State Veterinary Diagnostic Laboratory Research Articles

Serotyping and Enzyme Characterization of Pasteurella haemolytica and Pasteurella multocida Isolates Recovered from Pneumonic Lungs of Stressed Feeder Calves

Ninety-one isolates of Pasteurella multocida (Pm)and 124 of Pasteurella haemolytica (Ph) were recovered from the lungs of calves that died of bovine respiratory tract disease (BRTD). Nine Pm enzyme profiles (A through I) and 9 Ph enzyme profiles (J through R) were determined for the Pasteurella isolates. The Pm isolates were relatively evenly divided among the enzyme profiles, with one exception, profile I. The Ph isolates were not evenly distributed among the profiles. Fifty of the 91 Pm isolates were serotyped. Forty-two Pm isolates were positive for capsule type A, and 8 were untypable. Five somatic type antigen profiles (3; 3,4; 3,7; 3,4,7; and 4) were identified among the 50 serotyped Pm isolates; one isolate was untypable. The Ph isolates were further divided through serotyping and grouped as follows: 74 (60%) Pasteurella haemolytica A1 (PhA1), 12 (10%) PhA2, 4 (3%) PhA5, and 34(27%) PhA6. Eighty-one percent of the Ph serotypes were clustered in the M and N enzyme profile. The P enzyme profile was almost unique to PhA2 (8 of 12, 67% of PhA2 isolates). Results of this study indicate a need to collect more data on Ph serotypes at the state veterinary diagnostic laboratories.

Specificity of Polymerase Chain Reaction Identification ofToxoplasma GondiiInfection in Paraffin-Embedded Animal Tissues

Acknowledgements. We thank Theresa Hunsaker and Dana Hill for technical assistance and Dr. Bill Layton (Montana State Veterinary Diagnostic Laboratory), Dr. Bruce Anderson (University of Idaho), and Dr. Patricia Blanchard (California Veterinary Diagnostic Laboratory System, Tulare) for samples. Dr. Pare was supported by the Austin Eugene Lyons Fellowship and by the Graduate Group in Epidemiology, University of California-Davis. This study was funded by the California Milk Advisory Board and Experiment Station funds through the Livestock Disease Research Laboratory and the School of Veterinary Medicine, University of Califomia-Davis.

Detection of Prion Protein in Formalin-Fixed Brain by Hydrated Autoclaving Immunohistochemistry for the Diagnosis of Scrapie in Sheep

4The diagnosis of scrapie has traditionally relied on histopatholog ic demonstration of characteristi c lesions in brain sections. In recent years, however, increasing attention has been given to the use of immunodiagnos tic methods that focus on detection of a specific protein known as the prion protein (PrP). 7 This protein, which is believed to play an essential role in the pathogenesis of scrapie, is present in normal brain as a protease-sensitive isoform (PrP-sen). In contrast, most of the PrP in scrapie brain is protease resistant (PrP-res), probably because of a posttranslational alteration affecting the conformation of normal PrP. An immunohistoch emical (IHC) method to detect PrP-res in periodate, lysine, paraformaldehyde (PLP)-fixed brains from sheep with scrapie has been described. 5 Researchers working on bovine spongiform encephalopathy have recently reported that PrP-res also can be detected in formalin-fixe d brain if tissue sections are autoclaved prior to immunostaining. 1 This novel method of antigen enhancement, termed hydrated autoclaving, was originally devised to improve the immunoreactivity of certain brain proteins. 10 Hydrated autoclaving has not been used to detect other antigens. However, antigen enhancement is believed to result from heat denaturation of the protein, and the same mechanism has been suggested as the basis for antigen retrieval methods that utilize microwave immunohistochemistry. 6 In the present report, we describe diagnosis of scrapie using hydrated autoclaving IHC on formalin-fixe d sheep brains. For comparative purposes, some brains also were tested for PrP-res by western blot assay or by IHC on PLP-fixed brain. Brain samples from 186 scrapie suspect sheep were submitted to veterinary medical colleges, state veterinary diagnostic laboratories, or the National Veterinary Services Laboratories. Additional brain samples from 10 sheep in a scrapiefree flock were used as controls. Tissue sections were cut from paraffin blocks of formalin-fixe d brain stem (usually obex or midmedulla) and mounted on positively charged

Congenital Defects in the Llama

When faced with the question of what you should do as a veterinary practitioner, when presented with a congenital defect in a llama, one answer is to seek qualified help from your state veterinary diagnostic laboratory. But most importantly, encourage the owners of such animals to report the defect to their llama association, including pedigree information. The better we understand the pathogenesis of such defects, the better we will be able to control their incidence.

Prevalence of Encysted Toxoplasma gondii in Raptors from Alabama

Little is known about the prevalence of encysted Toxoplasma gondii in wild birds. We examined the hearts and breast muscles from 101 raptors for encysted T. gondii. All of the raptors had been submitted for necropsy to the State Veterinary Diagnostic Laboratory, Auburn, Alabama. Tissues were digested in acid-pepsin solution and inoculated into groups of 3-5 laboratory mice. Toxoplasma gondii was isolated from 27 of 101 (26.7%) raptors: 8 of 12 (66.7%) red-shouldered hawks (Buteo lineatus), 13 of 27 (41.1%) red-tailed hawks (Buteo jamaicensis), 1 of 4 (25%) Cooper's hawks (Accipiter cooperi), 1 of 5 (20%) great horned owls (Bubo virginianus), 4 of 15 (26.7%) barred owls (Strix varia), and 1 of 3 (33.3%) kestrels (Falco sparverius). Toxoplasma gondii was not isolated from 3 broad-winged hawks (Buteo platypterus), 3 sharp-shinned hawks (Accipiter striatus), 6 barn owls (Tyto alba), 9 screech owls (Asio otus), a Mississippi kite (Ictinia misisippiensis), 2 golden eagles (Aquila chrysaetos), a bald eagle (Haliaeetus leucocephalus), 4 ospreys (Pandion haliaetus), 4 turkey vultures (Cathartes aura), or 2 black vultures (Coragyps atratus). No significant difference (P > 0.05) in prevalence was detected based on sex using chi-square analysis. Chi-square analysis of the data demonstrated that adult raptors had encysted stages of T. gondii significantly (P < 0.05) more often than did immature raptors.

Survey of state veterinarians and state veterinary diagnostic laboratories for selenium deficiency and toxicosis in animals

Summary: Surveys were performed in which the state veterinarian and the major state veterinary diagnostic laboratories in every state were asked about selenium (Se) deficiency and toxicosis in livestock and wildlife in their state. Selenium-deficiency diseases were diagnosed in 46 states and were reported to be an important livestock problem in regions of 37 states; deficiencies were diagnosed in wildlife in 10 states. Natural Se toxicosis was a rare problem, with only 7 states reporting naturally developing Se toxicosis. Oversupplementation with Se was reported as a cause of toxicosis in 15 states. Toxicoses in aquatic environments were reported from 4 states; in all cases, high-Se content in the water had resulted from agricultural irrigation or natural rainfall causing leaching of Se from high-Se soils. Current amounts of supplemental Se that can be fed to our major food-producing animals are being reviewed by the FDA because of speculation of potential environmental problems caused by Se supplementation in animals. Amounts of supplemental Se allowed in the western United States (states west of and including North Dakota, South Dakota, Nebraska, Kansas, Oklahoma, and Texas) are proposed by the FDA to have more potential for environmental Se enrichment and toxicosis. This premise was not supported by our data. Selenium supplementation in domestic livestock was not involved in wildlife toxicosis found by these surveys.

Multifocal polioencephalomyelomalacia in Simmental calves with elevated tissue aluminum and decreased tissue copper and manganese.

A spectrum of related malacic disorders called Simmental multifocal symmetrical encephalopathy have been reported in Simmental and Simmental crossbred cattle in Canada, Australia, and New Zealand. 3 The cause of this condition is unknown. We describe multifocal polioencephalomyelomalacia in Simmental calves reared in Oregon, USA, with morphologic lesions similar to those described in Simmental multifocal symmetrical encephalopathy. These calves also had elevated tissue concentrations of aluminum and deficient hepatic copper and manganese concentrations, which may be related to this condition. Four 7-month-old bull calves from a herd of 135 Simmental cows developed paresis, ataxia, and knuckling over of both hind limbs that progressed over a 2-month period to inability to rise. During the previous 2 years, 3 calves similarly affected died. Affected calves remained bright and alert and maintained the ability to suckle. Antibiotic therapy was not beneficial, and complete blood count and serum chemistry profiles were normal in the 3 calves evaluated. Two of the calves were presented to the Oregon State Veterinary Diagnostic Laboratory for postmortem examination. Significant lesions were limited to the central nervous system, with secondary mild symmetrical atrophy of the rear limb muscles. Grossly, oblong to circular foci of malacia of various sizes (up to 1 cm diameter) were seen throughout the gray matter of the brain. Similar gross lesions were within the gray matter of the lumbar spinal cord in 1 of the calves. Microscopically, these lesions were cavitated and were composed almost entirely of necrotic debris (Fig. 1). Scattered viable neuroglial cells were within these foci but neurons were not observed. Microgliosis, astrocytosis (including gemistocytes), active neuronal necrosis, ischemic neuronal changes, and vasculitis were not observed. Vessels were prominent within the lesions, principally because of hypertrophy of endothelial nuclei. Neurofibrillary tangles and/or extensive demyelination were not observed in multiple Bodain- and Luxol fast blue-stained histologic sections. The histopathologic changes were markedly uniform throughout the affected foci. The distribution of brain lesions was essentially identical in the 2 affected calves. The gray matter lesions of the brain involved numerous nuclei but invariably extended beyond nuclear boundaries. Individual lesions often involved more than 1 nucleus with some extension into the adjacent white

Diagnostic Characteristics of Bovine Spongiform Encephalopathy

Bovine spongiform encephalopathy (BSE) is a progressive disease affecting the central nervous system (CNS) of adult cattle, ultimately resulting in death of the animal. Since 1986 when the disease was first characterized, >25,000 cattle in Great Britain have been diagnosed as having BSE. The disease has also been found in the Republic of Ireland, Oman, Switzerland, and France. The occurrence of BSE in Great Britain has had an adverse impact on the cattle industry in that country from the loss of export markets and public health concerns regarding the safety of beef for consumption. The best evidence from epidemiologic studies indicates that BSE originated from exposure of cattle to a scrapie-like agent in ruminant-derived protein by-products from rendered animal carcasses. Because of this finding, the rendering industries in Great Britain have also been affected adversely because of lack of product marketability. Similar effects could be expected for the US cattle and rendering industries should the disease be found in this country. There has been speculation that BSE or a scrapielike disease may already be present but undetected in the US cattle population. Because of this possibility, a BSE survey was implemented through a cooperative effort between the US Department of Agriculture and the Iowa State University. 19 This survey involves histologic examination of brains from adult cattle showing signs of CNS disease. Samples are submitted from a variety of sources, including state veterinary diagnostic laboratories, federally inspected meat packing plants, and public health laboratories. No evidence of BSE has yet been found in cattle brains submitted for the survey. Prompt detection of BSE, should it occur in the United States, is important because it would allow control and eradication measures to be implemented imme-

Evaluation of breed as a risk factor for sarcoid and uveitis in horses.

The relationship between breed and the risk of developing sarcoid tumours or uveitis of unknown etiology was evaluated in a retrospective study of 16242 equine cases admitted between 1975 and 1987 to the New York State College of Veterinary Medicine Large Animal Hospital, and 3198 equine tissue samples sent to the New York State Veterinary Diagnostic Laboratory between 1977 and 1987. Of 120 sarcoid cases from the Large Animal Hospital, sarcoids were twice as likely to develop in Quarter Horses (odds ratio, OR = 1.8, P less than 0.05) relative to Thoroughbreds and less than half as likely to develop in Standardbreds (OR = 0.2, P less than 0.001) relative to Thoroughbreds. Similarly, of 376 sarcoid cases from the Diagnostic Laboratory, Quarter Horses had a significantly higher risk of developing sarcoid (OR = 2.0, P less than 0.001) relative to Thoroughbreds, while Standardbreds had a significantly lower risk of developing sarcoid (OR = 0.5, P less than 0.05) relative to Thoroughbreds. In the uveitis diagnosis category, Appaloosas had a significantly higher risk of developing uveitis (OR = 6.4, P less than 0.001) relative to Thoroughbreds, while Standardbreds had a significantly lower risk of developing uveitis (OR = 0.4, P less than 0.05) relative to Thoroughbreds.

Use of the API 20E system to identify veterinary Enterobacteriaceae.

A total of 503 veterinary enteric bacterial pathogens obtained from state veterinary diagnostic laboratories were tested on API 20E strips to determine whether this rapid microidentification system could be utilized for veterinary clinical microbiology. The API 20E strip accurately identified 96% of the veterinary isolates and misidentified 3%. Identifications by the API system and the diagnostic laboratories were in agreement in 85% of the isolates, disagreement on 16% of the isolates, and 1% were not identified by the API strip. Differences in identification occurred primarily in distinguishing between Klebsiella and Enterobacter and between Enterobacter and Escherichia coli. These disagreements were most often due to incorrect identifications by the diagnostic laboratory rather than by the API system. Biotype differences between human and veterinary isolates were compared. Significant differences were noted in several biochemical reactions. The main differences observed for E. coli isolates were in ornithine decarboxylase production and melibiose fermentation. The largest differences for Salmonella occurred in arginine dihydrolase production, citrate utilization, and inositol fermentation, whereas for Klebsiella pneumoniae the main differences were noted in urease production and nitrate reduction. These biotype differences, however, did not affect the accurate identification of organisms on the API strip.

Biotelemetry as a Technique in Disease Ecology Studies

Disease ecology studies frequently demand a detailed knowledge of movements and activity patterns of infected animals, base-line data from apparently healthy individuals and/or inter-action rates between hosts. Additionally, some investigations require periodic examinations of hosts and the recovery of dead animals for necropsy. Until quite recently these data have been obtained primarily by live trapping and marking animals, systematic collections of specimens, or, as in the case of rabies, reports of state public health or veterinary diagnostic laboratories. All of these approaches present difficulties which may limit the quality or quantity of the information gained. For example, the technique of live trapping requires the assump- tion that movement and activity parameters are not affected by the experimental procedures. Balph ( 1968 ) , however, has shown that ground squirrels may become trap happy in response to a ready source of food or trap shy as a result of the noxious experience of being captured and handled. Similarly information obtained from systematic collections of specimens and from laboratory diagnostic reports frequentlyprove to be of limitedvalue in unravelingthe ecologicalrelationships of disease complexes. The technique of attaching miniature radio transmitters to free ranging wild animals, or biotelemetry, may prove to be quite useful in disease investigations by reducing or eliminating the above limitations and by providing more data per unit effort than are provided by current techniques. A properly designed biotelemetry system enables the investigator to determine, with minimal disturbance, behavior patterns of unconfined wild animals. It also provides for determination of the time of death of radio-marked animals and thus permits immediate collection of the remains for necropsy. Houseknecht (1969) used this technique to determine the denning habits of striped skunks (Mephitis mephitis) and the resulting exposure potential for disease. By quickly retrieving carcasses of succumbed radio-marked fawns (Odocoileus virginianus), Cook et a!. (1967) determined the cause of death in 49 of 58 cases. The capability of continuously monitoring the location of radio-marked animals also permits examination of hosts or the collection of specimens at will. Montgomery (1968) attached radio collars to a white-tail deer fawn then recaptured the animal at predetermined intervals in order to investigate the rate of tick attachment. Simi- larly one could recapture radio-marked striped skunks for monthly bladder traps for an investigation into the ecology of leptospirosis. The technique of biotelemetry has been employed in animal movement studies since 1959. These first transmitter packages (Le Munyan et a!., 1959) measured 7.5 x 4.0 x 1.4 cm, weighed 122.5 gm and were designed for implantation into the