Terry W. Lehenbauer

Monoclonal Antibody-Based Competitive Enzyme-Linked Immunosorbent Assay for Detection of Morbillivirus Antibody in Marine Mammal Sera

ABSTRACT A competitive enzyme-linked immunosorbent assay (cELISA), using two monoclonal antibodies (MAbs), was developed and compared with the standard virus neutralization test (VNT) for detecting antibodies against canine distemper virus (CDV) and phocine distemper virus (PDV) in sera from dogs and various species of marine mammals. The test depends on the blocking of MAb binding to solid-phase antigen in the presence of positive serum. Test conditions were optimized by using control VNT-negative and -positive sera specific for CDV and PDV. A positive cutoff value of 30% inhibition, which represents the mean cutoff of a VNT-negative population (n = 623) plus 2 standard deviations, was adopted for the test. A total of 736 serum samples were tested by the new cELISA and by the VNT as the “gold standard.” An unexpected but useful finding was the ability of this CDV- and PDV-specific cELISA to also detect antibodies against the related pair dolphin morbillivirus and porpoise morbillivirus. Based on a subpopulation of 625 sera used in statistical analyses, the overall sensitivity and specificity of cELISA relative to those of the VNT were 94.9 and 97.7%, respectively. Because the cELISA proved to be nearly as sensitive and specific as the VNT while being simpler and more rapid, it would be an adequate screening test for suspect CDV or PDV cases and would also be useful for epidemiological surveillance of morbilliviral infections in marine mammal populations.

Canine hepatozoonosis: comparison of lesions and parasites in skeletal muscle of dogs experimentally or naturally infected with Hepatozoon americanum

We report previously undescribed, early lesions in skeletal muscle of dogs experimentally infected with Hepatozoon americanum by ingestion of laboratory-reared, infected Amblyomma maculatum. The earliest muscle lesion was recognized at the first interval of examination 3 weeks following exposure. The lesion consisted of a large, modified host cell whose cytoplasm frequently contained a demonstrable parasite. In skeletal muscle, the cell was consistently located between muscle fibers or in loose connective tissue adjacent to those fibers. Evidence suggesting that the parasite arrives in muscle and other tissue within the host cell cytoplasm is presented. Mucopolysaccharide encystment of the host cell, absent at this early stage, was acquired gradually and approached maximal development 26 weeks post exposure. Completion of the asexual cycle as evidenced by the presence of parasites entering vascular lumens within granulomas and also by the presence of gamonts in peripheral blood leukocytes, occurred within 28-32 days postexposure. Progression of the parasite cycle from meront to passage of zoites into vessel lumens of granulomas can occur in 11 or fewer days. The density with which parasitic lesions occur in one named skeletal muscle compared to other named muscles, although somewhat variable, was not significantly different in either experimentally induced or natural infections. The distribution of developmental stages of the parasite/lesion in four experimental infections (969 lesions) is compared with those in eight dogs with natural infections (557 lesions).

Comparison of tepoxalin, carprofen, and meloxicam for reducing intraocular inflammation in dogs.

OBJECTIVE To compare effects of orally administered tepoxalin, carprofen, and meloxicam for controlling aqueocentesis-induced anterior uveitis in dogs, as determined by measurement of aqueous prostaglandin E(2) (PGE(2)) concentrations. ANIMALS 38 mixed-breed dogs. PROCEDURES Dogs were allotted to a control group and 3 treatment groups. Dogs in the control group received no medication. Dogs in each of the treatment groups received an NSAID (tepoxalin, 10 mg/kg, PO, q 24 h; carprofen, 2.2 mg/kg, PO, q 12 h; or meloxicam, 0.2 mg/kg, PO, q 24 h) on days 0 and 1. On day 1, dogs were anesthetized and an initial aqueocentesis was performed on both eyes; 1 hour later, a second aqueocentesis was performed. Aqueous samples were frozen at -80 degrees C until assayed for PGE(2) concentrations via an enzyme immunoassay kit. RESULTS Significant differences between aqueous PGE(2) concentrations in the first and second samples from the control group indicated that aqueocentesis induced uveitis. Median change in PGE(2) concentrations for the tepoxalin group (10 dogs [16 eyes]) was significantly lower than the median change for the control group (8 dogs [16 eyes]), carprofen group (9 dogs [16 eyes]), or meloxicam group (9 dogs [16 eyes]). Median changes in PGE(2) concentrations for dogs treated with meloxicam or carprofen were lower but not significantly different from changes for control dogs. CONCLUSIONS AND CLINICAL RELEVANCE Tepoxalin was more effective than carprofen or meloxicam for controlling the production of PGE(2) in dogs with experimentally induced uveitis. Tepoxalin may be an appropriate choice when treating dogs with anterior uveitis.

An indirect enzyme-linked immunosorbent assay for diagnosis of American canine hepatozoonosis.

American canine hepatozoonosis (ACH), caused by Hepatozoon americanum, is an emerging tick-borne disease of dogs. An indirect enzyme-linked immunosorbent assay (ELISA) that should facilitate diagnosis of infection and study of the epidemiology of ACH has been developed using H. americanum sporozoites as antigen. Efficacy of the new test as a diagnostic tool was compared with that of skeletal muscle biopsy, the current gold standard for confirming H. americanum infection. Results show that the test is sensitive (93%) and specific (96%) and that it is as reliable as histopathologic examination of skeletal muscle for detecting infection. The ELISA would be suitable as a routine laboratory test for diagnosis of ACH.

Long-term outcomes of thigh circumference, stifle range-of-motion, and lameness after unilateral tibial plateau levelling osteotomy.

Our study evaluated thigh circumference (TC), stifle range of motion (ROM), and lameness in dogs one to five years after unilateral tibial plateau levelling osteotomy (TPLO). We hypothesised that TC, stifle ROM, and lameness would not be different to the unoperated limb (control), one to five years after surgery. Patients that were one to five years post-TPLO were reviewed and were included if they had a unilateral TPLO, and no additional clinical evidence of orthopaedic disease. Standing mid-thigh TC measurements and stifle extension and flexion angles were made in triplicate. Clinical lameness was graded blindly. Data were evaluated statistically using paired t-tests for TC and stifle flexion and extension. Significance was set at p <0.05. Twenty-nine dogs met the inclusion criteria. Mean results for the surgery limbs and control limbs were 39.5 +/- 5.5 cm and 40.1 +/- 5.6 cm for TC, 36.6 +/- 6.8 degrees and 28.6 +/- 4.3 degrees for stifle flexion, and 155.2 +/- 6.6 degrees and 159.8 +/- 4.9 degrees for stifle extension, respectively. The mean TC for the operated limb was 98.5% of the control limb. A significant difference was found between the operated and the control limbs for all measurements. Time after surgery had no apparent affect on outcome. Four of 29 dogs (14%) exhibited some lameness in the TLPO limb during evaluation (one dog was 1 to 2 years postoperative and three dogs were 2 to 3 years postoperative). These results indicate that TC and stifle ROM in the TLPO limb do not return to control-limb measurements one to five years after a TPLO surgery. The clinical significance is unknown as TC returned to 98.5% of control, and the source of lameness in the lame dogs was not identified.

Comparison of genotypic and phenotypic characterization methods for Pasteurella multocida isolates from fatal cases of bovine respiratory disease

Bovine respiratory disease (BRD) is the most costly disease of beef cattle in North America. Because Pasteurella multocida is a commensal of the upper respiratory tract, it is generally considered an opportunistic pathogen. However, studies in swine indicated that there may be a limited number of strains associated with disease, suggesting that some are more virulent than others. Although this may also be true of isolates from cattle, appropriate typing methods must be established before this possibility can be investigated. The purpose of this study was to compare effectiveness of polymerase chain reaction (PCR) fingerprinting to more traditional approaches for typing bovine P. multocida isolates. Isolates were obtained from 41 cases of fatal BRD and subjected to random amplified polymorphic DNA PCR (RAPD-PCR), whole cell protein (WCP) profiles, outer membrane protein (OMP) profiles, and serotyping. The discrimination index was calculated for each typing method and combinations of each using Simpsons index of diversity. Correlation coefficients were calculated to assess concordance between classification results achieved through genotypic (RAPD-PCR) and phenotypic (WCP, OMP, and serotyping) approaches. All characterization methods were capable of discriminating between isolates. However, there was poor concordance between techniques. There were also few significant associations between typing results and epidemiologic data. Random amplified polymorphic DNA PCR was validated as being a repeatable and reliable means of discriminating between P. multocida isolates obtained from cattle. Isolates obtained from fatal cases of BRD in calves in a commercial feedlot demonstrated significant diversity, justifying additional investigation into whether P. multocida is a strictly opportunistic pathogen in cattle.

Neosporal abortion in Oklahoma cattle with emphasis on the distribution of brain lesions in aborted fetuses

a preliminary placentitis from which the organism is distributed through the blood to fetal tissues. In field cases of neosporal abortion, the organism has been identified in brain, spinal cord, striated muscle, liver, kidney, lung, adrenal gland, and adipose tissue, and it is associated with either granulomatous or nonsuppurative inflammation. Since late 1995, a small number of cases of neosporal abortion have been positively identified in Oklahoma cattle. In cases in which the entire fetus was submitted to the Oklahoma Animal Disease Diagnostic Laboratory for examination, microscopic lesions were similar to those reported by others, with the brain being the most commonly affected organ. However, all areas of the brain were not uniformly affected, and the current literature does not detail the specific distribution of brain lesions. 5 The purpose of this report is to document the occurrence of neosporosis in Oklahoma cattle and to describe the distribution of brain lesions in these diseased bovine fetuses. Six cases of bovine neosporal abortion in which the complete fetus was submitted for diagnostic examination were reviewed from medical records at the Oklahoma Animal Disease Diagnostic Laboratory. Diagnosis of neosporal abortion was based on the presence of typical histological lesions for fetal neosporosis and the identification of tachyzoites and tissue cysts in lesions by immunohistochemistry with antiNeospora antiserum. Moreover, bacteriologic and viral isolation procedures failed to identify any significant agents that may produce similar histological lesions, and serological profiles for infectious bovine rhinotracheitis virus, Brucella abortus, bovine virus diarrhea virus, and 6 serovars of Leptospira spp. were negative. Representative samples of lung, heart, liver, kidney, spleen, and abomasum from each fetus were fixed in 10% buffered formalin. The entire brain was placed in either 10% or 30% buffered formalin. In this series of cases, spinal

Computed tomographic evaluation of the canine intercondylar notch in normal and cruciate deficient stifles.

In the human and veterinary orthopaedic literature it has been implied that intercondylar notch stenosis is a mechanical factor in cranial cruciate ligament rupture and intraarticular graft failure. The patients in this study were classified as normal (32), unilateral cruciate rupture (23), or bilateral cruciate rupture (17). The dogs were placed under general anaesthesia and both stifles were scanned via computed tomography (CT) as previously described. Three CT slices at predetermined levels were evaluated within the notch. Measurements included opening notch angle, notch width and height, condyle width, and notch width index (notch width/condyle width) at two different heights within the notch. Intercondylar notch measurements at the most cranial extent were significantly more narrow in unilateral and bilaterally affected stifles when compared to the normal population. Significant differences were noted in the opening notch angle (ONA), notch width index (NWI), NWI at two thirds notch height (NWI2/3), and tibial slope index (TSI). No significant differences were noted between unilateral and bilateral affected stifles. Increased mechanical contact of the cranial cruciate ligament with a stenotic intercondylar notch may predispose the ligament to mechanical wear and structural weakening. Intercondylar notch measurements have been used as a tool to predict the risk of anterior cruciate ligament injury in young human athletes, and to assess the risk factors for intra-articular graft replacements. Our findings may be useful in developing similar predictive models using stifle CT scans.

Refractive states of eyes and associations between ametropia and age, breed, and axial globe length in domestic cats

OBJECTIVE To determine the refractive states of eyes in domestic cats and to evaluate correlations between refractive error and age, breed, and axial globe measurements. ANIMALS 98 healthy ophthalmologically normal domestic cats. PROCEDURES The refractive state of 196 eyes (2 eyes/cat) was determined by use of streak retinoscopy. Cats were considered ametropic when the mean refractive state was ≥ ± 0.5 diopter (D). Amplitude-mode ultrasonography was used to determine axial globe length, anterior chamber length, and vitreous chamber depth. RESULTS Mean ± SD refractive state of all eyes was -0.78 ± 1.37 D. Mean refractive error of cats changed significantly as a function of age. Mean refractive state of kittens (≤ 4 months old) was -2.45 ± 1.57 D, and mean refractive state of adult cats (> 1 year old) was -0.39 ± 0.85 D. Mean axial globe length, anterior chamber length, and vitreous chamber depth were 19.75 ± 1.59 mm, 4.66 ± 0.86 mm, and 7.92 ± 0.86 mm, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Correlations were detected between age and breed and between age and refractive states of feline eyes. Mean refractive error changed significantly as a function of age, and kittens had greater negative refractive error than did adult cats. Domestic shorthair cats were significantly more likely to be myopic than were domestic mediumhair or domestic longhair cats. Domestic cats should be included in the animals in which myopia can be detected at a young age, with a likelihood of progression to emmetropia as cats mature.

Clinical efficacy of fenbendazole against gastrointestinal parasites in llamas

Llamas or South American Camelids (SACs) are increasingly popular in the United States, as a source of fiber, livestock guard, and pack animals. Gastrointestinal parasites have been identified as a major health problems in all classes of livestock including llamas. Currently there are no approved anthelmintics available for use in llamas. In this study, fenbendazole was evaluated for its clinical efficacy in the control of gastrointestinal parasitism in llamas. Twelve, healthy, young adult llamas of both sexes naturally infested with Nematodirus, Strongyloides, Trichuris, and Capillaria were randomly divided into two groups. One group received a single oral dose of fenbendazole paste at 5 mg/kg. The second group received a comparable dose of water as a placebo. Fecal samples were obtained per rectum from each animal prior to administration of either the drug or placebo treatment and weekly thereafter. These samples were analyzed for total fecal egg burden using a modified Wisconsin sugar floatation technique. The fenbendazole treated group had a significant reductions in total fecal egg counts of 95%, 84%, 89% and 76%, respectively, for each week of the four-week sampling period. Nematodirus, Strongyloides, and Trichuris all had significant reductions in egg counts during the study period All animals were observed twice daily during the experiment. All animals remained healthy and showed no adverse effects related to treatment. These results indicate that fenbendazole is a safe and an effective anthelmintic for the treatment of naturally occurring gastrointestinal parasite infestations of Nematodirus, Strongyloides, and Trichuris in llamas. # 2000 Published by Elsevier Science B.V. All rights reserved.