclusion criteria for a β-γ bridge. Of these cases, 8/25 (32%) had hepatic disease based on
biochemistry, cytology, histopathology, and/or necropsy findings, while 9/25 (36%) had infectious
diseases. The positive predictive value of β-γ bridging for hepatic disease was determined to be
32.0% with a 95% confidence interval of 15.0-53.5% (p<0.001). The positive predictive value of
this phenomenon for infectious disease was determined to be 36.0% with a 95% confidence
interval of 18.0-57.5% (p<0.001). Though β-γ bridging can be found in some cases of hepatic
pathology, it is not “pathognomonic” for liver diseases and is as frequently found with infectious
diseases.
UNDERSTANDING THE INTRODUCTION OF ANTIMICROBIAL RESISTANCE
THROUGH THE PET TRADE: USING THE TOKAY GECKO (Gekko gecko) AS A MODEL.
C L Casey, S M Hernandez, M J Yabsley, S Sanchez. University of Georgia College of Veterinary
Medicine, Athens, GA
Wildlife traded for the pet market has the potential to contribute to the threat of increased
prevalence of multidrug-resistant bacteria (Ahmed et al. 2007). We propose the pet trade,
particularly the conditions under which wildlife is captured and handled, has the potential to
influence the rate of antimicrobial resistance development. Through experimental manipulations,
using the Tokay gecko as a model for the pet trade, we will investigate 1) the antimicrobial
resistance of Enterobacteriacae and Salmonella sp. isolated in their fecal bacteria, 2) how
antimicrobial resistance changes after mimicking conditions under which reptiles are imported for
the pet trade, and 3) the antimicrobial genes responsible for conferring phenotypic resistance.
To accomplish this we will import wild Tokay geckos from their native range in Central Java,
Indonesia. Geckos will be housed individually after collection and during import. Fecal samples
will be collected upon arrival, prior to experimental manipulation. We will use four treatments
where founder populations exist at varying densities. We will collect fecal samples over time as
we increase animal density by adding new individuals. We will determine the phenotypic
antimicrobial resistance using Minimum Inhibitory Concentration (MIC) plates which are
impregnated with antibiotics. We will use statistical analysis to determine multiple relationships
that concern antimicrobial resistance development and conditions modeling the pet trade. We will
utilize PCR, to determine the presence of resistance genes that confer antimicrobial resistance.
In a group of geckos received from Indonesia in March of 2009, preliminary data showed that:
1) individually housed geckos harbored Enterobacteriacae that were resistant to tetracycline,
ampicillin, cephalothin, amoxicillin w/ clauvulinic acid and ticarcillin. This provides evidence that
antibiotic resistance of commensal, non-pathogenic bacteria is present; 2) the prevalence of
Salmonella sp. of these geckos indicates that these animals harbor both Salmonella sp. types
associated with livestock (Serogroups D, F), in addition to Salmonella sp. previously reported in
healthy reptiles (Serogroups H, O). This supports our theory that these Tokay geckos have indeed
come in contact with, and have acquired enteric bacteria. There is evidence that suggests stressful
conditions of captivity increases the shedding of salmonella in reptiles (Richards et al. 2004) and
the exchange of antimicrobial resistance genes in non-pathogenic bacteria (Sorum and Sunde
2001). From this evidence we concluded that animals that experience similarly stressful conditions
associated with the pet trade also have a higher probability of shedding fecal bacteria and acting as
reservoirs for exchange of antimicrobial resistance. This conclusion with an understanding of
mobile genetics is essential for determining the potential impact the pet trade has on the
development of antimicrobial resistance. This research is novel because it explores a new
mechanism by which antibiotic resistance is dispersed globally.
EFFECT OF ALDOSTERONE BLOCKADE ON CHRONIC ALLOGRAFT NEPHROPATHY IN RATS
COGAR SM; SCHMIEDT CW; CHESSMAN CH; BROWN CA; and HURLEY DJ
College of Veterinary Medicine, University of Georgia
The long-term efficacy of human and feline renal transplantation is adversely impacted by the occurrence of
chronic allograft nephropathy (CAN), a common condition which ultimately results in deterioration of graft function
and resurgence of systemic signs of kidney disease. Recent studies suggest aldosterone potentiates renal
parenchymal damage in patients with kidney disease; however its role in modulating CAN is unknown. The purpose
of this study was to evaluate the effect of an aldosterone receptor blockade on the severity of lesions associated with
CAN in a F344 to Lewis rat renal transplantation model. We hypothesized that the inhibition of aldosterone will
result in a reduction in renal allograft damage evaluated by allograft function, characteristic histopathologic lesions,
and activation of proinflammatory and profibrotic genes.
Lewis rats were divided into 4 groups. Two groups underwent heterotropic kidney transplantation followed by a
bilateral native nephrectomy during 2 staged procedures approximately 10 days apart. Donors for all transplanted
kidneys were male F344 rats. Kidney transplantation was performed in an end-to-side anastomosis. The cold
ischemia and anastomosis time was also recorded for each rat. Following renal transplantation, 1 group received
spironolactone (Group 1, 10 mg/kg/day) and the other received water (Group 2, 0.25 ml/day). Groups 3 and 4 were
right sided nephrectomy control groups to control for reduction in nephron number. Of these 2 groups, 1 received
spironolactone at the same dose and the other received water. Each group was medicated daily for 112 days. All
rats received cyclosporine daily for 10 days following surgery. All medication was administered via gavage. Serum
creatinine and urine protein : urine creatinine (UP:UC) was measured at set intervals and at the conclusion of the
study. Kidneys were also obtained for histopathologic scoring for the lesions associated with CAN based on the
Baniff 97’ scoring system. The remained of the kidney was stored in a -80°C freezer. These samples were thawed
at a later date and underwent RT-qPCR to quantitate the up regulation of pro-inflammatory and pro-fibrotic genes.
Statistical analysis was performed to determine significance of measured parameters between groups.
Cold ischemia time (p=0.33) and anastomosis time (p=0.34) were not statistically different between transplant
groups. In general there was an increase in UP:UC values following transplantation in both groups. When the 16
weeks end-point UP:UC levels were compared to the baseline there was a significant increase in both transplant
groups, but a difference between groups was not observed at any time point. Regarding serum creatinine
concentration there were significant differences between weeks (p<0.001) and groups (p<0.001). Notably, serum
creatinine was significantly different between the nephrectomy and the transplant groups 1 day after transplantation,
but was not significantly different between the two transplant groups at any time points. In general the
transplantation groups had an increase in serum creatinine compared to baseline following transplantation at
multiple points during the 16 week study when compared to baseline. There were no differences in the nephrectomy
groups at any time. Histological and gene activation data analysis between groups are pending.
In conclusion, the data analyzed from the urine protein : urine creatinine and serum creatinine suggests that
aldosterone inhibition with spironolactone does not result in improved allograft function over 16 weeks when
compared to either nephrectomy and transplant control rats. However, analysis of histopathologic and RT-qPCR
data is still pending.
INFLUENCE OF SALINE, ACEPROMAZINE OR MIDAZOLAM AS PREMEDICATION ON
THE QUALITY OF ANESTHESIA IN ISOFLURANE ANESTHETIZED NEW ZEALAND
WHITE RABBITS Cremer J1, Allworth L2, Harvey S2, Hofmeister E3.
1
Department of Large Anima Medicine, 2University Research Animal Resources and Department
of Population Health, 3Department of Small Animal Medicine, University of Georgia College of
Veterinary Medicine, Athens, Georgia
Certain procedures in laboratory animals (i.e. blood sampling), require short term anesthesia or
sedation. Acepromazine and midazolam are commonly used sedatives in small animals, however,
their sedative effects have not been evaluated in rabbits. The purpose of this study was to
investigate the effects of premedication with acepromazine, midazolam, or saline on sedation,
induction, and recovery in normal New Zealand White (NZW) rabbits.
Twelve adult female NZW rabbits were randomly assigned to one of three groups in a crossover
design: sterile saline 0.2 ml/kg, acepromazine 1mg/kg, and midazolam 0.5 mg/kg, all given IM.
Thirty minutes after premedication the rabbits were anesthetized by mask with 5% isoflurane in
2L/min oxygen. Induction was considered completed when the animal could be placed in lateral
recumbency. Quality of sedation, induction, recovery, and anesthesia were evaluated using a VAS,
and data was analyzed with a one-way ANOVA.
No significant difference was seen in quality of sedation (mean VAS scores: saline: 2.7+/- 3.0,
midazolam: 3.1+/- 3.8, acepromazine: 5.8+/- 3.0) and quality of induction (saline: 4.6+/- 3.9,
midazolam: 5.2+/- 3.0, acepromazine: 4.2+/- 2.4). Induction time was similar for all three
treatments (saline: 280s+/- 98, midazolam: 230s+/- 232, acepromazine: 264s+/- 215). No
difference was seen in quality of recovery (saline: 8.0+/- 1.7, midazolam: 7.0+/- 2.7,
acepromazine: 8.1+/- 1.2) or overall quality of anesthesia (saline: 6.8+/- 2.5, midazolam: 7.4+/2.5, acepromazine: 7.5+/- 1.1).
There was no effect of premedication with acepromazine or midazolam on sedation, induction
time or quality, or recovery time or quality in laboratory-used NZW rabbits.
CANINE SALMONELLOSIS: PUBLIC HEALTH IMPLICATIONS
KOREN M. CUSTER1*, LAURA ADAMS1, STEVEN VALEIKA4, SUSAN SANCHEZ2,1,
ERIN K. LIPP3, JAMES MORGAN1, YING CHENG1, JOHN J. MAURER1
Department of Population Health1, Department of Infectious Disease2, Department of
Environmental Health Sciences3, Department of Epidemiology and Biostatistics4, University of
Georgia, Athens, GA
Salmonellosis is a disease that affects a diverse array of species, including dogs. Salmonellosis
has not been as intensively studied in canines as in other animals, but is potentially of great
importance as dogs are intricately linked with humans in everyday life. It is possible that dogs
may pose a zoonotic risk to humans, and inversely may also be susceptible to a “reverse”
zoonosis. Dogs may also serve as an amplifying host for environmental Salmonella serovars that
could affect human populations. Salmonella isolates were collected from several different
species, including dogs, passerines (songbirds), cattle, horses, chickens and wildlife species in
Georgia and were compared using pulsed field gel electrophoresis (PFGE). In addition, the data
were uploaded to the Center for Disease Control and Prevention’s PulseNet database, a
collection of isolate patterns that can be compared nationwide. It appears that dogs are exposed
to the same Salmonella serotypes and strains as those found in other animal species and the
environment. The public health implications of our findings are noteworthy as the data indicate
that canines may be useful as an indicator species for environmental Salmonella contamination
and for the risk of infection in humans and other species.
POTENTIAL ROLE OF HEME OXYGENASE-1 IN THE ENHANCEMENT
INFLAMMATION AND ENSUING NEUROTOXICITY CAUSED BY MANGANESE
OF
CA Dodd, II Georgieva, and NM Filipov. Physiology and Pharmacology, University of Georgia,
Athens, GA.
In humans, excess exposure to manganese (Mn) is associated with a Parkinsonian type
neurological disorder. Several studies provide evidence for the role of glial-derived (microglia and
astrocytes) inflammatory products in the development of Mn neurotoxicity. In fact, Mn was found
to potentiate the production of inflammatory cytokines induced by the inflammagen
lipopolysaccharide (LPS) in microglia cells. Inducible heme oxygenase (HO-1), an enzyme
responsible for the cleavage of the oxidant heme into biliverdin, carbon monoxide, and iron (Fe),
is increased in response to oxidative stress caused by various toxicants, including metals, and has
been reported to play a role in the regulation of inflammation. While the initial function of HO-1
appears to be anti-inflammatory, excessive induction of HO-1 can deregulate Fe homeostasis and
contribute to the increased production of free radicals and potentially inflammatory mediators.
Expression of HO-1 is increased in response to LPS; however, at present the effect of Mn on HO1 has yet to be determined. This study was designed to examine the effect of Mn on HO-1
induction and on LPS induced HO-1 in microglia (N9) and dopaminergic neuronal (N27) cells. N9
microglia and N27 neuronal cells were exposed to either LPS (100 ng/ml), Mn (100 µM), or
combined Mn+LPS for 24 hours. In microglia, while Mn had minimal effects on its own,
induction of HO-1 (protein and mRNA) by LPS was potentiated by Mn. The increase in HO-1
was not due to increased intracellular Mn, but was accompanied by a small but significant increase
in Fe concentration and an increase in mRNA for iNOS, and the inflammatory cytokines TNF-α
and IL-6. In contrast, neither LPS nor Mn had any effect on HO-1 in the N27 neuronal cells.
Moreover, combined Mn+LPS treatment caused a small, but significant decrease in HO-1. These
results indicate that Mn potentiates the induction of HO-1 by LPS in microglia, but not in neuronal
cells. Because the microglial increase in HO-1 is accompanied by an increase in inflammatory
mediators as well as Fe, there is a possibility that in the presence of Mn, HO-1 acts as a prooxidant and is involved in the enhanced cytokine production by Mn-exposed activated microglia
that has been reported previously. Future studies incorporating inhibition of HO-1 will enable us
to uncover whether induction of HO-1 is important for the increase in inflammatory cytokines
observed with Mn+LPS treatment in microglia cells.
THE IMPACT OF A SALMONELLA VACCINATION PROGRAM ON SALMONELLA
TRANSMISSION AND CHICKEN CARCASS CONTAMINATION IN COMMERCIAL
POULTRY INTEGRATORS
Fernanda Dorea1*, Dana Cole2, Charles Hofacre1, Demetrius Mathis1, Katherine Zamperini1, and
John J. Maurer1
Department of Population Health1, The University of Georgia, Athens, GA 30602
Centers for Disease Control and Prevention2, Atlanta, GA
Consumption of poultry meat and eggs are recognized risk factors for foodborne outbreaks of
salmonellosis and campylobacteriosis. Due to the integrated nature of the poultry industry,
Salmonella can be introduced at any point within this system. The poultry industry is very
interested in implementing “on-farm” HACCP but do not have sufficient information as to which
strategies are effective at reducing Salmonella carriage in breeder flocks. We currently possess
very little information about which particular management practices reduce or promote
Salmonella carriage by breeder flocks. Two commercial poultry companies in the southeastern US
participated in this study. One poultry integrator (Company C) had initiated a Salmonella
vaccination program of their chicken pullets. Salmonella positive pullet flocks were followed to
broiler-breeder farms and their progeny were followed onto broiler farms and ultimately the
processing plant, sampling the farm environment, as well as chicken carcasses for Salmonella.
Chi-square and Fisher’s exact test were used to identify statistically significant associations in
comparisons within and between companies, sample type, and Salmonella prevalence. We
identified statistically significant differences in Salmonella prevalence between the two poultry
integrators, with lower Salmonella prevalence in the company with the Salmonella vaccination
program. There was also a significant difference in Salmonella prevalence in broiler chicks at
placement from unvaccinated vs. vaccinated broiler-breeders. While significantly more broiler
farms with Company C were contaminated with Salmonella, this did not translate into increased
carcass contamination. It appears that vaccinating broiler-breeders significantly reduces broiler
chicken carcass contamination with Salmonella.
Combination of molecular and bioinformatics tools for detection of antigenic variant
strains of Infectious Bursal Disease Virus
Vijay Durairaj & Egbert Mundt
Poultry Diagnostic and Research Center,
College of Veterinary Medicine,
University of Georgia, Athens.
Infectious bursal disease (IBD) is a highly contagious and immunosuppressive disease in
chickens and is a worldwide problem. It is caused by Infectious Bursal Disease Virus (IBDV)
belonging to family Birnaviridae. Antigenic different strains of IBDV are isolated in poultry
flocks. This is likely caused by antigenic drift due to existing vaccination programs. These new
antigenic variant strains can break through current vaccination programs and cause devastating
losses to the poultry industry. The aim of our research is to detect, identify and characterize new
antigenic variant strains of IBDV. We apply reverse genetics as a diagnostic tool in identifying
these strains and characterize them based on their reaction with panel of monoclonal antibodies.
By applying this technique, we were able to identify strains of IBDV with an unusual reaction
pattern based on their reactivity with the panel of monoclonal antibodies. Our studies indicate
that IBDV strains are circulating in poultry flocks which are antigenic different from known
IBDV isolates. These new isolates might become candidates for future IBDV vaccines to induce
protection of young chickens against this important disease in poultry.
PHARMACOKINETICS OF A NOVEL INTRANASAL MIDAZOLAM GEL IN DOGS.
JS Eagleson,1 M Kent,1 AC Freeman,1 AC Haley,1 P Nghiem,1 AC Durden,1 D Strong,2 S White,2
SJ Schatzberg,1 SR Platt.1 University of Georgia Colleges of Veterinary Medicine1 and Pharmacy,2
Athens, GA.
The pharmacokinetics of a novel bioadhesive gel formulation of midazolam administered
intranasally (IN) were compared to those of parenteral midazolam solution administered IN,
intravenously (IV) and rectally.
Ten healthy beagles were randomly assigned to treatment groups in a crossover design.
Parenteral midazolam solution was administered IV to group 1, rectally to group 2, and IN to
group 3 (0.2 mg/kg). A 0.2 % carbopol midazolam gel formulation was administered IN to group
4 (0.2 mg/kg). Blood was collected from the jugular vein of each dog into tubes containing
lithium heparin before and 3, 6, 9, 12, 15, 20, 30, 60, 120, 240, and 480 minutes following
midazolam administration. The protocol was repeated 4 times with a 7-day wash out period
between administrations. Each dog received all 4 forms of midazolam administration. Plasma
concentration of midazolam was determined using high performance liquid chromatography.
Mean (+/- SD) peak plasma concentrations (Cmax) of midazolam were 1.82 (+/- 0.278) μg/mL
(IV), 0.45 (+/- 0.09) μg/L (IN gel), 0.21 (+/- 0.02) μg/mL (IN solution) and 0.15 (+/− 0.01) μg/mL
(rectal). The Cmax following IN gel administration was significantly higher than IN solution and
rectal administration (p<0.0001). Mean plasma concentrations of midazolam following IV
administration were significantly higher at 3, 6 , 9 and 12 minutes post-delivery when compared to
all other methods of delivery (p<0.0001). Mean plasma concentrations of midazolam following
IN gel administration were significantly higher at 6, 9 and 12 minutes post-delivery when
compared to IN solution and rectal administration (p<0.0001). Time (+/- SD) to peak
concentration (Tmax) was <3 minutes (IV), 11.70 (+/- 2.63) minutes (IN gel), 17.50 (+/- 2.64)
minutes (IN solution) and 39 (+/- 14.49) minutes (rectal). The Tmax following rectal
administration was significantly longer than both IN gel and IN solution administration
(p<0.0001). Mean bioavailability of midazolam was 70.4% (IN gel), 52.0% (IN solution) and
49.0% (rectal). Bioavailability following IN gel administration was significantly higher than IN
solution and rectal administration (p<0.0001). The half lives (+/- SD) were 121 (+/- 25.74)
minutes (IV), 119.61 (+/- 22.83) minutes (IN gel), 118.66 (+/- 18.84) minutes (IN solution) and
115.83 (+/- 15.56) minutes (rectal).
Intranasal midazolam gel was superior to IN solution administration and rectal administration
with respect to peak plasma concentration and bioavailability. Although IV administration of
benzodiazepines is still considered the treatment of choice for short acting anti-convulsant therapy,
intranasal administration of this novel midazolam gel may be useful for treatment of seizures in
dogs by owners or when intravenous access is not readily available.
IN VITRO ATTENUATION OF LPS-INDUCED TNF PRODUCTION BY BLOOD PRODUCTS
Epstein KL, Pellegrini-Masini A, Fortes BP, Pate AK, Moore JN
University of Georgia, Athens, GA, USA
The administration of blood products designed to contain high concentrations of anti-LPS antibodies is frequently
included in recommended therapies for horses with endotoxemia. However, only a limited number of studies have
evaluated the use of hyperimmune blood products in horses with experimental and clinical endotoxemia, and the results
have been inconsistent. This study was designed to compare the attenuation of in vitro TNF production in whole blood
induced by three types of LPS by three “anti-endotoxic” blood products (two hyperimmunized plasma products and one
concentrated hyperimmunized serum product) and pooled plasma at three clinically relevant doses
Heparinized whole blood was obtained from 11 healthy horses. The whole blood was exposed to four conditions—
unstimulated (negative control) and stimulated by three sources of LPS at a 1 ng/ml final concentration (Salmonella
LPS alone, E. coli LPS alone, and a combination of Salmonella, E. coli, and Klebsiella LPS). Following stimulation,
the blood was exposed to 13 treatments—saline (positive control) and three doses [1:80 dilution (simulating a 1 ml/kg),
1:40 dilution (simulating 2 ml/kg), and 1:20 (simulating 4 ml/kg)] of four blood products (pooled plasma from normal
horses, E. coli hyperimmunized plasma, Salmonella hyperimmunized plasma, or concentrated Salmonella
hyperimmunized serum). Samples were incubated for 6 hours and the plasma was harvested for TNF quantification
with an ELISA. TNF production was compared between LPS stimulations for each dose of each treatment. For each
type of LPS stimulation, percentage change in TNF production from saline for each blood product was compared.
Comparisons were made using a one-way ANOVA or Kruskall-Wallis analysis of ranks (significance p<0.05).
There was no difference in TNF concentrations in unstimulated whole blood treated with any dose of saline or blood
product and stimulation with all three LPS sources resulted in a similar and significant increase in TNF in saline treated
samples. When stimulated with the combination of LPS, there was a significant decrease in TNF production with a 1
and 4 ml/kg dose of pooled plasma from normal horses, a 4 ml/kg dose of Salmonella hyperimmune plasma, and a 4
ml/kg dose of Salmonella. When stimulated with E. coli LPS alone, there was a significant decrease in TNF production
with a 4 ml/kg dose of Salmonella hyperimmune serum. When stimulated with Salmonella LPS alone, no treatment
resulted in a significant reduction in TNF production. No treatment at any dose returned TNF concentrations to
unstimulated levels.
Treatment with blood products results in an inconsistent decrease in LPS-induced TNF production in vitro. The
source of the inconsistency is unclear. Further studies are warranted to determine the active component(s) of the blood
products and the effect of the blood products on other mediators of the effects of endotoxin (neutrophil ROS production,
macrophage procoagulant activity, and cytokine gene expression).
IN VITRO EVALUATION OF VISCOELASTIC COAGULATION TESTING FOR MONITORING LOW
MOLECULAR WEIGHT HEPARIN
Epstein KL†, Whelchel DD†, Chaffin MK‡
†
University of Georgia, Athens, GA, USA ‡Texas A&M University, College Station, TX, USA
Administration of low molecular weight heparin (LMWH) to horses at risk for coagulopathy may prevent
thromboembolic complications. Currently, the most accurate method for therapeutic drug monitoring, quantification of
anti-Xa activity, is not widely available. This study was designed as a preliminary evaluation of two viscoelastic
coagulation tests [thrombelastography (TEG) and Sonoclot®] as potential methods for therapeutic drug monitoring.
Citrated whole blood samples were obtained from 10 healthy horses. Dalteparin was added to result in three plasma
anti-Xa concentrations within the range recommended for prophylactic treatment (0.1 and 0.2 IU/ml plasma) and
achieved at following administration of currently recommended doses (0.3 IU/ml plasma) five minutes prior to
performing TEG (tissue factor activated) and Sonoclot® (glass bead activated). An untreated sample was tested
concurrently as a control. Coagulation parameters [TEG (R, K, α, and MA) and Sonoclot® (ACT, CR)] were compared
(significance p<0.05) using a one-way ANOVA (normally distributed) or Kruskall-Wallis one-way ANOVA on ranks
(not normally distributed). TEG and Sonoclot parameters were correlated with anti-Xa concentrations using linear
regression.
Compared to baseline TEG and Sonoclot parameters, there was an increased time for clot formation [TEG R (0.2 and
0.3 IU/ml anti-Xa activity), Sonoclot® ACT (0.1, 0.2, and 0.3 IU/ml anti-Xa activity)], decreased rate of clotting [TEG
α (0.3 IU/ml anti-Xa activity), Sonoclot® CR (0.1, 0.2, and 0.3 IU/ml anti-Xa activity)], and decreased clot strength
[TEG MA (0.3 IU/ml anti-Xa activity)]. No significant changes in TEG-K was detected.
These results suggest that TEG and Sonoclot® may be valuable point-of-care methods for monitoring therapy with
LMWH. It appears that Sonoclot® may be more sensitive to low anti-Xa activity consistent with prophylaxis. Based
on these preliminary results, in vivo testing is warranted and will be required to validate the use of viscoelastic
coagulation test for LMWH drug monitoring.
THE EFFECTS OF EXTUBATION WITH AN INFLATED VERSUS DEFLATED
ENDOTRACHEAL TUBE CUFF ON ENDOTRACHEAL FLUID VOLUME IN THE DOG. AR
Farmer, EH Hofmeister, C Laas, J Williams. University of Georgia College of Veterinary
Medicine, Athens, GA.
The purpose of this study was to investigate the effect of extubation with the endotracheal tube
(ETT) cuff inflated versus deflated on endotracheal fluid volume in normal canine cadavers.
Sixteen adult beagle cadavers were orotracheally intubated in lateral recumbency, and the ETT
cuffs were inflated to a closing pressure of 20 cm H2O before barium was introduced orad to the
cuff. The dogs were randomly assigned to an ETT cuff extubation condition of deflated or
unchanged from the original closing pressure. After extubation, lateral thoracic radiographs of the
cadavers were obtained and scored by 3 independent blinded reviewers. Each reviewer ordered all
16 lateral radiographs from most to least intratracheal contrast and also estimated residual
intratracheal contrast volume.
Dogs extubated with a deflated ETT cuff had a median rank of 13 and dogs extubated with an
inflated ETT cuff had a median rank of 4.5 (P<0.0001). Dogs extubated with a deflated ETT cuff
had a mean ± standard deviation estimated intratracheal volume of fluid of 1.8 mL ± 0.7 mL and
dogs extubated with an inflated ETT cuff had a volume of 0.9 mL ± 0.5 mL (P<0.0001). Fleiss
Kappa for agreement among evaluators was 0.875.
Extubation with the cuff inflated removed more liquid contents from the trachea than extubation
with the cuff deflated and may assist in the prevention of pulmonary aspiration.
5-LIPOXYGENASE EXPRESSION IN HYPERPLASTIC, INFLAMED, AND NEOPLASTIC
CANINE PROSTATE TISSUES
Laura Goodman, Carla Jarrett, Thomas Krunkosky, Steve Budsberg, Nicole Northrup, Corey
Saba, Bruce LeRoy
5-lipoxygenase (5-LO) metabolizes arachidonic acid to produce lipid mediators of inflammation.
5-lipoxygenase is overexpressed in human prostate carcinomas and 5-LO inhibition induces
apoptosis in prostate cancer cell lines. We hypothesized that as in human prostate carcinoma, 5LO would be overexpressed in canine prostate carcinoma and may be important in the
pathogenesis of the disease. We evaluated 5-LO expression in hyperplastic, inflamed, and
neoplastic canine prostate tissues.
Western blots were performed to validate the antibody in canine tissue and to evaluate 5-LO
expression in prostate carcinomas compared to benign prostatic hyperplasia (BPH) tissues.
Subsequently, immunohistochemistry was performed to evaluate 5-LO expression in BPH,
suppurative prostatitis, prostate carcinoma, and metastatic tissues. Stain distribution and intensity
were determined for the epithelial and stromal components of all samples.
Western blot analysis validated the antibody and demonstrated 5-LO expression in both prostate
carcinoma and BPH tissue. 5-lipoxygenase staining was not significantly different between
epithelial and stromal cells in BPH, prostatitis, and prostate carcinoma. However, 5-LO
immunoreactivity was markedly decreased in intensity and distribution within the epithelial and
stromal components of metastatic lesions compared to primary prostate carcinomas (p<0.015).
5-lipoxygenase is expressed in hyperplastic, inflammatory, and neoplastic lesions of the canine
prostate. The similarities in 5-LO expression between BPH, prostatitis, and prostate carcinoma
suggest that there is not differential expression of the enzyme in these conditions. Decreased
expression of 5-LO in metastatic lesions may indicate down-regulation or altered expression of the
enzyme with progression of canine prostate carcinoma to a metastatic phenotype.
RISK
FACTORS
FOR
SEPTIC
PERITONITIS
AND
SURVIVAL
AFTER
GASTROINTESTINAL SURGERY IN DOGS: 225 CASES. JA Grimes, CW Schmiedt, K
Cornell, M Radlinsky. University of Georgia College of Veterinary Medicine, Athens, GA.
Gastrointestinal surgery is commonly performed in small animal surgical practice. The purpose
of this retrospective study is to identify risk factors for survival and development of septic
peritonitis (SP) following a full thickness gastrointestinal incision in different populations of dogs.
Our hypothesis is that hypoalbuminemia and hypoproteinemia are risk factors for both postoperative SP and mortality and that risk of mortality is affected by the presence of pre-operative
SP.
Medical records of dogs undergoing full-thickness intestinal incisions between 1998 and 2007
were reviewed. Data was collected on pre-, intra-, and post-operative variables and significance as
it related to survival and development of SP was evaluated using logistic regression analysis.
Two hundred and twenty five cases were identified; 28 (12.4%) cases developed post-operative
SP and 29 (12.9%) cases did not survive to discharge. Forty four (19.5%) cases had pre-operative
SP, of those 16 (36.4%) had post-operative SP and 14 (31.8%) died. In cases without pre-operative
SP, 11 (6.0%) developed post-operative SP and 20 (11.0%) died. When all cases are considered,
common significant risk factors include presence of pre-operative SP, pre-operative and postoperative albumin and protein concentrations, and intra-operative hypotension. The presence of a
foreign body was frequently a protective factor.
Multiple factors are involved in survival rates and the development of SP post-operatively.
Dogs with decreased plasma protein and albumin concentrations are at risk for decreased survival
and development of SP. Aggressive peri-operative attempts to increase protein concentrations and
intra-operative surgical strategies to minimize morbidity and mortality may be indicated in
patients with risk factors identified here.
RISK
FACTORS
FOR
SEPTIC
PERITONITIS
AND
SURVIVAL
AFTER
GASTROINTESTINAL SURGERY IN DOGS: 225 CASES. JA Grimes, CW Schmiedt, K
Cornell, M Radlinsky. University of Georgia College of Veterinary Medicine, Athens, GA.
Gastrointestinal surgery is commonly performed in small animal surgical practice. The purpose
of this retrospective study is to identify risk factors for survival and development of septic
peritonitis (SP) following a full thickness gastrointestinal incision in different populations of dogs.
Our hypothesis is that hypoalbuminemia and hypoproteinemia are risk factors for both postoperative SP and mortality and that risk of mortality is affected by the presence of pre-operative
SP.
Medical records of dogs undergoing full-thickness intestinal incisions between 1998 and 2007
were reviewed. Data was collected on pre-, intra-, and post-operative variables and significance as
it related to survival and development of SP was evaluated using logistic regression analysis.
Two hundred and twenty five cases were identified; 28 (12.4%) cases developed post-operative
SP and 29 (12.9%) cases did not survive to discharge. Forty four (19.5%) cases had pre-operative
SP, of those 16 (36.4%) had post-operative SP and 14 (31.8%) died. In cases without pre-operative
SP, 11 (6.0%) developed post-operative SP and 20 (11.0%) died. When all cases are considered,
common significant risk factors include presence of pre-operative SP, pre-operative and postoperative albumin and protein concentrations, and intra-operative hypotension. The presence of a
foreign body was frequently a protective factor.
Multiple factors are involved in survival rates and the development of SP post-operatively.
Dogs with decreased plasma protein and albumin concentrations are at risk for decreased survival
and development of SP. Aggressive peri-operative attempts to increase protein concentrations and
intra-operative surgical strategies to minimize morbidity and mortality may be indicated in
patients with risk factors identified here.
RESPONSE GENE TO COMPLEMENT 32 INTERACTS WITH SMAD3 TO PROMOTE
EPITHELIAL-MESENCHYMAL TRANSITION OF RENAL TUBULAR CELLS
Xia Guo and Shi-You Chen
Department of Physiology & Pharmacology, College of Veterinary Medicine, University of
Georgia, Athens, GA
Abstract: Epithelial-Mesenchymal Transition (EMT) is an important process in carcinogenesis
and organ fibrogenesis. Transforming growth factor-β (TGF-β) plays a major role in inducing
EMT, largely through Smad signaling pathway. Emerging evidence indicates that response gene to
complement 32 (RGC-32) mediates TGF-β-induced EMT of human renal proximal tubular cells.
However, the mechanisms underlying RGC-32 function remain largely unknown. In this study, we
found that RGC-32 function in EMT is associated with Smad3. Co-expression of RGC-32 and
Smad3, but not Smad2, induced a higher protein expression of myofibroblast marker α-SMA as
compared to RGC-32 or Smad3 alone, while knockdown of Smad3 using short hairpin interfering
RNA blocked RGC-32-induced α-SMA expression. These data suggest that RGC-32 interacts
with Smad3, but not Smad2, in the regulation of EMT. Indeed, RGC-32 colocalizes with Smad3 in
the nuclei of HPTC upon TGF-β induction. Co-immunoprecipitation assay showed that Smad3,
but not Smad2, physically interacts with RGC-32 in renal tubular cells. RGC-32 and Smad3
appear to work together to regulate α-SMA gene transcription. RGC-32 and Smad3 synergistically
upregulated α-SMA promoter activity. Blockade of RGC-32 significantly inhibited Smad3 activity
in activating α-SMA promoter, while knockdown of Smad3 inhibits RGC-32-indced α-SMA
mRNA expression. In addition to α-SMA, RGC-32 and Smad3 also synergistically activated the
expression of extracellular matrix proteins fibronectin and downregulated an epithelial marker Ecadherin. Mechanically, RGC-32 and Smad3 coordinate the induction of EMT by regulating the
EMT regulators Slug and Snail. Smad3 knockdown significantly inhibited RGC-32-induced Slug
and Snail expression. Taken together, our data demonstrate for the first time that RGC-32 interacts
with Smad3 to mediate TGF-β-induced EMT of human renal tubular cells.
EVALUATION OF THREE IN VITRO BIOASSAYS FOR MEASURING THE
ANTHELMINTIC ACTIVITY OF A CONDENSED TANNIN EXTRACT FROM SERICEA
LESPEDEZA
SB Howell, BE Storey, RM Kaplan, Department of Infectious Disease, College of Veterinary
Medicine, University of Georgia, Athens, GA.
Certain forages high in condensed tannins (CT) demonstrate anthelmintic activity, and appear to
be a useful non-chemical adjunct to parasite control in small ruminants. In addition to feeding
trials, in vitro bioassays have been used to evaluate this antiparasitic effect against several species
of trichostrongyle nematodes. However, it remains unclear which in vitro assay is the most
appropriate evaluation tool. The goal of this research is to evaluate the repeatability and sigmoidal
dose response characteristics of different in vitro methods, and determine which are most suitable
for measuring the effective concentration (EC50) of these extracts. In this project, 3 in vitro
methods (larval migration inhibition assay (LMIA), egg hatch assay (EHA), and larval
development assay (LDA) were performed using the CT extract of sericea lespedeza (Lespedeza
cuneata) with Haemonchus contortus eggs or larvae. The concentration ranges used were 1.5 1560 µg/ml. The LMIA was repeated 11 times in triplicate, whereas the EHA and LDA were
repeated 14 times in triplicate. The LMIA yielded inconsistent values for EC50, ranging from 9.3 470.4 µg/ml. In contrast, the LDA yielded much more consistent results, with EC50 ranging from
26.5 - 66.2 µg/ml. The EC50 for the EHA was > 1560 µg/ml in all cases, thus could not be
measured. Our results indicate that the LDA yields the most consistent and repeatable EC50 and
dose response curves. Therefore, the LDA appears to be the most appropriate bioassay for
measuring the antiparasitic activity of CT plant extracts in trichostrongyle nematodes of small
ruminants.
BROADLY REACTIVE PAN-VIRAL PCR OF CEREBROSPINAL FLUID IN CANINE
MENINGOENCEPHALITIS OF UNKNOWN ETIOLOGY. Q Li1, SJ Schatzberg1, SR Platt1, M
Kent1, R. Barber1, JM Levine2, GJ Levine2, K Chandler3, LJ Anderson4, S Tong4. 1. University of
Georgia, College of Veterinary Medicine (CVM), Athens, GA. 2. Texas A&M University, CVM,
College Station, TX. 3. Royal Veterinary College, Hatfield, UK. 4. Centers for Disease Control
and Prevention, Division of Viral Diseases, Atlanta, GA.
Definitive etiologies for canine meningoencephalitis are unknown in 75%-95% of cases. This is
due largely to limitations in the spectrum of diagnostic tests utilized in the clinical setting. In this
investigation, broadly reactive consensus-degenerate hybrid oligonucleotide (CODEHOP) PCR
assays for 11 viral families were implemented to evaluate canine meningoencephalitis of unknown
etiology (MUE).
Total nucleic acids were extracted from cerebrospinal fluid (CSF) from 146 dogs with
neurological disease, including 60 cases of MUE. Nucleic acids from all samples were evaluated
by CODEHOP PCR for an extremely diverse group of viruses, including adeno-, herpes-, alpha-,
flavi-, bunya-, corona-, borna-, rhabdo-, paramyxo-, polyoma-, and picornaviruses. For each PCR
reaction, rigorous positive and negative controls were included. Nucleic acid integrity of all
samples was confirmed by PCR or reverse transcriptase-PCR for GAPDH or beta-actin,
respectively. PCR reactions were evaluated by 2% agarose gel electrophoresis, with amplicons
assessed under ultraviolet light after the addition of ethidium bromide. All positive and negative
controls, including no template PCR and PCR of water extracted in parallel to clinical samples,
yielded expected results. Pan-viral PCR was predominantly negative aside from positive results
using pan-bunyavirus and pan-polyomavirus PCR. Sequence analysis of PCR amplicons thus far
has confirmed LaCrosse virus and Merkel cell polyomavirus (MCV) in sporadic CSF samples.
Serological investigations for LaCrosse virus and MCV are underway to evaluate for the
presence of antibodies in the PCR positive dogs, and specific PCR screens will be designed to
evaluate for the presence of nucleic acids from LaCrosse and MCV in the present and future cases.
The ability to detect known and potentially novel pathogens should help to determine the etiology
of a subset of cases of canine meningoencephalitis, may identify emerging (eg MCV) and zoonotic
(eg LaCrosse) pathogens, and in a clinical setting should improve survival with the subsequent
implementation of more directed anti-viral therapies.
PREVENTION AND TREATMENT OF ANTERIOR UVEITIS BY TARGETING THE
COMPLEMENT SYSTEM
Balasubramanian Manickam1 and Nalini S Bora2
1.
Department of Veterinary Pathology, College of Veterinary Medicine, University of
Georgia, Athens, GA
2. Department of Ophthalmology, University of Arkansas for Medical Sciences,
Little Rock, AR
Experimental Autoimmune Anterior Uveitis (EAAU) is an animal model for Idiopathic Anterior
Uveitis in humans. We have previously shown that complement activation plays a critical role in
the development of experimental autoimmune anterior uveitis (EAAU). Additionally, it has been
reported that suppression of complement regulators (CRegs) exacerbated EAAU. However, the
pathway of complement activation involved in the induction of EAAU, the role of recombinant
soluble CRegs and complement activation products C3a, C5a and membrane attack complex
(MAC) in EAAU are unknown. Therefore, we investigated to delineate the pathway of
complement activation involved in the induction of EAAU. We next hypothesized that exogenous
administration of recombinant soluble complement regulatory protein; Crry-Ig will ameliorate
EAAU. Finally, we investigated the role of complement activation products C3a, C5a and
membrane attack complex (MAC) in the induction of EAAU. Our findings from these studies
indicate for the first time that the alternative pathway of complement activation is crucial for the
induction of EAAU. Most importantly, our studies indicate that Crry-Ig can be used both
prophylactically and therapeutically in amelioration of EAAU which is of high clinical
significance. Our results also reveal that complement activation products C3a, C5a and MAC are
not the major players in the pathogenesis of EAAU. Together, the evidence from the present study
clearly demonstrates that complement system and CRegs play a key role in the pathogenesis of
EAAU. In conclusion, inhibition of complement activation via the alternative pathway and
blocking at the C3 level is crucial in inhibiting EAAU. We believe that therapy based on
complement inhibition has great potential in future for the treatment of idiopathic anterior uveitis
(AU), untreated AU leads to blindness.
ESTABLISHING A REPRODUCIBLE METHOD FOR THE CULTURE OF PRIMARY
EQUINE CORNEAL CELLS (RL Mathes 1, UM Dietrich 1, TM Krunkosky 2, DJ Hurley
3, AJ Reber 3) 1 Department of Small Animal Medicine and Surgery, College of
Veterinary Medicine, University of Georgia; 2 Department of Anatomy and Radiology,
College of Veterinary Medicine, University of Georgia; 3 Department of Population
Health, College of Veterinary Medicine, University of Georgia
Purpose. To establish a reproducible method for the culture of primary equine corneal
epithelial cells, keratocytes and endothelial cells and to describe each cell’s
morphologic characteristics, immunocytochemical staining properties and conditions
required for cryopreservation.
Methods. Corneas from eight horses recently euthanized for reasons unrelated to this
study were collected aseptically and enzymatically separated into three individual layers
for cell isolation. The cells were plated, grown in culture and continued for several
passages. Each cell type was characterized by morphology and immunocytochemical
staining.
Results. All three equine corneal cell types were successfully grown in culture.
Cultured corneal endothelial cells were large, hexagonal cells with a moderate growth
rate. Keratocytes were small, spindloid cells that grew rapidly. Epithelial cells had
heterogenous morphology and grew slowly. The endothelial cells and keratocytes
stained positive for vimentin and were morphologically distinguishable from one
another. The epithelial cells stained positive for cytokeratin. Keratocytes and
endothelial cells were able to be cryopreserved and recovered. The cryopreserved cells
maintained their morphological and immunocytochemical features after
cryopreservation and recovery.
Conclusion. This work establishes reproducible methods for isolation and culture of all
three equine corneal cells. Cell morphology and cytoskeletal element expression for
each cell type is also described. This has not previously been reported for equine
corneal cells. This report also demonstrates the ability to preserve equine keratocytes
and endothelial cells for extended periods of time and utilize them long after primary cell
collection, a feature that has not been reported for veterinary corneal cell culture.
University of Georgia Veterinary Ophthalmology Research Fund (VORF). None.
Mortality in Dogs from 1984-2004: An Investigation into Breed, Gender, and Age Related Causes
of Death. JM McGill, KE Creevy, D Promislow. University of Georgia College of Veterinary
Medicine, Athens, GA.
The purpose of this study was to investigate trends in disease occurrence and determine if breed
or sex influences the likelihood of mortality from certain diseases. Throughout the history of
veterinary medicine, beliefs regarding increased risk of certain diseases within specific breeds
have been postulated, yet seldom documented. Within the veterinary literature, there is limited
evidence that certain breeds are more commonly diagnosed with specific conditions; this project
further defines the prevalence of these breed-related conditions. The objective of this study was to
describe the occurrence of disease categories among over 80,000 cases of canine mortality
reported to the Veterinary Medical Database (VMDB) between 1984-2004, and the search criteria
were limited to hospital visits which resulted in death of the dog. Examining any breed with
greater than 100 entries in the dataset, we investigated the influence of age and gender as well as
breed, and their relationship to the frequency of broad categories of diseases.
Data for the study came from the VMDB, which contains information on hundreds of thousands
of dogs submitted from a variety of Veterinary Teaching Hospitals. Cases were included in this
study if the visit resulted in the death of the animal. Any breed with greater than 100 entries in the
dataset was included in the investigation. Cases were excluded if the only cause of death was
euthanasia, or if the gender of the animal was not given. Using these criteria, a total of 50,096
cases representing 70 different dog breeds were included. The dataset was sorted by 2 individual
investigators, who used a predetermined set of criteria to identify the most likely cause of death if
multiple simultaneous diagnoses were given. For our study, we categorized the diagnoses into 12
organ systems (musculoskeletal, neurologic, cardiovascular, behavioral, respiratory,
gastrointestinal, endocrine, ophthalmologic, hematopoetic, genito-urinary, dermatologic, hepatic,
and other), and 11 disease processes (anomalous/anatomic/congenital, neoplastic, infectious,
inflammatory/immune-mediated, metabolic, toxic, traumatic, vascular, degenerative, healthy, and
other).
Ongoing data analysis confirms that certain breeds are more often afflicted with specific
diseases, suggesting a genetic basis for certain diseases. Results thus far have revealed that
Newfoundlands die of cardiovascular disease more than all other breeds. In addition, St. Bernards
die of musculoskeletal disease more frequently than all other breeds. In addition, some breeds may
be more closely related than once thought, based on the common diseases that affect them.
SEROLOGIC AND MOLECULAR EVALUATION OF CATS FROM GEORGIA FOR
HEPATITIS E VIRUS. JL Mobley, EW Howerth. University of Georgia College of Veterinary
Medicine, Athens, GA.
Causes of hepatitis are poorly studied in cats, particularly lymphocytic portal hepatitis (LPH).
LPH is similar clinically and histologically to hepatitis seen in humans and pigs infected with
Hepatitis E Virus (HEV). HEV is an enterically transmitted non-enveloped, positive single
stranded RNA virus in the new virus family Hepeviridae; it is endemic in humans in developing
countries with sporadic disease also occurring in developed countries. HEV has only been
identified in humans, chickens, and pigs; however, we hypothesized that HEV may be a cause of
LPH in cats. The objective of this study was to determine if cats have evidence of a HEV, using
PCR, serology, and correlating viral presence or antibodies to either histopathologic or clinical
evidence of hepatitis. Bile, liver, and intestinal contents from cats (n = 69) submitted to the Athens
Veterinary Diagnostic Laboratory for necropsy were screened for HEV using a universal nRTPCR and qRT-PCR, both known to amplify all four HEV genotypes. Serum from 18 of these
cats—as well as serum from 93 cats submitted to the University of Georgia Clinical Pathology
Laboratory—were screened for HEV antibodies using immunoblotting. HEV RNA was not
detected but 2 of 93 cats had serologic evidence of antibodies to a HEV-like agent. Of the
necropsy cats, 18 of 40 had hepatitis but only 5 had LPH. For one of the cats with evidence of
HEV antibodies we had formalin-fixed liver tissue available, but there was no histopathologic
evidence of hepatitis. Twenty of the 93 cats for which only serum was available had serum
biochemical evidence of hepatic disease. We have detected little evidence of HEV in cats, possibly
because they do not have a related virus. Alternatively, cats may have a HEV, but due to low
prevalence the sample size was too small for detection of the virus, or the HEV in cats has a
dissimilar genome that cannot be detected by the PCR techniques used. It is also possible that
these cats originated from a non-endemic area decreasing the likelihood of exposure. The zoonotic
potential of HEV warrants further studies of this virus in cats.
DEVELOPMENT OF AN IN VITRO BIOASSAY TO DETECT THE PRESENCE OF
ANTHELMINTIC RESISTANCE IN DIROFILARIA IMMITIS
A. R. Moorhead, M.T. Dzimianski, P. Supakorndej and R. M. Kaplan
Heartworm disease is a significant threat to canine health. Over the past few decades,
the prevalence of heartworm infection in pet dogs has been greatly reduced by monthly
prophylaxis using anthelmintics of the avermectin/milbemycin class. However, recent
reports of heartworm infections in dogs receiving documented monthly prophylaxis are
cause for concern. Two possible explanations for this observation are failure of owner
compliance in ensuring proper administration of prophylaxis, and the development of
anthelmintic resistance. However, it is currently not possible to distinguish these two
scenarios because compliance is not possible to verify, and there are no validated assays
for detecting resistance in D. immitis. In order to address this problem, we developed a
larval migration inhibition bioassay (LMIA) for D. immitis, using a 96-well plate format.
Dirofilaria immitis L3 obtained from mosquitoes fed on microfilaremic dog blood were
incubated for 3 h in the presence of increasing concentrations of ivermectin. Following
incubation, L3 were transferred to wells containing a 20-micron mesh filter, and numbers
of larvae migrating through the mesh were measured after 12 h. Preliminary results
indicate that the LMIA produces a sigmoidal dose response with D. immitis L3. We are
currently in the process of further optimizing this assay and testing the dose response
characteristics of a variety of avermectin/milbemycin drugs. Our goal is to validate this
assay so it can be used to screen D. immitis L3 produced from microfilaremic blood
samples obtained from dogs for which a failure of anthelmintic prophylaxis has been
reported.
JOINT CONTRACTURES IN GOLDEN RETRIEVER MUSCULAR DYSTROPHY: A
MODEL FOR DUCHENNE MUSCULAR DYSTROPHY.
Nghiem PP1,2, Schatzberg SJ1, Hoffman EP2, Wang Z2, Ghimbovschi S2, Rayavarapu S2,
Knoblach S2, Nagaraju K2, and Kornegay JN3. 1University of Georgia, College of Veterinary
Medicine, Athens, GA, 2Children’s National Medical Center, Center for Genetic Medicine
Research, Washington, D.C., 3University of North Carolina, School of Medicine, Chapel Hill, NC.
In both Duchenne muscular dystrophy (DMD) patients and Golden retriever muscular dystrophy
(GRMD) dogs, joint contractures are the result of the dystrophic process and are extremely
debilitating. In DMD, both muscle weakness and joint contractures are thought to contribute to
postural instability and ultimately the loss of ambulation. Conventional thought is that joint
contractures are driven by progressive skeletal muscle wasting and weakness. However, in
GRMD, our functional phenotypic data suggest differently, as joint contractures seem to be driven
by an imbalance of skeletal muscle extensor and flexor forces and skeletal muscle hypertrophy. In
an attempt to correlate functional abnormalities (i.e. increased Cranial sartorius (CS) m.
circumference, increased tetanic flexor force, decreased tetanic extensor force, and decreased
tibiotarsal joint angle) with skeletal muscle molecular pathways, we have performed genome-wide
mRNA expression profiling on archived biopsy samples from three muscles (CS, Long digital
extensor, and Vastus lateralis) that were collected at 6 months of age from 8 GRMD and 4 normal
Golden retriever dogs. We have identified a major cytokine, osteopontin (OPN) that may be
associated with the inflammatory and hypertrophic changes in the CS muscle. We also have
identified three genes that are over-expressed in the hypertrophied CS muscle including DAG1,
LARGE, and SNTA1, that code for α- and β-dystroglycan, like-glycosyltransferase, and
syntrophin alpha 1, respectively. To validate the expression profiling results, quantitative PCR
and immunohistochemical studies of these gene transcripts and proteins, respectively, are
underway in variably affected GRMD and control dogs. OPN, DAG1, LARGE, and SNTA1
ultimately may prove to be important therapeutic targets for GRMD dogs and DMD patients.
MOLECULAR ANALYSIS OF A NOVEL DYSTROPHIN INSERTIONAL MUTATION IN
THE CAVALIER KING CHARLES SPANIEL. Nghiem PP1,2, Kornegay JN3, Li Q1, Platt SR1,
Kent M1, Smith J1, Piercy RJ4, Hoffman EP2, and Schatzberg SJ1.1University of Georgia, College
of Veterinary Medicine, Athens, GA, 2Children’s National Medical Center, Center for Genetic
Medicine Research, Washington, D.C., 3University of North Carolina, School of Medicine, Chapel
Hill, NC, 4Royal Veterinary College, Department of Veterinary Clinical Sciences, London, UK.
Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder originating from
mutations of the dystrophin gene. It is the second most common genetic disorder in people and
occurs in 1 of 3,500 male births. The dystrophin gene encompasses 2.4 megabases in the p21
region of the X chromosome and is transcribed into the dystrophin protein, a cytoskeletal protein
that links the cytoskeletal components of the muscle cell to the muscle sarcolemmal membrane.
Dystrophin-deficient muscular dystrophy is reported rarely in the veterinary literature; therefore,
its true prevalence in dogs and cats is unknown. Genetic mutations have been fully characterized
in a murine (mdx mouse), feline, and canine models of dystrophinopathy including: a premature
stop codon in exon 23 of the mdx mouse, a deletion of the muscle and Purkinje dystrophin
promoter region in the dystrophic cat, a splice site mutation in intron 6 of the Golden retriever, a
complete deletion of the dystrophin gene in the German short haired pointer, a premature stop
codon in exon 58 in the Rottweiler, an intronic insertion in the Welsh Corgi, an insertion mutation
in a Labrador retriever, and an exon deletion and frameshift mutation in a Cavalier King Charles
Spaniel (CKCS). We have recently identified a new CKCS that presented for a chronic history of
dysphagia, decreased exercise performance, poor skeletal muscle mass, and coughing.
Dystrophin-deficient muscular dystrophy was diagnosed on the basis of skeletal muscle
histopathology, immunohistochemistry, and immunoblotting using monoclonal antibodies to the
dystrophin rod and carboxy termini. To date, RT-PCR studies of overlapping regions of the
dystrophin cDNA have disclosed an insertional mutation within exons 26-34. We are currently
further characterizing this novel mutation by sequence analysis. This novel dystrophin insertional
mutation provides a unique canine model to study therapies for similar mutations in DMD.
CKCS
Large band not present
in control muscle
CONTROL
1500 BP
DYSTROPHIN GEL
MAGNETIC RESONANCE IMAGING EVALUATION OF HEAD TRAUMA IN 32 DOGS;
ASSOCIATIONS WITH MODIFIED GLASGOW COMA SCORE AND PATIENT OUTCOME
S.R.Platt,1 V. Adams,2 F. McConnell,2 M. Kent,1 SJ Schatzberg,1 L. De Risio.2 1College of
Veterinary Medicine, University of Georgia, Athens. 2The Animal Health Trust, Newmarket, UK.
The role of magnetic resonance imaging (MRI) in human head trauma offers distinct advantages
over computed tomography in the recognition of parenchymal lesions, but its use has not been
evaluated in veterinary medicine.
The aims of this study were to investigate whether MRI assessment of head trauma is associated
with severity of neurolgical dysfunction and could be predictive of patient survival.
Dogs presenting with evidence of head trauma and which were imaged with a 1.5T MRI were
retrospectively evaluated. Criteria necessary for inclusion in the study were (i) imaging performed
within 7 days of the trauma (ii) neurological examination at time of MRI enabling a modified
Glasgow coma score (MGCS) to be estimated (iii) survival at 1 and 6 months after MRI (iv) T1,
T2, T2* gradient echo and FLAIR weighted images. All images were blindly evaluated for (i)
extra-axial hemorrhage (ii) intra-axial hemorrhage (iii) fractures (linear, comminuted, compound
and/or depressed) (iv) degree of parenchymal shift (mm) (v) single or multiple parenchymal
lesions and (vi) MRI grade of severity (I-IV) modified from established criteria in human head
trauma imaging.
The MRI parameters were individually evaluated with the estimated MGCS and survival at 1
and 6 months. Cross-tabulations and Fishers exact tests or Goodman and Kruskal’s gamma were
performed to identify associations between MRI characteristics and outcome and MGCS. The
effect of the MGCS on outcome was examined using Wilcoxon rank sum tests.
Thirty-two dogs fulfilled the criteria. The median MGCS was 15 (range 7-18). A linear trend
was demonstrated between survival at 1 and 6 months and the MGCS (P=0.01 and 0.0002).
Nineteen of 32 dogs (59%) had an abnormal MRI which was not associated with outcome at 1 or 6
months. MRI grade was significantly associated with outcome at 1 and 6 months (P=0.04); a
higher class was associated with a reduced probability of survival. The presence of intra- or extraaxial hemorrhage was not associated with outcome at 1 or 6 months. Dogs with no midline shift
were more likely to survive to 1 month than dogs with any evidence of midline shift (P=0.02).
Whether a dog had a skull fracture or not and type of fracture were not associated with survival.
The MGCS was not associated with the presence of extra-axial hemorrhage, skull fractures or
fracture types. There were significant associations between the MGCS and abnormal MRI, MRI
grade, presence of intra-axial hemorrhage and the degree of midline shift (P<0.0001 in each case).
The use of the MGCS in conjunction with MRI can help determine prognosis in dogs with head
trauma. This study may provide MR indicators for surgical therapy.
PRELIMINARY MORPHOMETRIC EVALUATION OF SYRINGOHYDROMYELIA IN
AMERICAN BRUSSELS GRIFFONS. AC Freeman, SR Platt, SJ Schatzberg, M Kent.
University of Georgia College of Veterinary Medicine, Athens, GA.
Syringohydromyelia (SM) has been described in the American Brussels Griffon (ABG) although
its morphometric associations with skull conformation and clinical signs have not been evaluated.
The aim of this study was to evaluate the size of the central canal (CC) and or SM in ABGs for an
association with cerebellar deviation and herniation, CSF pleocytosis and clinical signs.
Twenty-eight ABGs, recruited as part of a larger epidemiological and genetic study, underwent
brain and spinal MRI evaluation (3.0T General Electric Signa HDx, Milwaukee, WI). All dogs
were evaluated neurologically, recording deficits and the presence of neck pain. Sequences
acquired included T2W, T1W pre- and post-contrast, and T2W FLAIR sagittal and transverse
(2mm thick; 0mm interspaced). Cervical spinal cord CC and/or syrinx size was measured using
OsiriX 3.6 (The OsiriX Foundation, Geneva, Switzerland); dorsoventral height (mm) on
transverse and sagittal images and length (vertebral bodies) were recorded. The presence of Charilike malformation (CM) was assessed by recording the presence of caudal cerebellar deviation
and/or vermal herniation. Fifteen dogs underwent atlanto-occipital cerebrospinal fluid tap at the
time of MRI and the analyzed WBC count was recorded. Student’s t-tests were used to compare
the means of syrinx dorsoventral size and length between groups with and without skull
abnormalities, neck pain and neurological signs. Simple Pearson’s correlation was used to test for
correlations of spinal fluid WBC and age with syrinx sizes and length. A chi-square test was used
to test for an association between cerebellar deviation and/or vermal herniation and the presence of
neck pain or neurological signs.
The mean age of the 7 males and the 21 females was 52 months (range 21-106). Neurological
deficits and neck pain were noted in 8 (29%) and 10 (36%) of dogs respectively; 3 dogs (11%)
exhibited both. Cerebellar deviation and vermal herniation were present in 12 (43%) and 24 (86%)
dogs respectively. Mean sagittal height of the CC was 2.3mm (0.6-7.2); mean transverse height
was 2.6mm (0.8-7.6). Fifteen (54%) CCs were greater than 2mm in height; the mean length of
these lesions was 3.4 vertebrae (1-7). Mean CSF wbc count was 5 (1-13). Syrinx heights were
significantly greater in dogs with cerebellar deviations than in those without (p=0.029). Syrinx
extent was significantly higher in dogs without cerebellar herniation (p=0.019) and in dogs with
neurological signs (p=0.035). There were no associations of syrinx height or extent with CSF wbc
and patient age or gender. Significantly less of the dogs with cerebellar herniations had
neurological signs (21%) than the dogs without cerebellar herniations (75%; p=0.0264).
This preliminary study suggests that SM and CM are prevalent in ABGs. Syrinx size is
associated with neurological signs and cerebellar deviation but may not be associated with the
classic form of CM (cerebellar herniation) in the ABG. EPIDEMIOLOGY OF SALMONELLA ENTERICA TYPHIMURIUM IN SONGBIRDS IN
THE SOUTHEASTERN UNITED STATES
Al W. Ray III1*, Susan Sanchez1, Kevin Keel2, Sonia Hernandez3, Ying Cheng3, and John J.
Maurer3
Departments of 1Infectious Diseases, 2Veterinary Pathology, and 3Population Health, the College
of Veterinary Medicine, the University of Georgia, Athens, GA, 30602
Salmonella enterica serovar Typhimurium outbreaks of unknown origin have plagued passerines
for years, causing significant mortality in the wild. The illness is marked by enteritis with
esophageal lesions, generally a clinical presentation not seen in other avian groups including
psittacines or gallinaceous birds. These outbreaks have a devastating ecological impact on bird
populations and might prove a significant threat to public health. These epizootic outbreaks are
seasonal, occurring most frequently in winter, early spring. We do not know how Salmonella
Typhimurium is transmitted in the wild bird population, the environmental reservoir, or factors
responsible for these wild bird die-offs. During 2009, we observed a significant epizootic
outbreak of S. Typhimurium in songbirds, especially in Pine Siskins. By Pulsed-Field Gel
Electrophoresis (PFGE), we identified the same S. Typhimurium strain isolated from Pine
Siskins and other passerines, in multiple Southeastern states including Georgia, Tennessee, and
North Carolina. In a retrospective comparison of this S. Typhimurium strain to an archival
collection of S. Typhimurium from multiple animal sources, we found PFGE matches with
songbird isolates from as far back as 1996. This strain appeared to be unrelated to S.
Typhimurium strains isolated from other avian species, most notably psittacines and gallinaceous
birds- chickens and turkeys; and cattle. Presently, we’re comparing the PFGE patterns for S.
Typhimurium songbird isolates against the Center for Disease Control and Prevention (CDC)
USA PulseNet database to determine if this songbird S. Typhimurium strain matches with any S.
Typhimurium associated with human illnesses in the US.
Multiple-anthelmintic resistance on a llama farm in the southeastern
United States
Daniel Zarate1, Bob Storey1, Lisa Williamson2, and Ray M. Kaplan1
University of Georgia, College of Veterinary Medicine, Department of Infectious Diseases1, and
Department of Large Animal Clinical Sciences2, Athens, Georgia USA
Documentation of anthelmintic resistance on goat and sheep farms in the southeastern United
States is substantial; however, little is known about its prevalence on llama farms. A study was
conducted on a llama farm in Florida to test for the presence of resistance to fenbendazole,
levamisole, ivermectin, and moxidectin using both fecal egg count reduction test (FECRT) and
larval development assay (LDA, DrenchRite®). Seventy-two llamas were allocated randomly
into six treatment groups (n=12/group): fenbendazole oral(FBZ 20 mg/kg), levamisole oral (LEV
12 mg/kg), ivermectin injectable (IVM 0.3 mg/kg), moxidectin oral (MOX 0.3 mg/kg), moxidectin
injectable (MOX 0.3 mg/kg), and untreated control. For the LDA, nematode eggs were isolated
from a pooled fecal sample collected at the time of treatment. FEC reductions were 0%, 96%,
0%, 90%, and 58% for FBZ, LEV, IVM, MOX PO, and MOX SC, respectively. Based on
WAAVP guidelines, these results indicate resistance to all drugs tested except levamisole. In
contrast, the LDA data indicated resistance for BZ and IVM, and sensitivity to LEV and MOX.
Based on coprocultures, the most common nematode was Haemonchus contortus, followed by
Nematodirus spp. and Trichostrongylus spp. These findings confirm the presence of multipleanthelmintic resistance on a llama farm in the southeastern US. Furthermore, the incongruent
results for MOX in the LDA and FECRT suggest that the dose applied may not be adequate,
and that optimal route of administration requires further investigation. . Further research is
required to assess the prevalence of anthelmintic resistance on camelid farms in US.
THE PRESENCE OF RESIDUAL DISC AND ITS INFLUENCE ON RECOVERY AND RECURRENCE
IN 43 CHONDRODYSTROPHIC DOGS WITH ACUTE THORACOLUMBAR INTERVERTEBRAL
DISC DISEASE. ROACH W, THOMAS M, WEH J, PLATT S, KENT M, BLEEDORN J, WELLS K.
The purpose of this study was to report the amount of residual disc material following
hemilaminectomy, to determine if certain hemilaminectomy techniques are associated with
less residual disc, and to determine if the amount of residual disc effects patient functional
outcome in chondrodystrophic dogs with acute intervertebral disc disease (IVDD).
Computed tomography (CT) was performed immediately pre-operatively and postoperatively on 43 dogs undergoing hemilaminectomy to determine the amount of residual
disc. Patient recovery and long-term functional outcome was recorded while in-hospital and
with two telephone interviews with the owners.
Residual disc was present in all of the dogs in this study, which has not been previously
reported. More ventral hemilaminectomies in this study were associated with more residual
disc. There was a positive correlation between maximum residual effacement percentage
and days until ambulation.
There was a significant effect of residual disc percentage on patient functional outcome with
respect to residual neurologic deficits at long-term follow-up.
The findings of this study reveal the presence of residual disc in all dogs, and suggest that
the amount residual disc is an important factor in patient functional recovery.
USE OF LOMUSTINE FOR FELINE VACCINE-ASSOCIATED SARCOMAS (VAS).
Corey Saba,1 Nicole Northrup,1 Douglas H. Thamm,2 Ruthanne Chun,3 and David Vail.3
1
University of Georgia, College of Veterinary Medicine, Athens, GA, 2Colorado State University,
College of Veterinary Medicine and Biomedical Sciences, Fort Collins, 3University of Wisconsin,
School of Veterinary Medicine, Madison, WI.
The purpose of this study is to evaluate the overall response rate (ORR), time to progression
(TTP), and toxicity for cats with measurable VAS treated with lomustine chemotherapy as a sole
treatment modality.
Cats with histologically confirmed, measurable VAS of any subtype are eligible for this
prospective, Phase II multi-institutional trial.
Fourteen cats have been enrolled to date. Eight tumors were fibrosarcomas, and 6 were softtissue sarcomas. Median longest diameter or sum of longest diameters of the primary tumor(s)
was 4.2 cm (range: 1.3-12.1 cm).
Five cats were treated at a starting “target” dosage of 60 mg/m2 (range: 30.1-60.8 mg/m2). All
cats were neutropenic after the first treatment. Neutropenia was severe in all 5 cats. All cats were
also thrombocytopenic after the first treatment. Thrombocytopenia was moderate in 2 cats and
severe in 3 cats. The median duration between treatments #1 and #2 was 4 weeks (range: 3-6.5).
Nine cats were treated at a starting “target” dosage of 48 mg/m2 (range: 31.3-51.1 mg/m2). Five
cats were neutropenic after the first treatment. Neutropenia was moderate in 1 cat and severe in 4
cats. Eight cats were thrombocytopenic after the first treatment. Thrombocytopenia was mild in 3
cats, moderate in 2 cats, and severe in 3 cats. The median duration between treatments #1 and #2
was 3 weeks (range: 3-4).
For all cats, the median number of treatments administered was 2 (range: 1-4). ORR was 29%.
Maximal responses included complete response (CR) in 1 cat, partial response (PR) in 3 cats, and
stable disease (SD) in 8 cats. Two cats experienced progressive disease (PD) following the initial
treatment, and 1 cat was unavailable for response evaluation due to toxicity related euthanasia.
The CR was not durable, lasting < 7 days. Median TTP was 59.5 days (range: 21-113 days).
Lomustine has some efficacy in the treatment of VAS. Although the response rate is relatively
low, the number of cats treated thus far is small and all cats had substantial gross disease. Based
on tumor biology, it is likely that this drug is more efficacious if administered in a microscopic
disease setting. The initial “target” dosages of 60 mg/m2 and 48 mg/ are intolerable, however the
ORR of 29% suggests further investigation into additional dosing modifications.
EVALUATION OF THE FAMACHA© SYSTEM IN SOUTH AMERICAN CAMELIDS
Bob Storey1, Lisa H. Williamson2, and Ray M. Kaplan1
Department of Infectious Diseases, University of Georgia College of Veterinary Medicine,
Athens, Georgia, USA and 2Department of Large Animal Medicine, University of Georgia
College of Veterinary Medicine, Athens, Georgia, USA
1
The FAMACHA© system was developed in South Africa as a means to apply a targeted and
selective based approach to anthelmintic treatment of sheep infected with Haemonchus contortus.
The FAMACHA© system evaluates the animals anemia status by comparing the lower conjunctiva
to a laminated card (the FAMACHA© card) depicting 5 illustrations of ocular membrane colors,
ranging from a score of 1 (red, non-anemic), to a score of 5 (white, severely anemic). The
FAMACHA© system was validated in the United States (US) in 2004 for use with both sheep and
goats, based on highly significant correlations between packed cell volumes (PCV), FAMACHA©
eye scores, and fecal egg counts (FEC). The FAMACHA© system has gained wide acceptance
since its validation in the US and is used extensively by sheep and goat producers throughout the
US. Recent identification of multiple anthelmintic-resistant H. contortus isolates on camelid
farms in the southeastern US has prompted interest in the applicability of the FAMACHA©
system with new world camelids.
FAMACHA© eye scores, PCVs, FECs, body condition scores, age, sex and species were
measured/obtained on 848 camelids on 25 southeastern US llama and alpaca farms with
documented H. contortus populations.
Animals with FAMACHA© scores of 1 to 5 had mean PCV of 31%, 28%, 27%, 22% and 16%,
respectively. Animals with FAMACHA© scores of 1 to 5 had mean FEC (EPG) of 139, 284, 567,
1238, and 4,047, respectively. Statistical analysis with GraphPad Prism 5.0 software yielded a
spearman correlation between FAMACHA© score and PCV of -0.427 (p < 0.0001), FAMACHA
score and FEC of 0.297 (p < 0.0001), and PCV to FEC of -0.441 (p < 0.0001).
These Preliminary results indicate that FAMACHA© scores demonstrate discriminatory value in
camelids for anemia and therefore H. contortus burden, in herds where the primary GI parasite is
H. contortus. A full statistical analysis is underway, which will enable us to make further
inferences on the accuracy and usefulness of FAMACHA© in South American camelids. COMPARISON BETWWEEN THE PROTECTION INDUCED BY DIFFERENT INFECTIOUS
LARYNGOTRACHEITIS VACCINES ALONE AND COMBINED WITH NEWCASTLE
DISEASE VIRUS VACCINE.
Vagnozzi, A., Riblet, S., Garcia, and M. Zavala, G.
Poultry Diagnostic and Research Center. Department of Health Population. College of Veterinary
Medicine. University of Georgia. Athens, GA.
The Infectious Laryngotracheitis (ILT) is a viral disease that generates a relevant economic impact
in the poultry production. The infectious agent is the Infectious Laryngotracheitis Virus (ILTV),
which belongs to the alpha-herpesvirinae subfamily. The control of the disease is mainly achieved
by vaccination, and two different attenuated ILTV vaccines have been used worldwide; the
chicken embryo origin (CEO) vaccines and the tissue culture origin vaccines (TCO). However, in
spite of the extensive use of the ILTV vaccination, the disease is still a problem in areas of intense
broiler production. Many factors could be involved in the lack of optimal protection in the
vaccinated flocks; some of them can be related to environment, nutrition, management, etc. One
factor that has been mentioned but no studied before is the possible action of other respiratory
diseases vaccines, such as Newcastle Disease Virus (NDV), which may impair the protection
induced by ILTV vaccines. The objective of this work was to determine the role that NDV play in
the protection induced by different ILTV vaccines. Protection induced by the ILTV vaccines
(CEO and TCO) was evaluated alone and in combination with NDV (B1 strain). Briefly, 14 daysold broilers in isolation units were vaccinated with a single, or combinations of vaccines via eyedrop. Two weeks after vaccination broilers were challenged with a virulent ILTV strain (Group V
genotype 63140 isolate) applied intratracheally and in the conjunctiva. Protection was evaluated at
5 and 7 days post-challenge by the following parameters: i) clinical signs; ii) body temperature);
iii) challenge virus shedding (quantified by real-time PCR). The results of this study indicate that
B1 NDV vaccination showed no significant interference with the CEO induced protection;
however, the NDV vaccinations strongly impaired the protection induced by the TCO vaccine (p >
0.05). Either alone or in combination with B1, protection efficiency of the TCO was lesser than
the CEO vaccine showing significant higher clinical signs and viral shedding after challenge.
DETERMINING EQUINE INTESTINAL TRANSIT TIME
Kruger M, Virgo J, Betts E, Ball BA, Pitts N, and Fayrer-Hosken RA
Veterinary Wildlife Services, Kruger National Park, Skukuza, RSA, Department of Population
Health and Reproduction, University of California, Davis, Davis, CA, Department of Large
Animal Medicine, College Veterinary Medicine, University of Georgia, Athens GA.
Kruger National Park (KNP) in South Africa is one of the world’s wellsprings of healthy
and genetically unique rhinoceroses. Annually in KNP, eighty to one hundred white rhinoceros
are captured and relocated. Of these, forty to fifty are boma-confined or boma-trained. The
relocation of white rhinoceros is critical to re-establish rhinoceros populations and to increase
genetic diversity of existing populations. The relocation is stressful to the animals and this
stress may be detrimental to their subsequent adaptation and reproduction. Because studies in
the rhinoceros are difficult, the availability of suitable domestic animal models, such as the
horse, allows the possibility to develop techniques to quantitate stress in white rhinoceroses.
The aim of this research was to develop techniques to measure gastrointestinal transit times in
horses at the University of Georgia, as a model for wild-caught rhinoceroses. Also these initial
techniques must prove the safety of the markers to ensure there is no injury to the rhinoceroses.
These transit times would be used subsequently to evaluate changes in fecal metabolites of
cortisol in the rhinoceros as a method to quantitate capture and handling associated stress. In
the current study, healthy horses that were barn acclimated and not stressed were used. To
determine the movement of ingesta a buoyant and non-toxic marker was sought. For the
research we placed a visual marker, beads or glitter (Sulyn Industries® Glitter and Beads (4 oz
jar)), in the food to determine the time from administration to the time of defaecation. Eight
horses were used and were dosed with a mixture of nontoxic glitter or beads in Kayro syrup.
Kayro syrup was used to provide a viscous and glutinous carrier to keep the marker in the
rhinoceros’s mouth until they were completely awake. After the dosing, each new pile of feces
was collected and evaluated to determine the time of the first passage of glitter. There were 13 replicates amongst various horses; the mean estimated intestinal transit time of a healthy
horse was 27.25 + SD 3.45 hours. The transit time for the beads could not be determined as
they did not emerge and were not used again. The horses were not anesthetized for this trial,
as would be the case in the wild. However, the quickest transit time would be 24 hours, so this
provides the expected time frame for wild rhinoceroses. Most importantly there are no
negative side effects from glitter or Kayro syrup administration. This is essential evidence, as
the dosing studies would not be permitted in white rhinoceroses without this safety validation.
NEMATODE ION CHANNELS AS TARGETS FOR ANTHELMINTIC COMPOUNDS
SM Williamson, HM Bennett, S McCavera, AJ Wolstenholme
Dept. Infectious Diseases, College of Veterinary Medicine, University of Georgia and Dept of
Biology & Biochemistry, University of Bath, Bath, U.K.
Nematode parasites have a huge impact on the health of both companion animals and livestock.
Antiparasitics are the largest-selling class of veterinary drugs. Many of these drugs act by binding
to the ligand-gated ion channels of the nematode, causing paralysis and cessation of feeding and
egg production by the parasite. Despite decades of routine administration of these compounds, the
molecular basis underlying the exact mode of action of these drugs is still poorly understood. The
aim of our work is to gain a more complete understanding of the drug targets and understand how
nematode parasites become resistant to dewormers. We use molecular biology techniques to study
the nematode ion channels targeted by ivermectin and the cholinergic anthelmintics such as
pyrantel and levamisole. We have studied the glutamate-gated chloride channels of Haemonchus
contortus, a gastrointestinal parasite of small ruminants, to determine the molecular basis of
ivermectin sensitivity; we have also studied the nicotinic acetylcholine receptors of Ascaris suum,
the large roundworm of pigs, to investigate the molecular basis of parasite sensitivity to
levamisole, pyrantel and oxantel. We have amplified and cloned the ion-channel subunit
sequences from the parasites, then used a heterologous expression system to produce recombinant
parasite ion channels which can be used for in vitro pharmacological studies. The anthelmintics
open the channels, resulting in uncontrolled ion fluxes that, in vivo, cause the paralysis and
expulsion or death of the worm. These studies have identified which ion channel subunits in these
parasites are targeted by the anthelmintic drugs, and have also demonstrated that heterologous
expression of parasite ion channels is an extremely useful method for both investigating the effects
of potential resistance mutations on drug sensitivity in vitro, and also as a tool for screening novel
compounds for potential anthelmintic activity.
NEMATODE ION CHANNELS AS TARGETS FOR ANTHELMINTIC COMPOUNDS
SM Williamson, HM Bennett, S McCavera, AJ Wolstenholme
Dept. Infectious Diseases, College of Veterinary Medicine, University of Georgia and Dept of
Biology & Biochemistry, University of Bath, Bath, U.K.
Nematode parasites have a huge impact on the health of both companion animals and livestock.
Antiparasitics are the largest-selling class of veterinary drugs. Many of these drugs act by binding
to the ligand-gated ion channels of the nematode, causing paralysis and cessation of feeding and
egg production by the parasite. Despite decades of routine administration of these compounds, the
molecular basis underlying the exact mode of action of these drugs is still poorly understood. The
aim of our work is to gain a more complete understanding of the drug targets and understand how
nematode parasites become resistant to dewormers. We use molecular biology techniques to study
the nematode ion channels targeted by ivermectin and the cholinergic anthelmintics such as
pyrantel and levamisole. We have studied the glutamate-gated chloride channels of Haemonchus
contortus, a gastrointestinal parasite of small ruminants, to determine the molecular basis of
ivermectin sensitivity; we have also studied the nicotinic acetylcholine receptors of Ascaris suum,
the large roundworm of pigs, to investigate the molecular basis of parasite sensitivity to
levamisole, pyrantel and oxantel. We have amplified and cloned the ion-channel subunit
sequences from the parasites, then used a heterologous expression system to produce recombinant
parasite ion channels which can be used for in vitro pharmacological studies. The anthelmintics
open the channels, resulting in uncontrolled ion fluxes that, in vivo, cause the paralysis and
expulsion or death of the worm. These studies have identified which ion channel subunits in these
parasites are targeted by the anthelmintic drugs, and have also demonstrated that heterologous
expression of parasite ion channels is an extremely useful method for both investigating the effects
of potential resistance mutations on drug sensitivity in vitro, and also as a tool for screening novel
compounds for potential anthelmintic activity.
ESTABLISHMENT OF HA6 MONOCLONAL ANTIBODIES TOWARDS THE
DEVELOPMENT OF A SPECIES-INDEPENDENT H6 cELISA
Lauren Gay1, Robert Hogan2, Frank Michel2, and Egbert Mundt1
1
Poultry Diagnostic and Research Center, Department of Population Health, University of
Georgia, Athens, GA. 2Department of Pathology, University of Georgia, Athens, GA.
Global surveillance of avian influenza viruses (AIVs) remains of paramount importance
given the ability of these viruses to cross species barriers and travel large distances in a relatively
short amount of time. Detection of AIV can be accomplished either by virus isolation or by
screening serum samples for antibodies. Since the virus is present in a given host for only a short
time, looking for longer lasting virus-specific antibodies is the more practical approach for piecing
together the epidemiology and ecology of AIV. Principal diagnostic tools which can be used to
detect AIV specific antibodies include the hemagglutination inhibition (HI) assay, the enzymelinked immunosorbent assay (ELISA), and the agar gel precipitation test (AGPT). Between these
tests, ELISA is advantageous because it is rapid, efficient, and it can be automated. Additionally,
with a competitive ELISA (cELISA), it is possible to detect antibodies in serum regardless of the
species from which the serum originated. This can be accomplished through the use of a
monoclonal antibody (mAb) that is specific for the protein of interest. In this study, the HA6
protein was expressed in a baculovirus system, purified, and used for the generation of four mAbs.
Prior to the selection of a single mAb for use in an H6-specific cELISA, the four mAbs were
characterized. It was observed that all four mAbs bind to a linear epitope within the HA1 portion
of the hemagglutinin protein. In addition, out of subtypes H1-H13 (H14-H16 were not tested),
the mAbs were found to be specific only for H6. Initial results indicate that one mAb will be of
value in the development of the H6-specific cELISA.
SONOCLOT EVALUATION OF WHOLE BLOOD COAGULATION IN HEALTHY ADULT
DOGS. D Babski, A Koenig, J Pittman, B Brainard. University of Georgia College of Veterinary
Medicine, Athens, GA.
The Sonoclot analyzer provides information on whole blood coagulation using both a qualitative
graph (Sonoclot Signature) and quantitative values: the activated clotting time (ACT), the clot
rate (CR), and the platelet function (PF). We hypothesized that the Sonoclot would provide an
accurate and rapid evaluation of canine whole blood coagulation. Our objectives were to establish
a standard procedure for assessment of canine whole blood samples and to generate a reference
range for the Sonoclot signatures of healthy dogs.
Blood was collected from 6 healthy adult dogs by jugular venipuncture. After a rest period at
room temperature of 30, 60, and 120 minutes, 340 μL of citrated blood was added to 20 μL of
0.2M CaCl2 in one of two cuvette types warmed to 37° C. Cuvettes contained either a magnetic
stir-bar with glass beads (gbACT) or only a magnetic stir-bar (nonACT). To generate a reference
range, blood was collected from 29 healthy adult dogs and analyzed in duplicate.
The ACT, CR, and PF were not significantly affected by duration of the rest period in either
gbACT or nonACT cuvettes at any time point. Although there was no difference between cuvette
type for CR and PF values, variability was significantly decreased when using gbACT cuvettes
when measuring ACT at all time points (p < 0.05). In normal dogs, ACT reference range (mean ±
2 SD) was 120 to 132 seconds, CR range was 11.3 to 41.7, and PF range was 2.2 to 4.8.
DETERMINATION OF IRRITANT THRESHOLD CONCENTRATIONS OF SIX MITES
THROUGH SERIAL DILUTIONS IN INTRADERMAL TESTING ON HEALTHY
CLINICALLY NON-ALLERGIC DOGS
CL Bauer1, P Hensel1, M Austel1, D Keys2
1
Department of Small Animal Medicine, University of Georgia College of Veterinary Medicine,
Athens, GA, USA
2
Statistical Consultant, Athens, Georgia, USA
Abstract: Intradermal allergy testing concentrations for dust and storage mites commonly induce
positive skin test reactions. One explanation for this could be the use of testing concentrations
which exceed an irritant threshold concentration inducing false positive reactions. The purpose of
this study was to determine the irritant threshold concentration (ITC) of six mites for intradermal
testing (IDT) in 37 healthy, clinically non-allergic dogs. Six allergens, two dust mites and four
storage mites, were tested at five variable concentrations ranging from 10-250 PNU ml-1. ITC’s
were determined by evaluating the lowest concentration to which no dogs (0% cut-off) and to
which at least 10% of dogs (≥10% cut-off) reacted. ITC’s at the 0% cut-off were: 10 PNU ml-1 for
Dermatophagoides farinae and Tyrophagus putrescentiae, 25 PNU ml-1 for Acarus siro and
Lepidoglyphus destructor, and 75 PNU ml-1 for Dermatophagoides pteronyssinus. For Blomia
tropicalis the 0% cut-off was < 10 PNU ml-1 and could not be determined. ITC’s at the ≥10% cutoff were: 50 PNU ml-1 for Blomia tropicalis, 75 PNU ml-1 for Acarus siro, Dermatophagoides
farinae, and Lepidoglyphus destructor, 100 PNU ml-1 for Tyrophagus putrescentiae and 200 PNU
ml-1 for Dermatophagoides pteronyssinus. Based on these results it is suggested that ITC’s for the
mites tested may vary and that testing at concentrations greater than 200 PNU ml-1 may induce
clinically non-relevant positive reactions. Further studies are needed to evaluate the benefit of
lower IDT concentrations for house dust and storage mites in atopic dogs.
THE NICOTINIC ACETYLCHOLINE RECEPTORS OF ASCARIS SUUM
HM Bennett1,2, SM Williamson1, AP Robertson3, RJ Martin3, T Williams4, DJ Woods4, DB
Sattelle5, AJ Wolstenholme1
1
Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, and
2
Dept of Biology & Biochemistry, University of Bath, Bath, U.K. 3Department of Biomedical
Sciences, Iowa State University, 4Pfizer Animal Health, Kalamazoo, Michigan, 5MRC Functional
Genomics Unit, Oxford, UK.
Parasitic nematodes are important pathogens of livestock and companion animals. Currently,
and for the foreseeable future, control of these infections is largely via treatment with chemical
anthelmintics. Nicotinic acetylcholine receptors (nAChRs) are important targets for these drugs,
with pyrantel and levamisole being notable examples, and a new class of compound, the aminoacetonitrile derivatives, currently in development. However, increasing resistance presents a
problem for sustainable helminth control. Despite their importance as drug targets in parasites,
most previous studies of nematode nAChRs have been carried out using the free-living species, C.
elegans. Using bioinformatics, we found that some parasites have fewer nAChR genes than C.
elegans, but some subunits of the levamisole-sensitive neuromuscular nAChR (unc-38, unc-29 and
unc-63) are well conserved. We have cloned cDNAs encoding UNC-29 and UNC-38 from Ascaris
suum, a large GI parasite of pigs; antibody labelling shows that Ascaris UNC-38 and UNC-29 colocalise on the muscle cell membrane. We have co-expressed Ascaris UNC-38 and UNC-29 in
Xenopus oocytes to form functional ACh-, nicotine and levamisole-gated ion channels, the first
successful heterologous expression of a parasite nAChR. Changing the subunit stoichiometry
produces receptor populations with different pharmacological properties: injecting different RNA
ratios to drive expression towards a 3:2 UNC-38:UNC-29 stoichiometry produces a receptor
population more sensitive to nicotine and oxantel, whereas driving expression to favour a 2:3
UNC-38:UNC-29 stoichiometry produces receptors more sensitive to levamisole and pyrantel.
The pharmacology of these receptors resembles the L- and N- subtypes previously observed in
native A. suum muscle cell membranes.
In addition we describe a novel nAChR subunit gene, acr-26, that is conserved in several
evolutionary distinct parasitic species of veterinary importance but, to date, not in any free-living
or plant parasitic species. An antibody against A. suum ACR-26 showed that it was expressed in
the head region of the nematode, but not body-wall muscle. Sequence similarities with other
nAChR subunits, such as the nematode ACR-16 and the vertebrate α7 and α9 subunits, combined
with a computer modelling approach, predicted that ACR-26 could form a homomeric receptor.
We injected cRNA encoding ACR-26 into Xenopus oocytes and observed that a novel homomeric
receptor was expressed in these cells, forming cation channels sensitive to both acetylcholine and
nicotine.
IN VIVO EFFECTS OF FIROCOXIB, MELOXICAM AND TEPOXALIN ADMINISTRATION ON
EICOSANOID PRODUCTION IN TARGET TISSUES OF NORMAL CATS. L Goodman, BT Torres, LR
Reynolds, SC Budsberg. University of Georgia College of Veterinary Medicine, Athens, GA.
The purpose of this study was to investigate the in vivo activity of firocoxib, meloxicam and tepoxalin in normal
cats by measuring eicosanoid production within target tissues. Eight normal adult neutered male cats were used in
this blinded, randomized, crossover study. Cats were treated with firocoxib (1 mg/kg PO q 24 h), meloxicam (0.05
mg/kg PO q 24 h), tepoxalin (5.0 mg/kg PO q 12 h), or placebo for 8 days. Samples of blood and gastric and
duodenal mucosal biopsies were collected on days 0 (baseline), 3, and 8 of each dosing period. Thromboxane
B2(TXB2)concentrations were measured in serum and prostaglandin E2(PGE2) and leukotriene B4 (LTB4) levels
were measured in plasma. PGE1 and PGE2 synthesis, and LTB4 levels were determined in the mucosal biopsy
specimens. A 21 day washout period was observed between treatments. Repeated measures analyses were
performed with significance set at p<0.05. In the blood, firocoxib and meloxicam decreased plasma PGE2 levels
compared to baseline on both days. Firocoxib and meloxicam decreased PGE2 compared to placebo on day 3.
Tepoxalin did not decrease PGE2 in the plasma compared to baseline or placebo on either day. Firocoxib showed no
difference in serum TXB2 compared to baseline or placebo on both days. Tepoxalin decreased TXB2 compared to
baseline and placebo on both days and compared to firocoxib on both days. No decrease was noted in plasma LTB4
concentrations in cats administered placebo, firocoxib, meloxicam, or tepoxalin at any time Firocoxib decreased
pyloric PGE1 synthesis on day 8 compared to baseline. Meloxicam showed no significant differences from baseline
or placebo on either day. Tepoxalin decreased duodenal PGE1 synthesis on both days compared to baseline. Neither
firocoxib or meloxicam decreased pyloric PGE2 synthesis compared to baseline or placebo on days 3 and 8.
Tepoxalin decreased pyloric PGE2 synthesis compared to baseline on days 3 and 8 and decreased pyloric PGE2
synthesis compared to placebo on day 3. Furthermore, tepoxalin decreased pyloric PGE2 synthesis compared to
firocoxib and meloxicam on days 3 and 8. Tepoxalin decreased duodenal PGE2 synthesis compared to baseline on
days 3 and 8. Meloxicam decreased pyloric LTB4 on day 3 compared to baseline and placebo. Tepoxalin decreased
pyloric
mucosal
LTB4
on
days
3
and
8
compared
to
baseline.
Firocoxib and meloxicam spared the cyclooxygenase-1(COX-1) enzyme while tepoxalin exhibited COX-1, COX2 and lipoxagenase-5 (LOX-5) inhibition at the dosages used in this study. Meloxicam may have LOX-5 inhibition
capabilities.
INITIAL CHARACTERIZATION OF MIRNA PROFILES IN CANINE LYMPHOMA
Erin L. Burdette1, Elizabeth W. Uhl1, S. Mark Tompkins1, Chantel L. Lester1, Steven E. Suter2
1
University of Georgia College of Veterinary Medicine, Athens, GA, 2North Carolina State
University College of Veterinary Medicine, Raleigh, NC
Lymphoma is one of the most common malignant cancers in dogs; however prognosis varies
with the specific type of lymphoma and classification as to cell of origin based upon morphology
can be difficult. MicroRNAs (miRNA) are small, non-coding RNAs that regulate gene expression
through mRNA repression or degradation and have vital roles in cellular processes including
growth, differentiation, and death. miRNA profiles are cell type specific and aberrant expression
has been linked to cancer development. miRNA are also highly conserved across species.
miRNA expression profiles in human cancers have been associated with clinopathologic features,
disease outcomes, and response to therapy. Our long term goal is to identify cancer-specific
miRNA profiles that can be used to improve diagnostics, classification, outcome predictions, and
treatment of canine lymphoma. For this study, we utilized a Real-time PCR miRNA array
originally designed for the characterization of human tumors to analyze miRNA expression
profiles in canine tumors. Our preliminary results indicate there are differences in the miRNA
profiles in both B and T cell lymphomas. Specifically, compared to samples of PBMC (n=4),
miR-17-92 were increased in B-cell tumor samples (n=8) while miR-17-92 and miR-146 were
increased in T-cell samples (n=4). We also developed an in situ hybridization protocol to detect
miRNA in paraffin-embedded samples, which can be used to further characterize canine tumors.
Given its cell and tissue specificity, and its critical role in cell differentiation and proliferation,
miRNA has immeasurable potential for improved characterization and understanding of both
human and animal cancers.
Caroline Colden
Undergraduate Student, Dr. Corrie Brown’s Lab
Abstract for 2009 Vet Med Research Day Symposium
Temporal Distribution of the Vesicular Stomatitis Virus in Coronary Bands of
Experimentally Infected Cattle: An Immunohistochemical Study
The Vesicular Stomatitis Virus (VSV) is a single-stranded, negative-sense
arbovirus in the Family Rhabdoviridae. VSV infects cattle, pigs, and horses and settles in
specific tissues, primarily the coronary bands of feet, tongue, snout, and teats, causing
vesicular (blistering) lesions. Infection can have debilitating effects on the animals,
posing a huge threat to the food and livestock industry. It is important to understand how
the virus spreads and damages cells. In this study, immunohistochemistry was used to
detect the presence of the virus in tissues of cattle infected with VSV through
scarification. Virus was present in the coronary bands 12, 24, 48, 72, 96, and 120 hours
post-VSV infection. Virus replicated predominantly in the very thick non-haired portion
of the coronary band and appeared to spread cell to cell, with evidence of most viral
protein just below the plasma membrane of the keratinocytes.
THE EFFECT OF GENETIC MAKE UP AND INCUBATION CONDITIONS ON
EXPRESSION OF TYPE I COLLAGEN, DECORIN AND TGFΒ IN GASTROCNEMIUS
TENDONS IN YOUNG BROILERS
Joshua Cummock, Abel Kusovschi, Jaroslava Halper. University of Georgia, Athens, Ga. Edgar
O. Oviedo-Rondón. North Carolina State University, Raleigh, N.C.
Commercial broilers undergo rapid growth, primarily due to increase in muscle mass, during the
first few weeks of their life. Their gastrocnemius tendons undergo tremendous growth as well. It
is known that changes in tendon structure result from growth, and mobilization. The role of
breeding and husbandry conditions in tendon growth and development has been studied to a lesser
degree. Leg problems have been well documented in rapidly growing broilers, and though bone
deformities have been described, it is unclear to what extent, if any, leg tendons are affected.
To study whether genetic makeup and/or incubation conditions contribute to tendon problems in
rapidly growing young broilers, we evaluated the expression of several proteins in gastrocnemius
tendons of broilers from several strains whose eggs were incubated under different conditions.
Two treatment groups each containing chickens from three genetic strains, labeled A, B and C,
were used for experiments. Eggs from one group were incubated under standard conditions (SS)
before hatching. Eggs assigned to the second group were incubated under conditions of
alternating low/high temperature (LH). Gastrocnemius tendons were collected at hatch (1 day of
age), 4, 14 and 21 days of age, and positioned longitudinally in casettes for histological
processing. Hematoxylin-eosin was used for basic visualization of tendon tissues. Using
immunohistochemistry, tissue sections were then stained for type I procollagen, decorin and
transforming growth factor β (TGFβ). Image ProPlus software was used for morphometric
evaluation of collagen fiber thickness in H&E stained sections and of crimp of collagen fibers in
type I procollagen stained section. Fibers in several different areas of tendons were evaluated. The
thickness of collagen between rows of tenocyte nuclei in longitudinal bundles and fascicles was
measured separately from the thickness of collagen between rows of tenocyte nuclei in
transversely cut smaller bundles of collagen located in the proximal end of the tendons.
As expected, the thickness of collagen fibers increased with age in all three strains and both
treatment groups. The collagen fibers were slightly thicker in tendons from SS chickens, than
fibers from the LH chickens H&E stained sections at all examined days (1,4,14, 21) by an average
of 0.1 µm (SD 0.14 µm). We evaluated striation pattern created by procollagen binding in fibers
in sections immunostained for type I procollagen. The striations were more pronounced in LH
tendon for day 21 (31.9 µm, SD 1.5 µm) than in the SS tendons (30.3 µm, SD 4.9 µm).
We conclude that incubation conditions of eggs before hatching may play a bigger role in
integrity of tendon structure and function than the strain of broiler.
PASSAGE OF LOW PATHOGENIC H5N1 AND H5N3 AVIAN INFLUENZA VIRUS
ISOLATES IN CHICKENS RESULTS IN ADAPTIVE MUTATIONS IN THE
HEMAGGLUTININ AND NEURAMINDASE GENES
Daniel Dlugolenski, Les John, Mark. S. Tompkins, Ralph A. Tripp, Egbert Mundt
Poultry Diagnostic and Research Center, Department of Population Health, University of Georgia,
Athens, GA 30602. Department of Infectious Diseases, University of Georgia, Athens, GA 30602
To better understand the mechanisms of LPAIV transmission, adaptation, and disease
pathogenesis six serial passages of LPAIV isolates in chickens were performed. The sequences of
the HA and NA gene were determined before passage and at each passage level. Three week old
SPF chickens were infected 10^6 EID50 of an LPAIV wild bird isolate H5N1 and chicken isolate
H5N3, respectively. At 1 day pi, 5 contact birds were added to each group. Tracheal and cloacal
swabs were taken at 2, 4, 7, 9, 11, and 14 d pi. Swab samples were passaged in 9 day-old
embryonated SPF eggs for virus isolation. The presence of virus in the allantoic fluid was tested
by HA. Existence of HI antibodies was tested on serum samples taken before infection and 21d pi.
The NA and HA genes of HA positive swab and allantoic fluid samples were sequenced and
amino acid sequences were deduced. During passage, virus was isolated only from the infected
birds. H5N1 showed no transmission where as H5N3 exhibited limited transmission as evidenced
from serological data. The sequence analysis revealed several mutations in the amino acid
sequences which mostly occurred during early passages of the viruses. The mutations in the HA
gene of H5N1 include amino acids I75V, D101N, T343K, and K395T. Here mutation T343K lead
to a more hydrophobic cleavage site in the HA protein. Additionally, at passage 5 a potential Nglycosylation site was introduced by the mutation A174T. Several mutations (C72Y, S75N) were
observed in the NA gene of the H5N1. Most interestingly a deletion in the stalk region (delta5675) was observed which eliminated four potential N-glycosylation sites. The observed mutations
of HA protein(N170D, Q174R) and NA protein (D100E) of the H5N3 isolate did not affect
potential glycosylation sites. The results of this study showed that the genetic makeup of the wild
bird isolate changed more rapidly in comparison to the chicken isolate during passaging in
chicken. This result indicates an adaptive process of the wild bird isolate to replicate more
effectively in a new host.
This work was funded by the contract NIH HHSN266200700006C.
DO PAX6-DEFICIENT ANIMALS HAVE CORNEAL EPITHELIAL STEM CELLS? JD Duncan*, J
Kim†, JD Lauderdale†, and PA Moore*. *University of Georgia College of Veterinary Medicine, Athens,
GA. †Department of Cellular Biology, University of Georgia, Athens, GA.
The cornea is the clear, fibrous tissue that accounts for approximately 2/3 of the eye’s optical power. It
has a specialized clear, stratified squamous epithelium that is composed of unique cell types. This
epithelium is replenished from a stem cell population found on the rim, or limbus, of the cornea. Limbal
stem cells (LSC) produce transient amplifying cells that proliferate, move centripetally, and usually
desquamate near the center of the cornea. A critical loss of LSC will cause keratopathy characterized by
fibrosis, vascularity and overgrowth of the cornea by conjunctival epithelial cells. PAX6, the master control
gene for the eye, is related to the development and maintenance of this epithelium. PAX6(+/−) humans
develop aniridia, an eye with a hypoplastic iris, and aniridia-related keratopathy (ARK). Small-eye mice are
haploinsufficient for Pax6 and develop a similar keratopathy. We are looking for evidence of the existence
of LSC in aniridic humans and small-eye, or Pax6(+/SeyNeu), mice. Finding LSC in these animals would
imply that stem cell dysfunction, rather than deficiency, is the cause of ARK. Human aniridic LSC culture
would allow cross validation of animal in vivo models to the animal and human cell culture models.
Frozen and paraffin histosections of normal human, rabbit and mouse eyes are compared to sections of
aniridic human and small-eye mouse eyes. The corneal limbal epithelial cells of normal rabbit have been
cultured after using Dispase to remove the epithelium from the stroma. Human corneal limbal epithelial
cells have been cultured from explants. Cell culture colonies have been evaluated for colony-forming
potential and immunofluorescent staining has been performed to identify colonies of corneal cells.
We are able to grow cells from normal rabbits in cell culture that appear to form stem-cell colonies with
features consistent with LSC. These techniques will be used to attempt to grow colonies of LSC from
normal and PAX6-deficient mice and humans.
MAPPING BINDING EPITOPES OF THE MORAXELLA CATARRHALIS HAG ADHESIN. J
Enners, R Balder, ER Lafontaine. University of Georgia College of Veterinary Medicine, Athens,
Ga.
Moraxella catarrhalis is a Gram-negative bacterium that is among the leading causes of otitis
media in children and lower respiratory infections in adults with Chronic Obstructive Pulmonary
Disease (COPD). The organism has acquired beta-lactamase activity in more than ninety percent
of isolates tested thereby rendering most strains resistant to commonly used antibiotics. This
growing antibiotic resistance coupled with the frequency of M. catarrhalis ear infections in
children has prompted research into vaccine development for the pathogen. Adherence to
epithelial cells is a key component in the pathogenesis of M. catarrhalis and therefore the proteins
that mediate this adherence could make excellent vaccine targets. Previous studies have shown
that the surface protein Hag is a key mediator in adherence of this bacterium to middle ear cells,
NCIH292 lung cells, and A549 type II pneumocytes. In order to further explore binding domains
of Hag, hag deletion constructs were manufactured with in-frame deletions removing specific
regions of the open reading frame and were designated 10.9, 10.32, 4.16, and 5.12. Previous
research has been performed with hag deletion construct 10.9 and showed that pLF.10.9 allowed
H. influenza to adhere to NCIH292 cells and HMEE cells. However, pLF.10.9 did not confer
adherence to A549 cells or NHBE cells. These results confirm that the Hag adhesin may have
different binding domains for different cell types. The goal of this project was to engineer
plasmids containing other hag deletion constructs to further map the binding epitopes of the
adhesin. Plasmids with the hag construct of interest were engineered using standard molecular
biologic techniques which included a standard plasmid preparation protocol using QIAprep Spin
Miniprep system (Qiagen), hag construct DNA isolation using BamHI restriction/digestion, DNA
purification from agarose gel using a High Pure PCR Product Purification Kit (Roche), hag
construct ligation with T4 DNA ligase into pWW115, and electroporation to incorporate plasmid
into Haemophilus influenza strain DB117. PCR and Western Blot techniques were used to screen
recombinant H. influenza strain DB117 clones for expression of Hag constructs. Results showed
that Hag insert 10.32 was successfully cloned into pWW115, and that electroporation allowed
incorporation of plasmid into DB117. However, The Western blot of DB117 clone D74 was
negative for expression of Hag 10.32. It was concluded that the hag insert was incorporated into
the plasmid in the wrong direction thus preventing expression of the protein. Future work will
involve reassessing the protocol described above and continuing to engineer plasmids containing
hag contstructs of interest in order to further characterize binding epitopes of the Hag adhesin.
ROLE OF CD18 IN THE PATHOGENESIS OF CYTAUXZOONOSIS
K. Frontera-Acevedo, D. S. Peterson, H. Brown, L. Sellers, and K. Sakamoto. Department of
Pathology and Department of Infectious Diseases, College of Veterinary Medicine, University of
Georgia, Athens, GA.
Cytauxzoonosis is a fatal hemoprotozoal disease of cats and wild felids in the Mid-Western, MidAtlantic, and Southeastern United States caused by Cytauxzoon felis. Although the causative
agent has been recognized since the seventies, no study has profiled the immune response of
infected cats and there is no definitive cure. One of the main histopathologic characteristics of this
disease is the presence of giant infected intravascular monocytes, many of which are adhered to
the vascular endothelium. We hypothesize that these infected monocytes have up-regulated
adhesion molecules, such as CD18, and that ligation of these molecules stimulate proinflammatory cytokines involved in the pathogenesis of the disease. In order to test this
hypothesis, immunohistochemistry was performed comparing CD18 surface expression between
C. felis-infected and uninfected cats, and showed markedly increased CD18 staining of infected
macrophages and interstitial neutrophils. Serum from critically-ill, but not surviving C. felisinfected cats contain high levels of pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-1β.
Neutralization of CD18 on peripheral leukocytes from C. felis-infected cats using anti-CD18
antibodies resulted in a significant to complete reduction of pro-inflammatory cytokine production
in response to lipopolysaccharide stimulation in vitro. These results suggest that neutralizing
antibody therapy against CD18 may be beneficial in the treatment of acutely-ill, C. felis-infected
cats.
MUTAGEN-EXPOSED FEMALE GERM CELLS MEDIATE DELAYED MUTAGENESIS IN EARLY
STAGE EMBRYOS
Gresham CS, Norris MB, and Winn R. Aquatic Biotechnology and Environmental Laboratory, Warnell
College of Forestry and Natural Resources, University of Georgia, Athens, Georgia
Analyses of mutations in offspring transmitted by female germ cells have been hampered by
numerous practical challenges. Further, genetic health risks based on analyses of male germ cells may not
be directly applicable to female germ cells. To address the need for improved approaches to study
mutagenesis mediated by female germ cells, we used a fish model that carries the cII mutation target gene.
We detected mutations in the cII genes carried by offspring of ethylnitrosourea (ENU)-treated λ transgenic
females and untreated, wildtype male medaka. Offspring that exhibited cII MF greater than 2-fold above
controls were scored as mutants, and comprised up to 16% of the offspring, comparable to frequencies of
mutant offspring derived from male germ cells. Mutant offspring exhibited a wide range of cII MFs, from
~2 to 500-fold (7.5 to 1585 X10-5) induction compared to controls. Characterization of mutational spectra
revealed both whole body and mosaic mutant offspring were produced, with mosaic mutants comprising
the vast majority (93%) of mutant offspring. Whole body mutants were generated from mutations fixed
either in gametes, or the one-cell stage, whereas mosaic mutants were generated from mutations fixed at or
after the two-cell stage of development. These data show fixation of persistent DNA damage in mature
oocytes was delayed until after fertilization. Consequently, as shown in male germ cells, post-fertilization
processes contributed by the maternal genome in the mutagenesis in early stage embryos. These data
illustrate this fish model provides new insights into germ cell mediated mutagenesis.
BREED SPECIFIC POLYMYOSITIS IN THE HUNGARIAN VIZSLA DOG
AC Haley1, SR Platt, M Kent, SJ Schatzberg, A Durham, S Cochrane, GD Shelton. 1. University
of Georgia, College of Veterinary Medicine, Athens GA. 2. University of Pennsylvania, School of
Veterinary Medicine, Philadelphia, PA. 3. Veterinary Emergency Clinic and Referral Centre,
Toronto, Ontario Canada, 4. University of California, San Diego, Department of Pathology,
School of Medicine, La Jolla, CA
Inflammatory myopathies are relatively common in dogs and may have a focal (masticatory
muscle myositis) or generalized (polymyositis) distribution. Recently, a breed specific myositis
presenting as pharyngeal dysphagia and masticatory muscle atrophy has been described in 14
Hungarian Vizsla dogs in the United Kingdom (Foale et al, BSAVA 2008). Here we report a
similar syndrome in 5 Vizsla dogs from the North America. This retrospective study provides a
preliminary clinicopathologic description and outcome in these 5 dogs.
Five Viszla dogs presented to 5 different veterinary hospitals (1 general practice, 2 referral
practices, 2 university practices) with clinical signs of dysphagia (3/5), regurgitation (3/5),
excessive salvation (3/5), masticatory muscle atrophy (4/5) and pain on opening the jaw (2/5). All
dogs were male (4/5 castrated, 1/5 intact) and ranged in age from 1 to 9 years (mean 5.2 years).
Creatine kinase activity was measured in 3 cases and was elevated (range 1061-9758 U/L; mean
5443). Antibodies against type 2M fibers and acetylcholine receptors were negative in those dogs
tested (3/3 and 2/2 respectively). Three dogs had radiographically evident megaesophagus (ME).
Two dogs had ME at the time of initial presentation while one dog developed ME 19 months after
onset of dysphagia. Serum antibody titers against Toxoplasma gondii and Neospora canis,
Borrelia burgdorferi, Ehrlichia canis and Ehrlichia equi were negative when tested.
Electromyography was performed in 2 dogs with no abnormalities. Histopathologic examination
of temporalis muscle biopsies was performed in 3 cases with multifocal areas of lymphocytic
infiltration in 2 cases. Although cellular infiltrates were not evident in one case, bilateral
multifocal hyperintensities in the temporalis muscle were observed on T2-weighted magnetic
resonance images (MRI). A complete necropsy was performed on the fifth case and chronic
lymphohistiocytic and plasmacytic myositis and fibrosis was evident in the esophagus,
myocardium and skeletal muscle. Immunosuppression with prednisone and azathioprine has not
resulted in clinical improvement at 2 and 3 months follow up in 2 dogs. Two dogs were lost to
follow up. In conclusion this study, in combination with the previous report from the UK, should
alert clinicians to the occurrence of a new breed associated polymyositis in the Vizsla dog which
warrants further investigation to elucidate pathogenesis, genetic associations, response to
treatment and prognosis.
SEROLOGICAL AND INTRADERMAL TEST REACTIVITY PATTERNS AMONG SIX
SPECIES OF HOUSE DUST AND STORAGE MITES
P Hensel1, CL Bauer1, M. Austel1, D Keys2
1
Department of Small Animal Medicine, University of Georgia College of Veterinary Medicine,
Athens, GA, USA
2
Statistical Consultant, Athens, Georgia, USA
Although the significance of dust mites as an important source for atopic dermatitis has been
recognized, the validation of positive results has not been elucidated in depth. Serology and
intradermal testing (IDT) were performed in 32 clinical healthy non-allergic dogs for two house
dust mites: Dermatophagoides farinae (DF), D. pteronyssinus (DP), and four storage mites:
Blomia tropicalis (BT), Tyrophagus putrescentiae (TP), Acarus siro (AS), and Lepidoglyphus
desctructor (LD). All dogs reacted to at least one allergen in the serology test, whereas 23/32 of
dogs had a positive IDT reaction to at least one allergen. Most common reactions (serology vs.
IDT) were observed for the following mites: DF (96.875% vs. 62.5%), AS (87.5% vs. 59.375%),
TP (87.5% vs. 43.375%), BT (65.625% vs. 53.125%). Less common reactions were observed for
DP (43.75% vs. 15.6%) and LD (6.25% vs. 34.375%). Concurrent positive reactions among
different mites were significant for most mites in IDT (except between DF and DP). Serology
revealed strong co-reactivity between DF and TP, TP and AS, as well as DP and BT. Skin test
positive dogs had significantly higher mean serology values for DP, DF, AS, and BT than dogs
with a negative skin test. In conclusion, positive reactions to dust mites in serology and IDT are
very common in clinically healthy, non-allergic dogs making a correct interpretation difficult.
Also, the dogs reacted in general to more than one mite indicating cross-reactivity or concurrent
exposure to different mites.
EFFECT OF DIFFERENT EXPOSURE TIMES TO TNF-α ON CELL RECEPTOR PROFILES
IN CULTURED CANINE ENDOTHELIAL CELLS
DN LoBato, BM Brainard, J Barber, and EW Howerth. College of Veterinary Medicine,
University of Georgia, Athens, GA 30602.
Endothelial changes in response to inflammatory cytokines can lead to potentially harmful
sequelae associated with systemic inflammation in veterinary patients. TNF-α activates endothelial
cells (EC) and leads to the expression of cell surface receptors (CSR). The sequence of CSR
expression is not fully documented in canine EC. Our objective was to determine how canine
endothelial CSR profiles change when EC are exposed to TNF-α. Cultured canine ECs were
incubated with human-derived TNF-α (50 ng/ml) for 0, 4, or 8 hours. Fluorescent-labeled
antibodies to specific canine CSR associated with EC activation (CD62P, CD62E, CD54) were
used to identify CSR profiles at different time points. CD-62E expression increased from baseline
median fluorescence intensity (MFI) of 201 to 654 at 4 hours and to 1445 at 8 hours. Baseline
CD54 expression was 712, and increased to 4948 at 4 hours and to 6212 at 8 hours. Interestingly,
a high level of CD62P was not demonstrated at any time point, with a baseline MFI of 142, 146 at
4 hours, and 202 at 8 hours. The progressive expression of CSR in stimulated canine ECs mirrors
the profile in EC from other species. CD62P was expected to have high expression soon after
stimulation because it is stored preformed in Weibel-Palade bodies of ECs. CD54 and CD62E,
conversely, require several hours for expression following induction, but persist within vessels for
up to 48h following exposure to TNF-α. The validation of these antibodies will allow future
studies evaluating endothelial activation and microparticle generation in the dog.
THE DISTRIBUTION OF LARGE MOLECULAR WEIGHT PLASMIDS AND
GENOMICS IN SALMONELLA
Karen Lyons, Margie Lee, John Maurer, Susan Sanchez, John Maurer
ABSTRACT
Salmonella remains one of the leading causes of foodborne illnesses; however
information regarding the distribution and genomics of plasmids other than the virulence
plasmid is limited. In this study, we characterized the diversity of large molecular weight
plasmids in Salmonella isolated from a diverse group of vertebrate hosts to determine if
distribution of large molecular weight plasmids is affected by the carriage of the virulence
plasmid. Few plasmids other than the virulence plasmid were detected among a diverse group
of Typhimurium isolates, although novel plasmids were detected among other serotypes.
DNA sequencing of novel plasmids isolated from Salmonella groups C1 and H revealed
genetic similarities to two newly characterized Salmonella plasmids, although novel
conjugation systems were detected from the same strains. In addition to detecting transposons
and colicin operons, we did not detect any antibiotic resistance genes. While some serotypes
may not exhibit the propensity to harbor multiple plasmids within their genome, other
Salmonella isolates have acquired unique plasmids from unknown hosts. These results
indicate that there are unknown genetic barriers to plasmid acquisition by some salmonellae.
GENERATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES
AGAINST THE NEURAMINIDASE 1 OF A HIGHLY PATHOGENIC AVIAN
INFLUENZA VIRUS
Rob Nichols1, Alice Mundt1, Jeff Hogan2, Frank Michel2, Egbert Mundt1, and Maricarmen García1
1
Poultry Diagnostic and Research Center, Department of Population Health, University of
Georgia, Athens, GA 30602. 2Department of Pathology, University of Georgia, Athens, GA
The gene encoding neuraminidase 1 subtype (N1) from the HPAI virus Indonesia/PA7/2003
(H5N1) was amplified and cloned. The N-terminal signal peptide sequence was replaced with a
signal peptide sequence from Leucania separata nucleopolyhedrovirus (LsNPV), and an RGS-His
tag sequence was added at its C-Terminus. A recombinant baculovirus was generated (N1-BV)
and used to infect Sf9 cells. The recombinant N1 protein (N1-Bac) was secreted into the cell
culture supernatant and was purified by affinity chromatography. Using N1-Bac, a panel of 16
mouse monoclonal antibodies (MAbs) specific to the recombinant N1 protein was generated. The
specificity of the MAbs was tested by indirect ELISA and an indirect immunofluorescence test
(IFA) using N1-BV infected Sf9 cells. Chicken cells infected with avian LPAI viruses encoding
for neuraminidase subtypes N1 – N9 were evaluated by IFA. 15 of the 16 MAbs demonstrated
specificity for the N1 subtype. One notable exception was MAb N1-27, which appears to have
broad reactivity for N1 – N9. In addition it was observed that only a limited number of the MAbs
reacted with A/WSN/1933 (H1N1) and A/Puerto Rico/8-SV14/1934 (H1N1) infected cells. To
determine the nature of the epitopes recognized by the MAbs, Western blot analysis was
conducted. This analysis demonstrated that while 14 of the 16 MAbs showed no reactivity to N1Bac, two of the MAbs, N1-8 and N1-9, remained reactive the recombinant protein.
IN-VIVO ROLE OF RAC-1 IN MAMMARY MORPHOGENESIS AND LACTATION.
V. Oduah, R. Wheeler, T. Nagy. Department of Pathology, University of Georgia College of
Veterinary Medicine, Athens, GA
This study was conducted to gain insight into the function of the Rac-1, a member of the Rho
family of GTPases, in mammary morphogenesis and lactational differentiation. To observe the
role of Rac-1 on morphogenesis and differentiation, we created a genetically engineered mouse
model with Rac-1 deletion specific to the epithelium of the mammary gland using the Cre-LoxP
system. To assess postnatal mammary development before pregnancy, we first analyzed ductal
elongation and branching of the mammary epithelium in virgin females. We found that virgin
female mice with mammary gland-specific Rac-1 deletion showed reduced ductal elongation and
branching in the mammary gland, in comparison to control virgin females. Lactating female mice
with Rac-1 deletion also had delayed mammary gland development, manifested by the reduction
in the size of the milk producing lobulo-alveolar units. We also observed that the offspring of
mutant females either died as a result of inanition or survived with reduced weight gain during
lactation, in comparison to the offspring of control females. Our immediate future goal is to
analyze the underlying signaling events that result in mammary gland hypoplasia in the mutant
females. Parallel with these investigations, we are currently also studying the role of Rac1- in
mammary carcinogenesis.
LESION ANALYSIS IN LEATHERBACK SEA TURTLES AT SANDY POINT NATIONAL
WILDLIFE REFUGE, ST. CROIX, USVI
Annie Page1, Justin Perrault2, Jeanne Garner3, and Debra Miller4
1
University of Georgia, CVM, Athens, GA USA
Florida Atlantic University, Boca Raton, FL USA
3
West Indies Marine Animal Research and Conservation Service, Frederiksted, St. Croix, USVI
4
University of Georgia, VDIL, Tifton, GA USA
2
Distribution of the principally pelagic leatherback sea turtle (Dermochelys coriacea) in the
Atlantic Ocean is considered temperate-north temperate, with adults entering tropical waters to
breed. Migrating leatherbacks often encounter the U.S. long-line fishery fleet that operates along
the U.S. Atlantic coast over the continental shelf and slope, and in distant areas including the
central North Atlantic. Leatherbacks nesting at Sandy Point National Wildlife Refuge (SPNWR)
frequently show evidence of human interaction, including open wounds, scars, and embedded fish
hooks with associated foreign body-type inflammation and granuloma formation. During 2009, we
documented the presence of lesions typical for fishhook granulomas on a subset of the nesting
population, and characterized them by diameter, height, consistency, and anatomic location. Forty
turtles were examined and five (12.5%) had lesions consistent with granulomas. The granulomas
had an average diameter of 44.12mm (± 26.03), and an average height of 18.61mm (± 6.68). Four
(80%) were firm, four (80%) were located on an anterior flipper, and three (60%) were located
next to a wound or scar. Four (80%) of the affected turtles were remigrants (i.e., individuals have
nested at SPNWR in previous years). Although these lesions often are not fatal, they represent
negative human interactions with these critically endangered animals. Thus, future studies should
attempt to verify the etiologies of these lesions and test their hypothesized link to long-line
fisheries. This information will help us evaluate the impact of long-line fisheries on leatherback
sea turtle populations.
EFFECT OF UNFRACTIONATED HEPARIN ON TEG R TIME IN HEALTHY DOGS
JR Pittman, A Koenig, BM Brainard.
University of Georgia College of Veterinary Medicine, Athens, GA, 30602
The objective of this study was to determine the effect of single and multiple doses of
subcutaneous (SQ) unfractionated heparin (UFH) at a dose of 200 U/kg on the thrombelastogram
reaction time (R time) of healthy dogs.
Six random-source famale dogs were studied. Baseline parameters, including a complete blood
count (CBC), prothrombin time (PT), activated partial thromboplastin time (aPTT), and
antithrombin (AT) were performed. Thrombelastography (TEG) and aPTT were performed hourly
for 12 hours after heparin dosing (200 U/kg SQ). Anti-Xa activity was assayed at 0, 3, 6, and 8
hours. Heparin was then administered every 8 hours for 3 days. Day 4 protocol was identical to
Day 1.
On Day 1, percentage change from baseline for TEG parameter R (reaction time) was evaluated.
Statistically significant (p < 0.01) prolongation of the R time was seen in all dogs by hour 3. R
was unmeasurable for most dogs between 3-5 hours. All TEG R time tracings returned to baseline
by 12 hours. Day 4 TEG tracings mimicked those on Day 1. Only one dog achieved aPTT values
outside the reference interval on both days. Anti-Xa activity levels increased on Day 4 but not on
Day 1. Based on post hoc in vitro analysis, prolongation of R-time occurred at plasma heparin
levels as low as 0.075 U/mL.
Administration of SQ heparin results in progressive prolongation of the TEG R time, with
maximal change occurring 3-5 hours after dosing. The extensive prolongation of the R time
indicates that thrombelastography may be too sensitive as a monitoring tool for UFH therapy.
As the heparin dose effect was waning, 4 of the 6 dogs displayed a shortened R time, indicative
of a state of transient hypercoagulability. While previously unrecognized, this phenomenon may
occur in dogs if heparin therapy is rapidly withdrawn. Further TEG investigation after
discontinuation of heparin therapy may give insight into the phenomenon of hypercoagulability
after abrupt cessation of therapy, and may provide guidelines for the most appropriate way to taper
the dose.
PREVALENCE OF AND RISK FACTORS FOR STATUS EPILEPTICUS OR CLUSTER
SEIZURES WITH CANINE IDIOPATHIC EPILEPSY
S. R. Platt1 R. Monteiro,2 V. Adams,2 SJ Schatzberg,1 M. Kent,1 K. Rogers.2
1
College of Veterinary Medicine, University of Georgia, Athens, 2Centre for Small Animal
Studies & 2Centre for Preventive Medicine, Animal Health Trust, Newmarket, Suffolk, UK. CB8
7UU
Status epilepticus (SE) represents a continuous series of two or more discrete seizures lasting at
least 5 minutes between which there is incomplete recovery of consciousness. Cluster seizures
(CS) represent two or more seizures within a 24 hour period. The aims of this study were to
investigate the prevalence of SE and CS in dogs with idiopathic epilepsy, whether there is any
relation ship between these two events and what risk factors exist for these events.
Medical records of dogs presenting to the Animal Health Trust from 2000 to 2004 with seizure
disorders were evaluated. All dogs included in the study were confirmed to have idiopathic
generalised tonic-clonic seizures based upon owner description of seizure events, normal physical
and neurological examination, normal blood work, normal thoracic and abdominal imaging,
normal cerebrospinal fluid analysis and normal cerebral magnetic resonance imaging. Signalment,
anticonvulsant therapy where given, occurrence of SE and occurrence of CS were recorded.
The proportion of cases with SE and with CS are reported with 95% confidence intervals (CI).
Logistic regression was carried out to examine the relationship between the occurrence of SE and
CS with potential explanatory variables (age, gender, neuter status and treatment). Variables were
selected for inclusion in the final model if they significantly improved the fit. Significance was set
at P < 0.05 for all final models.
Data for 407 cases were included in the analysis. The mean age at diagnosis was 4 years (SD
2.6, range 2 months to 14 years and 5 months). There were a total of 10 cases with SE (2.5%, 95%
CI: 1.0 – 4.0) and 166 cases with cluster seizures (41%, 95% CI: 36 – 46). There was no
association between SE and cluster seizures (P=0.46). Treatment was associated with the
occurrence of SE and CS while gender was also associated with CS. Entire dogs were 1.9 times
more likely than neutered dogs to suffer from CS. There were no breed influences on the
occurrence of SE but Labrador Retriever, German Shepard Dog, Crossbreed and Border Collies
were the most frequently identified breeds of dog with CS.
This study confirms a relatively low prevalence of SE and moderately high prevalence of CS in
dogs with idiopathic epilepsy, which is important information for owners. Seemingly neutering
may reduce the risk of CS in dogs.
CEREBROSPINAL FLUID GLUTAMATE LEVELS IN DOGS WITH INTRACRANIAL
NEOPLASIA. SR Platt1, D Marlin2, M Kent1, SJ Schatzberg1 V Adams3. 1. College of Veterinary
Medicine, University of Georgia. 2. Centre for Equine Studies, 3. Centre for Preventative
Medicine, Animal Health Trust, Newmarket, Suffolk, UK.
Cerebrospinal fluid (CSF) glutamate levels may reflect central nervous system (CNS) glutamatemediated excitotoxicity and may be involved with the pathogenesis of CNS neoplasia and
associated seizure activity. Although CSF glutamate levels have been evaluated in dogs with
epilepsy and spinal disease, the alterations of this excitatory neurotransmitter in the presence of
cerebral neoplasia have not been documented. The objective of this study was to compare
glutamate levels in the CSF of dogs with cerebral neoplasia to those with a normal central nervous
system.
Twenty-three dogs with intracranial neoplasia (Meningiomas - group Ia; intra-axial tumours group Ib) and 21 dogs with no evidence of intracranial disease, based upon magnetic resonance
imaging (MRI) and CSF analysis (group II), were included in the study. Histological tumor type
and the presence of clinical seizure activity were recorded for group I dogs. All dogs had a CSF
tap performed at the cisterna magna under general anesthesia after an MRI; 0.3ml of CSF from
each dog was stored in fluoride oxalate containers and randomly assigned to -20 or -80°C freezers
until analysis. CSF samples (100ul) were deproteinised with methanol (100ul) and centrifuged at
14,000g for 2 minutes. Glutamate analysis was performed using high performance liquid
chromatography with fluorescence detection. Derivatised amino acids were resolved by binary
gradient elution. Glutamate levels were compared using non-parametric Kruskal-Wallis one-way
analysis of variance with post-hoc comparisons of mean ranks and Wilcoxon rank sum tests.
Significance was set at P≤0.05.
Comparison of the glutamate levels indicated that there was a difference in the three groups of
dogs (P=0.001). Median glutamate levels were significantly higher in dogs with brain tumors
(4.77 µmol/L) compared to the control dogs (3.05µmol/L). There was no difference in median
glutamate levels between dogs with meningiomas (5.65 µmol/L, n=11) and dogs with other brain
tumors (4.73 µmol/L, n=12); group Ib tumors included gliomas and metastatic disease. There was
no difference in glutamate levels with seizure activity (P=0.4). There was also no effect of freezer
storage temperature on results (P=0.7).
The results of this study suggest that glutamate-mediated excitotoxicity may be involved in the
pathogenesis of CNS neoplasia in dogs but may not be involved in the pathogenesis of associated
seizure activity. Glutamate elevation is a potential future treatment target for dogs with CNS
neoplasia.
CEREBROSPINAL FLUID NEUROTRANSMITTER CONCENTRATIONS IN DOGS
WITH ISCHEMIC INFARCTION OF THE BRAIN. SR Platt,1 R Barber,1 L De Risio,2
M Kent,1 J Eagleson,1 AC Freeman,1 AC Haley,1 SJ Schatzberg.1 1. University of
Georgia, College of Veterinary Medicine, Athens, GA USA. 2. Animal Health Trust,
Newmarket, UK
Cerebrospinal fluid (CSF) concentrations of excitatory (glutamate) and inhibitory
(GABA) neurotransmitters may reflect central nervous system excitotoxicity.
Excitotoxicity is a component of the purported pathophysiology of cerebrovascular
accidents, specifically ischemic infarction. Although CSF glutamate levels have been
evaluated in dogs with epilepsy, spinal disease and brain tumors, neurotransmitter
concentrations have not been documented in dogs with ischemic infarction (stroke) of the
brain. Such information could provide therapeutic targets for clinically affected dogs. The
objective of this study was to compare CSF neurotransmitter levels in dogs with
clinically confirmed ischemic infarction to those of normal dogs.
Cerebellomedullary cisternal CSF was collected from 10 healthy beagles with normal
brain MRI and from 12 various dog breeds which had clinical signs and cerebral MRI
consistent with ischemic infarction. The medical records of the clinical dogs were
searched to determine duration of disease at time of CSF tap and prior use of antiinflammatory medications. The CSF samples (20µl) were analyzed for glutamate, GABA
and glycine concentrations with electrochemical high performance liquid
chromatography following derivitisation with o-phtaldialdehyde/2-mercaptoethanol. A
student’s t-test was used to test for differences between the levels of the 3
neurotransmitters in the following groups: clinical dogs vs. normal; duration of signs <=5
days vs. >5 days; anti-inflammatory medicines administered vs. none given. Student’s ttest was implemented in PROC GLM in SAS and significance was set at P≤0.05.
There were no significant differences between the mean glutamate concentrations of all
clinically affected dogs and normal dogs. However, there were significant differences
between the mean CSF glutamate levels for dogs clinically affected for <5 days
(0.28µmo/l) when compared to those affected > 5 days (0.09µmol/l) and to normal dogs
(0.1µmol/l) (P=0.03). There were also significant differences in the mean glycine
concentrations detected between normal dogs (1.64µmol/l) and those which had
experienced ischemic infarction (1.09µmol/l) P=0.0099. There were no differences
detected between the GABA concentrations of any of the groups assessed. There was no
effect of the use of anti-inflammatory medications on any of CSF neurotransmitters.
The results of this preliminary retrospective study suggest that glutamate –mediated
excitotoxicity may be involved in the pathogenesis of canine cerebral ischemic infarction
within the first 5 days of disease. The reduction of glycine concentrations may also play
an important role. Glutamate elevation is a potential future treatment target for dogs with
cerebral ischemic infarction.
DYNAMIC CONTRAST ENHANCED MAGNETIC RESONANCE IMAGING OF CANINE
BRAIN TUMORS: A PRELIMINARY INVESTIGATION SR Platt,1 S Lee2, AC Freeman,1 AC
Haley,1 J Eagleson,1 M Kent1, SJ Schatzberg1, Q Zhao2 1Department of Small Animal Medicine &
Surgery, University of Georgia, Athens, GA. 2Department of Physics & Astronomy, BioImaging
Research Center, University of Georgia, Athens, GA.
Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) is an imaging technique
which can be used to investigate the physiology of tissue microvasculature and therefore serves as
a more objective method of brain tumor assessment, both pre and post therapy. For DCE-MRI, a
series of MR images are taken before, during, and after the injection of a paramagnetic contrast
agent and so represents a continuous evaluation through a series of temporally sequential images.
The aims of this study were to describe and evaluate the preliminary use of DCE-MRI for canine
brain tumors. More specifically, we aimed to assess pharmacokinetic contrast differences between
histopathologically diverse brain tumors that would subsequently allow for the development of a
prospective study to more accurately assess the potential relevance of this modality
Magnetic resonance datasets were collected on 7 canine intracranial tumors with a 3-Tesla
magnet using a T1-weighted fast spin echo fluid attenuated inversion recovery (T1 FLAIR)
sequence after an intravenous bolus injection (0.1mMol/lb) of Gd-DTPA (Magnevist; Bayer
Healthcare, Wayne, NJ.). The data were analyzed using two methods: a two-compartment
pharmacokinetic model for estimation of 3 enhancement parameters, ER (rate of enhancement), Kel
(rate of elimination), and Kep (rate constant); and a model-free phenomenological parameter
IAGUC (initial area under the Gd-concentration curve) defined over the first 90 seconds postenhancement. Pearson correlations were calculated between parameters of the two methods.
Statistical t-test was performed with α=0.05 to differentiate the tumor types. The tumors were
confirmed histopathologically as meningiomas (3 dogs), pituitary adenocarcinomas (2 dogs),
ependymoma (1 dog) and oligodendroglioma (1 dog). There were statistical differences (P<0.001)
between each tumor type’s 3 enhancement parameters as well as their IAGUCs.
This pilot study showed that the kinetic parameters and the model-free phenomenological
parameters derived from DCE-MRI present complementary information and they may be
appropriate to non-invasively differentiate canine brain tumors although a larger prospective study
is necessary.
CEREBROSPINAL
FLUID
GLUTAMATE
CONCENTRATIONS
AFTER
SUBARACHNOID HEMORRHAGE IN DOGS: EFFECT OF TREATMENT.
S.R. Platt1, J Coates2, D.M. Eifler2, R. Barber,1 G Edwards1, S.J. Schatzberg1, M Kent,1
K.R. Bulsara3. 1University of Georgia, College of Veterinary Medicine, Athens, GA,
USA; 2College of Veterinary Medicine and 3School of Medicine, University of Missouri,
Columbia, MO, USA
Subarachnoid hemorrhage (SAH) is characterized by decreased cerebral blood flow,
subsequent cerebral vasospasm and ischemia, and high mortality in people. Impaired
endothelial and neuronal nitric oxide (NO) release further lead to inflammation and
excitoxicity triggered by excitatory amino acids, glutamate and aspartate.
Immunosuppression using cyclosporine A ameliorates cerebral vasospasm, as well as
upregulation of NO synthase using statin treatment. We hypothesized that dogs with SAH
have alterations in CSF concentrations of glutamate, aspartate, GABA and glycine which
are indirectly but positively affected by use of cyclosporine and simvastatin. CSF
concentrations of neurotransmitters were investigated as biomarkers of neuronal injury
and indicators of beneficial treatments for CNS ischemia.
A double SAH model was induced in dogs by 2 injections (3 mls) of autologous blood
into the cerebellomedullary cistern (CMC) 24 hours apart. Dogs were assigned to one of
three groups: Control-untreated (n=4); simvastatin (Zocor, Merck Inc., 20 mg/kg SID
PO) only (n=4); simvastatin (20 mg/kg SID PO) and cyclosporine A (Sandimmune,
Sandoz Inc., 6 mg/kg SID PO) (n=4). Drugs were administered 24 hours after the second
injection for 10 days. CSF was collected from the CMC before each injection and on days
3, 7, and 10 and immediately stored at -80°C. The CSF samples were reacted to produce
1-cyanobenz[f]isoindole derivatives. Derivatives were analyzed with electrochemical
HPLC. Data analysis used repeated measures model and included factors for treatment,
time and a treatment time interaction term (PROC MIXED in SAS; p<0.05). Multiple
comparisons were adjusted for using Tukey's test.
In the control group, glutamate significantly increased to highest levels by day 3 and
then returned to baseline, whereas glycine, GABA and aspartate were not significantly
altered from baseline at any time point. There was significant decremental effect of
simvastatin alone (p=0.0004) and in combination with cyclosporine (p=0.0007) on day 3
glutamate concentrations when compared to the control group. A significant incremental
effect of combination treatment on day 3 glycine levels was noted compared to control
group (p=0.006). No significant differences in GABA and aspartate levels were noted
between treatment groups on any of the sample days.
Although precise roles of these neurotransmitters have not been elucidated in
pathophysiology of canine CNS ischemia, their alterations from baseline suggest further
investigation. A combination of immunosuppression and NO synthase upregulation may
be useful in ameliorating elevated glutamate levels in CNS ischemia.
EVALUATION OF AN AUTORADIOGRAPHY TECHNIQUE USING [3H] MK801 TO ASSESS THE DISTRIBUTION OF CANINE CEREBRAL GLUTAMATE
RECEPTORS. Platt SR, Edwards GL, Schatzberg SJ, Kent M. College of Veterinary
Medicine, University of Georgia, Athens, GA
The objectives of this study were to develop a reliable technique to determine
glutamate (NMDA) receptor distribution in the canine brain and establish a normal
cerebral regional distribution of glutamate (NMDA) receptors in the dog. [3H]MK801, which is a potent noncompetitive antagonist at the NMDA receptor, has been
extensively used in a multitude of autoradiographic models but has not been used in
intact canine brain.
Six canine fresh-frozen canine brains were sectioned serially, ensuring that
representative 20-μm slices of the frontal, parietal and occipital lobes were cut. The
sections were thaw-mounted onto gelatin-coated (0.5%; porcine 300 bloom)
microscope slides and dessicated at 4°C under vacuum for 24 hours and then stored
at -20°C for up-to 3 weeks. MK-801 sites were labeled with [3H]MK-801in the
presence of 100 μM glutamate and 30 μM glycine to open the channel. Nonspecific binding was determined in the presence of 100 μM MK-801 and ketamine
in adjacent brain sections. The slides were apposed to Hyperfilm-3H and exposed
for 30 days at 20°C and then developed with Kodak D-19 developer. Analysis of
the binding was done using a digital densitometry system with an image analysis
system. Radioactivity of a given region was determined by comparison to a
standard curve constructed from the optical densities of the radioactive standards.
The glutamate (NMDA) receptors were identified in all areas of the cortex of the
canine brain. There was negligible radioactivity in the white matter tracts. There
was reproducible and dense labeling of the caudate nuclei and the hippocampal
formation similar to that seen in rat brain. The addition of chromium to the slide
coats was vital to achieve tissue adherence. The association rate of the radioligand
with its binding site was accelerated by the addition of glycine and glutamate. The
results obtained confirmed that this technique was a reliable and reproducible
method of evaluating the glutamate receptor density and distribution in the canine
brain. This technique will be used to compare the distributions of canine cerebral
glutamate receptors in disease and after medical and surgical therapies.
BATRACHOCHYTRIUM DENDROBATIDS IN RED-SPOTTED NEWTS (NOTOPTHALMUS
VIRIDESCENS VIRIDESCENS) FROM NORTHEASTERN GEORGIA. M Purdee, B Rothermel,
DL Miller, and MJ Yabsley. The University of Georgia College of Veterinary Medicine, Athens,
GA
Batrachochytrium dendrobatidis (Bd), a worldwide emerging pathogen of amphibians, is
responsible for the extinction of numerous species. Based on previous data of Bd in northeastern
Georgia, we hypothesized that (1) Bd prevalence/burden will decrease as ambient temperatures
increase, (2) Bd infection will not be correlated with body size or sex, and (3) PCR of swabs will
be more sensitive for Bd detection compared to PCR or histology of toe clips.
Newts were collected by hand, net, or funnel traps from two ponds in northeastern Georgia.
Newts (n=96 unique individuals, n=26 recaptures) were collected every two weeks between May
and July 2009. Newts were weighed, measured, sexed, swabbed along their ventral surface and
hind limbs, toe-clipped based on an individual marking scheme, and released at the capture site.
DNA extracted from the swab and a toe was tested for Bd by PCR and quantitative real-time PCR.
The other toe was tested for Bd by histologic examination (H&E).
To date, we have noted that prevalence of Bd decreased as ambient temperatures increased
(90.5% in June and 30.5% in July). Several newts changed infection status from positive to
negative during subsequent samplings. No correlation between infection status and sex, body
length, and weight was noted. PCR on skin swabs demonstrated higher sensitivity for Bd detection
(70.8%) as compared to PCR and histology on toe clips (64.5% and 7.3%, respectively).
A decrease in Bd infection prevalence (using PCR methods) among Red-Spotted Newts was
observed as ambient temperatures increased over three months. When finalized, qRT-PCR data
will be used to determine if Bd loads among newts decrease with changes in ambient
temperatures.
Abstract:
THE ROLE OF TH17 CELLS, A SUBSET OF CD4+ HELPER T CELLS, IN THE
PATHOGENESIS OF EQUINE RECURRENT UVEITIS. DP. Regan, PA. Moore, KP
Carmichael, ML. Vandenplas. University of Georgia College of Veterinary Medicine, Athens, GA
Equine recurrent uveitis is a spontaneous disease that is the most common cause of blindness in
horses, affecting up to 15% of the horse population. Multiple hypotheses as to the etiology of ERU
have been formulated, but research conducted on these hypotheses, have failed to provide any
definitive answers. Recent investigations have led to the concept that recurrent uveitis in horses is
an autoimmune disorder. Th17 cells have been shown to be the major cell population driving the
pathogenesis in several mouse models of autoimmune inflammation, including experimental
autoimmune encephalomyelitis (EAE), and experimental autoimmune uveitis (EAU).The
hypothesis of this study is that a Th17 cell-mediated autoimmune response is involved in the
pathogenesis of ERU. We evaluated banked equine globes from 3 horses with ERU, and 4 horses
with no history of ocular disease (controls). Immunohistochemical staining using the horseradish
peroxidase method was used to demonstrate the presence of the cytokines IL-17 (Santa Cruz
Biotech., 1:100) and IL-23 (AnaSpec, 1:500,000), essential players in the Th-17 axis of
inflammation. Staining was evaluated on a positive or negative basis. Preliminary results indicate
that these cytokines are present in the iris, ciliary body, choroid, and retina of 3 horses with ERU,
but not in 4 normal controls.
Determination of Shedding and Survivability of Avian Influenza (AI) Viruses
in Experimentally Infected Chickens and Survivability of AI in Feces and
Poultry Litter.
Aline R. Reis1 , Casey W. Ritz1 , David E. Stallknecht1 , and Maricarmen García1
1University of Georgia
To predict the risk of human infections associated with exposure to the
poultry environment further knowledge on the survivability of AI viruses in the
environment will be necessary. The objective of this study was to determine the
shedding of poultry adapted virus A/Ck/CA/431/00(H6N2) and duck isolate
A/Mallard/MN/355779/00(H5N2) in chicken feces and to determine the
survivability of these viruses in poultry litter and feces. To determine the
shedding rate of these viruses broilers and layers were infected by the
intravenous and intranasal routes with a dose of 107.2EID50. Feces and cloacal
swabs were collected at 3, 7, 11, 12, 13 and 14 days post infection (PI) and virus
isolation and titration were performed in 9 to 11 day old chicken embryos.Both
type of birds shed H6N2 viruses in the feces and the cloaca up to day 7 PI, with
average titers in the feces of 102.8 EID50 for birds inoculated intravenously, and
101.5 EID50 for intranasaly inoculated birds. At 12 and 14 days PI the H6N2 virus
was recovered only from feces of layers inoculated intravenously at titers of 102.5
to 102.7 EID50. Birds inoculated intravenously with H5N2 shed virus up to day 7 PI
at an average titer of 102.1 EID50, while birds inoculated intranasally only shed
virus at day 3 PI at an average titer of 101.5EID50. The viruses H6N2 and H5N2 in
direct contact with poultry litter (broiler house) survived up to 48 hours at 40C and
for 24 hours at 250C.
CHARACTERIZATION
OF
EQUINE
SYNOVIAL
MEMBRANE
DERIVED
MESENCHYMAL STEM CELLS. Sarah Ricco’1, Michel Vandenplas2, David Hurley2, Maurizio
del Bue1, J. Peroni2 1.Universita’ degli Studi di Parma (Italy) 2.University of Georgia Athens GA
The overall goal of this project was to isolate and characterize mesenchymal stem cells (MSCs)
from equine synovial membranes as a basis for their eventual use in the repair of injured articular
cartilage, synovial membranes or menisci. Our primary objective in addressing this goal was to
attempt to isolate MSCs from equine synovial membranes, determine their ability to differentiate
into specific cell type, and to characterize their gene expression profile. We hypothesized that cells
isolated from equine synovial membranes would undergo osteogenic, adipogenic and
chondrogenic differentiation, and express genetic markers characteristic of MSCs when exposed to
the appropriate stimuli. Synovial membrane was obtained from the femoropatellar joints of 6
middle aged horses. Tissue was processed until uniform fibroblast cell cultures were obtained.
Subsets of cells were appropriately identified and differentiated into adipocytes, osteoblasts and
chondrocytes identified using respectively Oil Red O, von Kossa and Alcian blue stains. The
phenotype of separate subset of cells was assessed by flow cytometry and RT-PCR using
antibodies previously validated for use in the horse.
After the first passage and in vitro expansion, synovial-derived cells appeared microscopically to
be a homogeneous population of fibroblast-like cells. However, under the appropriate conditions,
adipogenic differentiation was demonstrated by the accumulation of lipid vacuoles stained with oil
red O, osteogenic differentiation was evident by calcium deposition on von Kossa staining, and
chondrogenic differentiation was evident by uptake of toluidine blue. (Figure 1).
Cells isolated from
synovial membranes
were determined to
be positive for gene
transcripts
previously ascribed
C
B
A
to the mesenchymal
stem cell phyenotype
Figure 1. A. Vacuolization typical of adipose differentiation (Oil Red O). B.
(CD90, CD45, CD44
Chondrocytic differentiation. Note intense toluidine stain uptake in the top portion of
the micromass culture. C. Osteogenic differentiation demonstrated by calcium
CD73, and CD105)
deposition. Dark stain as detected by von Kossa staining.
and negative for
transcripts (CD45 and CD34) that characterize cells of the hematopoietic lineage. Collectively,
these findings indicated that MSCs can be successfully isolated from equine synovial membranes.
Future studies will determine the regenerative potential of MSCs derived from synovial
membranes in the treatment of joint diseases in horses.
SALMONELLA LOAD AT STAGES OF POULTRY PRODUCTION. R. Berghaus, S. Thayer,
A. Roebling. University of Georgia College of Veterinary Medicine, Athens, GA.
From June to August of this summer, I assisted with an ongoing project monitoring Salmonella
serotypes and levels in poultry facilities near the Athens area. The goal of this extensive sample
collection project is to obtain a large data pool that quantitatively evaluates Salmonella and
Campylobacter loads from poultry at various stages in the production chain. These early efforts
will later be used to evaluate the efficacy of intervention strategies in reducing bacterial loads in
the final product. It is significant that this project focuses on load rather than prevalence because
the authors believe that load is a more reliable indicator of infection risk. Because this project will
collect data over the course of a year, it will be very informative for many researchers hoping to
glean analytical correlations concerning production stages and disease prevalence.
At the point of the project where I was involved, work with Campylobacter had been put on
hiatus and the focus was only on Salmonella. We began by collecting litter samples at poultry
farms under contract with a local poultry production company. Samples were then collected at the
poultry plant in Athens. The farms were tracked numerically by house so that the information
about the samples could be organized based on the chickens' source. Plant samples were collected
by doing carcass rinses in peptone water. First, chickens were killed outside the plant and rinsed.
Then, carcasses were collected from the production line at rehang, the production stage in which
the carcasses have been defeathered. Next, samples were collected before the carcasses entered
the chiller (ante-chill) and after (post-chill). These samples were iced and returned to the lab.
At the lab, samples were loaded from the peptone rinses into Tetrathionate (TT) Broth and serial
diluted. After they were incubated, the samples were transferred to Rappaport Vassiliadis (RV)
Broth and incubated again. Then, samples were transferred to xylose lysine tergitol (XLT-4) agar
gel and incubated again. Finally, gels were evaluated for black colonies, indicative of Salmonella
growth. The results were recorded based on presence of Salmonella in the dilution, which could
then be used to describe Salmonella load. These colonies were presumptively serotyped in house
with agglutination testing, then sent to another lab for definitive serotyping.
This project will continue through the rest of this year. The results gathered thus far illustrate the
anticipated decrease in Salmonella load chronologically through the production process. When
the project is concluded, this data will be compared to other poultry plants that use other methods
of decreasing bacterial load, allowing evaluation of protocols to determine if significant
differences exist in efficacy.
The conclusion at the moment is simply that the methods employed by poultry production at one
plant in Athens effectively achieve load reduction in Salmonella. When more data is collected as
per the overall goal of this project, more conclusions will be drawn comparing different poultry
plant hygiene management styles.
CONSTRUCTION OF AN UNMARKED RIBOFLAVIN REQUIRING DELETION MUTANT
OF RHODOCOCCUS EQUI. AS Rogovskyy, MM Tatum, MK Hondalus. The University of
Georgia, Athens, GA.
Rodococcus equi, a facultative intracellular bacterium, is a causative agent of serious pneumonia
of neonatal foals and is an opportunistic pathogen of immune-compromised people. No safe and
effective vaccine is yet available for R. equi disease prevention. The purpose of this work was to
generate an unmarked in-frame riboflavin-requiring deletion mutant of R. equi for use as a
potential vaccine. The approach used was based on a mutant construction method (van der Geize
et al., 2008) that centers around a 5-fluorocytosine-based conditionally lethal selection system. A
suicide plasmid was constructed to carry flanking regions homologous to the sequences adjacent
to three members of a putative riboflavin biosynthesis operon. Homologous recombination
occurred between these regions resulting in deletion of orf 3179, encoding a potential riboflavin
synthase, and substantially truncating orfs 3177 and 3181, additional members of this operon,
yielding an unmarked strain which was auxotrophic for riboflavin.
The mutant was unable to propagate in minimal acetate medium (MMAc) without riboflavin
supplementation but showed wild type growth in the presence of exogenously added riboflavin.
Furthermore, complementation of the mutant with wild type sequence restored the ability of the
strain to grow in MMAc medium confirming that the observed MMAc-specific growth defect of
the mutant was indeed due to the deletion of the operon genes. To examine the effect of the
deletion on intracellular growth, mouse-macrophage-like cells (J774A.1) were infected with the
riboflavin auxotroph and its growth kinetics was compared to that of wild type R. equi strain 103+
and the intracellular replication deficient, avirulent, virulence plasmid-cured strain 103-. Infected
macrophages were lysed at various time points post infection and the lysate plated to determine
the number of colony forming units associated with each monolayer overtime. As expected, wild
type R. equi replicated intracellularly, increasing approximately 50-fold in number by 48 hours
post infection, whereas the virulence plasmid-cured 103- failed to replicate. The growth of the
riboflavin auxotroph was approximately 10-fold reduced in magnitude as compared to that of wild
type. Thus, deletion of riboflavin biosynthesis genes attenuates R. equi by compromising the
bacterium’s capacity to replicate intracellularly. Given the paucity of riboflavin in mammalian
cells, the strain is expected to be attenuated in vivo and could be utilized as vaccine. Since the
mutant possesses no antibiotic resistant marker, there is no concern about the spread of antibiotic
resistance via vaccination.
METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS INCIDENCE IN GEORGIA AND
KENTUCKY, 1995-2003.
Ha-Jung Roh, Chrissy Still, Stephanie Beavers, Susan Sanchez. College of Veterinary Medicine,
University of Georgia, Athens, GA
Ever since the first equine Methicillin-resistant Staphylococcus aureus (MRSA) infection was
reported in Wisconsin in 1997, the importance of MRSA in horses has increased. However,
knowledge about the epidemiology of MRSA in horses is still very limited.
In this study, we investigated 152 equine Staphylococcus aureus isolates from Kentucky and
Georgia, collected from 1995 to 2003 to observe the trend of MRSA in horses and compare the
genetic relationship of these MRSA strains between the two states.
All samples were obtained from sick animals at both Kentucky and Athens Veterinary
Diagnostic Laboratories. All equine S. aureus isolates were confirmed to the species level with
catalase test and nuc gene PCR, the presence of mecA gene and staphylococcal cassette
chromosome mec (Scc mec) typing were also determined by PCR. Strain typing was achieved
using repetitive-sequence based PCR (rep-PCR), and a PCR for the Panton-Valentine leukocidin
gene detection were performed.
The results showed that out of total 152 samples, 62 (40.8%) were MRSA and 48(64%) of
Kentucky samples, and only 14(18.2%) of Georgia samples were MRSA strain. More than 45%
isolates obtained from1996 to 2000 in Kentucky were MRSA stains. Scc mec typing showed that
type IVd was predominant (58 out of 62, 93.5%) in our isolate collection and none of equine
MRSA carried the Panton-Valentine leukocidin gene. More than 90% (56 isolates out of 62) of the
MRSA strains were clustered in the group of rep-PCR pattern 11 with >95% similarity. Most of
MRSA strains were isolated from reproductive or respiratory disease presentation.
These data provide evidence for that, at least in some states, MRSA already had the high
prevalence as late as 1996 that this is not a recent phenomenon. Also it seems that one equine
MRSA strain supposedly had a clonal expansion through two separate states and present with
different clinical picture.
THE USE OF XENON¹³³ TO MEASURE LAMINAR BLOOD FLOW IN THE HORSE
C Sherlock, J Moore, D Hurley, L Young, J Peroni
University of Georgia, Department of Large Animal Medicine, Athens, GA.
Alterations in laminar blood flow have been implicated in the pathogenesis of laminitis in horses
however controversy exists regarding the nature of these changes. We hypothesised that injection of
intra-arterial Xenon¹³³ could be used as a safe and repeatable method to assess laminar blood flow
within the equine digit.
Four horses free of lameness and radiographic signs of laminitis were injected repeatedly at 5
minute intervals with 0.25mCi of Xenon¹³³ in solution via the randomly assigned previously
catheterised medial palmar artery. The gamma counts emitted (counts per minute (CPM)) were
recorded at the hoof with a sodium iodide scintillator.
All horses tolerated the catheterisation and Xenon¹³³ solution injection and no alterations in
temperature, pulse, respiration, blood pressure, complete blood counts or serum biochemistry
profiles were noted. Counts per minute increased rapidly and peaked within 22.8+/- 7.8 seconds
after intra-arterial injection. The peak CPM recording was 37190+/- 4322. After the peak value,
there was a less rapid decline in CPM. Subsequent intra-arterial injections produced comparable
CPM peaks.
In conclusion, Xenon¹³³ solution could be safely injected intra-arterially in horses. After intraarterial injection in horses without laminitis, there were similar peaks in CPM recorded at the foot.
This repeatable safe technique may provide useful information about alterations in laminar blood
flow in horses with laminitis, and may provide useful information about alterations in blood flow
caused by pharmaceuticals that are currently used, and may assist in design of pharmaceuticals for
future use.
GEOGRAPHIC DISTRIBUTION AND PREVALENCE OF CYTAUXZOON FELIS IN WILD
FELIDS. Barbara C. Shock1,2, Staci M. Murphy1, Laura L. Patton3, Philip M. Shock4, Colleen
Olfenbuttel5, Jeff Beringer6, Suzanne Prange7, Daniel M. Grove8, Matt Peek9, Jay Butfiloski10,
Daymond W. Hughes11, Mitch Lockhart12, Victor F. Nettles1, Holly M. Brown13, David S.
Peterson2, and Michael J. Yabsley1,14. 1Southeastern Cooperative Wildlife Disease Study, College
of Veterinary Medicine, University of Georgia, Athens, GA.2Department of Infectious Diseases,
College of Veterinary Medicine, University of Georgia, Athens, GA.3Kentucky Department of
Fish and Wildlife Resources, Frankfort, KY.4West Virginia Division of Natural Resources,
Charleston, WV.5North Carolina Wildlife Resources Commission, Apex, NC.6Missouri
Department of Conservation, Columbia, MO.7Ohio Department of Natural Resources, Athens,
OH.8North Dakota Game and Fish Department, Bismarck, ND.9Kansas Department of Wildlife
and Parks, Pratt, KS.10South Carolina Department of Natural Resources, Columbia, SC.11Wildlife
Services, APHIS, United States Department of Agriculture.12College of Arts and Sciences,
Valdosta State University, Valdosta, Georgia.13Department of Pathology, College of Veterinary
Medicine, University of Georgia, Athens, GA.14Warnell School of Forestry and Natural
Resources, University of Georgia, Athens, GA.
Cytauxzoon felis, a tick-borne protozoal parasite of wild and domestic felids, is the causative
agent of cytauxzoonosis in domestic and some exotic felids. C. felis can be transmitted by two
tick species, Dermacentor variabilis and Amblyomma americanum. These ticks have overlapping
distributions throughout the Southern US, but D. variabilis ranges further into northern states.
The objective of the current project was to determine the distribution and prevalence of C. felis in
bobcats, Lynx rufus, and other wild/exotic felids from ten eastern states (Georgia, Kansas,
Kentucky, Louisiana, Missouri, North Carolina, North Dakota, Ohio, Oklahoma, and West
Virginia). The bobcat is believed to be the primary reservoir for C. felis, but few studies have
investigated the distribution and prevalence of the parasite within wild felids. Blood and/or spleen
samples from hunter/trapper-killed felids (n=467) were tested for C. felis by PCR, targeting the
ribosomal internal transcribed spacer region 1 (ITS-1) region. Prevalences were higher in southern
states where both tick species are present. We detected prevalences of 79% in Missouri (39
bobcats), 63% in North Carolina (8 bobcats), 60% in Oklahoma (20 bobcats), 57% in South
Carolina (7 bobcats), 55% in Kentucky (74 bobcats), 33% in Louisiana (1 bobcat, 1 cougar [Felis
concolor], 1 serval [Leptailurus serval]), and 27% in Kansas (41 bobcats). The prevalences were
lower in Georgia (8%, 89 bobcats), North Dakota (2.4%, 124 bobcats, 5 cougars), Ohio (0%, 19
bobcats), and West Virginia (0%, 37 bobcats). These data indicate that C. felis is widespread in
bobcat populations, but the spatial differences in prevalence may relate to differences in the
distributions of the two tick species. The ultimate goal of this project is to investigate intraspecific
variability of C. felis throughout the Eastern U.S. by comparison of ITS sequences present in wild
felids with those detected in domestic cats and ticks.
THREE-DIMENSIONAL KINEMATICS OF THE CANINE STIFLE DURING WALKING AND TROTTING
GAITS. BT Torres, SC Budsberg. University of Georgia College of Veterinary Medicine, Athens, GA
The canine stifle has been commonly used as a model of osteoarthritis. Joint function is an area of interest that can
be objectively quantified through the collection of kinetic and kinematic gait data. Collection and analysis
methodologies have varied and focused primarily on sagittal plane flexion and extension angles. The objective of
this study was two-fold. First, to model the kinematics of the canine stifle in three dimensions (3-D) via the Joint
Coordinate System (JCS); and secondly, to compare the waveforms produced during stifle joint range-of-motion
using a classic frequency spectrum reconstruction methodology (Fourier) with a newer method of waveform
analysis known as Generalized Indicator Function Analysis (GIFA) that is a multivariate vector waveform analysis
method. The hypotheses being tested were that JCS would provide the ability to asses stifle joint motion in 3-D and
that GIFA would provide comparable results to Fourier with the potential of evaluating the waveform in a time
function method.
Six adult dogs weighing 20 to 30 kgs were used in this study. Gait waveforms were produced by the application
of ten spherical retroreflective markers affixed to the right rear leg. Gait was captured at 200Hz by 6 infrared
cameras (Vicon MX03, Vicon Motion Systems, Inc.). Data was recorded and analyzed by a motion-analysis
program (Peak Motus 8.5, Vicon Motion Systems, Inc.).Dogs were walked at a velocity of 0.9-1.2 m/s and trotted at
1.7-2.1m/s. Each gait was recorded 4 times. The exact procedure was repeated 5 days following the first, providing 8
trials for analysis. Sagittal flexion/extension, internal/external rotation, and abduction/adduction data were collected.
Sagittal flexion/extension waveforms were then compared using two analysis methods. The data was analyzed
independently for each method. First, a Fourier transformation was performed for each waveform. Ten Fourier
coefficients were produced and compared with a repeated measures ANOVA. Significance was set at p < 0.05.
Then, the same data set was analyzed with GIFA, a multivariate statistical method designed to determine the vector
that best separates groups of vectors measured under different conditions. Significance was set at p < 0.05.
Fourier Analysis : Significant inter-dog differences (p<0.05) were found between dogs for both the walk and trot.
Significant inter-day differences (p<0.05) were found for the walk. No inter-day differences were found between
dogs at a trot.
GIFA : Significant inter-dog differences (p<0.05) were found between dogs at a trot. No inter-dog differences
were found between dogs at a walk. Significant inter-day differences (p<0.05) were found for both the walk and trot.
Both hypotheses in this study were accepted. The use of the JCS marking system allowed for the collection of 3-D
stifle motion. It produced sagittal flexion/extension waveforms that were consistent with previous kinematic studies
of the canine stifle, and provided information regarding the additional axes of joint motion. Furthermore, GIFA and
Fourier both provided the ability to assess differences in gait waveforms. Variation in the results between both
methods may be attributed to the fundamental differences in the two analysis methodologies. Given that GIFA gives
rise to eigenvectors that are functions of time, it may prove beneficial in temporally isolating gait differences.
EFFECTS OF CLOPIDOGREL ON PLATELET FUNCTION IN THE HORSE.
Whelchel DD, Brainard BM, FortesB, Moore JN.
College of Veterinary Medicine, University of Georgia, Athens, Georgia.
Clopidogrel (Plavix®), an antagonist at the platelet P2Y12 ADP receptor, significantly reduces
platelet aggregation in many species. This study provides preliminary data regarding the
pharmacodynamics of clopidogrel on platelet aggregation in the horse. A compounded paste
formulation of Clopidogrel was administered (2 mg/kg) orally once a day in the morning to 3
healthy horses for 3 days. Blood was collected before and at specified time intervals after the first
oral dosing (t= 0, 3, 6, 24, 48, 72, and 96 hours). A complete blood count and serum chemistry
were performed at the start and conclusion of the dosing interval (t = 0 and t = 72). Serum
serotonin (5-HT) concentrations were measured at each time point using a commercial ELISA.
Mean platelet volume increased at 72 hr (9.1 ± 0.8 fl, p = 0.012) from baseline values (8.0 ± 0.6
fl). ADP-induced platelet aggregation decreased from baseline values (mean ± SD; 72.7 ± 23%) at
72 h (23.7 ± 4%; p = 0.002) and 96 h (28.0 ± 3%; p = 0.003). Collagen-induced platelet
aggregation decreased from baseline values (86.3 ± 10%) at 3 h (19.7 ± 4%, p < 0.0001), 6 h (20.7
± 5% p = 0.0001), 24 h (18.3 ± 5%; p< 0.0001) and 72 h (33.3 ± 13.9%; p< 0.0001). Serum 5-HT
concentrations decreased from baseline (1580.9 ± 164.6 ng/mL) to 48 h (126.8 ± 44.3 ng/mL, p =
0.045). Clopidogrel given at 2 mg/kg PO q 24 h appears to decrease both platelet aggregation and
secretion of 5-HT in healthy horses.
AN IVERMECTIN-SENSITIVE ION CHANNEL FROM HAEMONCHUS CONTORTUS
S McCavera, S Glendinning, TK Walsh, AJ Wolstenholme
Dept Infectious Diseases, College of Veterinary Medicine, University of Georgia and Dept of
Biology & Biochemistry, University of Bath, Bath, U.K.
We wish to understand how ivermectin and related compounds, the biggest-selling class of
veterinary drugs, work to kill nematode parasites. Genetic and other experiments have shown that
ivermectin acts on a novel class of ion channel present in invertebrate nervous systems, the
glutamate-gated chloride channels, and one gene, avr-14, that codes for two alternatively spliced
channel subunits, AVR-14A and AVR-14B, is widely conserved throughout the Nematoda,
including the important parasite of small ruminants, Haemonchus contortus. We have cloned
cDNAs from H. contortus that encode these two subunits and expressed them in three systems to
study their ability to mediate the anthelmintic effects of ivermectin. In Xenopus oocytes, AVR14B, but not AVR-14A, formed ivermectin-sensitive channels: the drug opened the channels
slowly and irreversibly at low concentrations (estimated EC50 = ~0.1 ± 1.0 nM). In COS-7 cells,
AVR-14B bound radiolabelled ivermectin with high affinity (Kd = 0.35 ± 0.1 nM), but expression
of AVR-14A did not produce any high-affinity ivermectin binding sites. In order to study the
effects of these subnits in the whole organism, we have transformed highly ivermectin-resistant C.
elegans (glc-1;avr-14;avr-15) with the H. contortus avr-14 cDNAs, under the control of the C.
elegans avr-14 promoter. These studies have confirmed that avr-14 is widely expressed in the
nematode nervous system, including sensory and motor neurons. The H. contortus AVR-14B
rescued the drug susceptibility phenotype, but AVR-14A had no effect. We conclude that
glutamate-gated chloride channels containing AVR-14B are important ivermectin targets; the role
of AVR-14A is less clear.
EXPRESSION OF HEAT SHOCK PROTEINS, KI67 AND VASCULAR ENDOTHELIAL
GROWTH FACTOR IN CANINE ASTROCYTOMAS AND OLIGODENDROGLIOMAS
A.B. Yanke, S.R. Platt, A.C. Freeman, A.C. Haley, J. Eagleson, S.J. Schatzberg, M. Kent, E.W.
Howerth. College of Veterinary Medicine, University of Georgia, Athens, GA
Human brain tumors express Heat Shock Proteins (HSP), which are associated with their degree
of malignancy. HSPs serve various roles in cell differentiation and proliferation during the normal
cell cycle. Their up-regulation during tumor cell growth helps keep tumor proteins stable and
therefore makes them a reasonable target for therapy. The aim of this study was to determine if
canine astrocytomas and oligodendrogliomas express HSP 27, 72 and/or 90. An additional aim
was to determine whether the expression of the HSPs was associated with Ki67 and/or Vascular
Endothelial Growth Factor (VEGF) expression. Ki67 expression and VEGF up-regulation have
been strong indicators of cell proliferation and angiogenesis during tumor growth, respectively.
Twenty-two formalin-fixed, paraffin-embedded canine brain tumors (8 astrocytomas, 2
oligoastrocytomas and 12 oligodendrogliomas) underwent immunohistochemical staining using
anti-HSP 27, 72 or 90 antibodies. These tumor samples were also immunohistochemically stained
for Ki67 and VEGF expression. Canine mammary carcinoma and squamous cell carcinoma
tissues served as the control samples, as both have previously been shown to express HSPs. Four
non-overlapping high power fields of each stained sample were selected and cell staining was
analyzed using a semi-quantitative method. All analyses were performed using SAS V 9.2 (Cary,
NC). Descriptive statistics of staining percentages were calculated for all tumors tested. Staining
percentages were compared between tumor types and tumor type/grade subgroups by the KruskalWallis test. All hypothesis tests were 2-sided and the significance level was α = 0.05.
All three HSPs and VEGF were expressed in all tumor types. For all tumors pooled (n=22), there
was a moderate positive correlation between HSP27 expression on tumor vessels and Ki67
expression on tumor tissue (r=0.46, p=0.0307). For astrocytomas (n=8) there was a positive
correlation between Ki67 and HSP 27 (r=0.73, p=0.0378). No such associations found with HSP
72 or 90. No associations were found with any HSP variant when all oligodendrogliomas were
grouped together. However, for grade 3 oligodendrogliomas (n=7) there were strong positive
correlations between Ki67 total % and HSP72 % tumor (r=0.84, r=0.0169), and between Ki67
total % and HSP72 total % (r=0.795, p=0.0325), and VEGF total % and HSP90 total % (r=0.79,
p=0.0362).
In conclusion, HSP expression was demonstrated in canine glial cell tumors. In the case of
astrocytomas, the expression of HSP27 was associated with a more aggressive tumor, as noted by
Ki67 expression. A similar association was found for aggressive oligodendrogliomas between
HSP72 and Ki67. This study suggests that HSPs may have a role in the maintenance of aggressive
canine gliomas and potentially represent a novel treatment target.