Patricia C. Schultheiss

Biocompatibility of a biodegradable in situ forming implant system in rhesus monkeys

Formulations of a polymeric delivery system containing a 75/25 poly(DL-lactide-co-caprolactone dissolved in either N-methyl-2-pyrrolidone or dimethyl sulfoxide were injected both subcutaneously (SC) and intramuscularly (IM) into rhesus monkeys. Each monkey received an SC and IM injection of each of the two formulations, for a total injection volume of 4 mL. The monkeys were observed daily for overt signs of toxicity, and after 4 weeks biopsies of each implant site were fixed, stained, and evaluated histologically for tissue reaction to the polymer system. Tissue response was graded upon the presence and level of fibrous connective tissue and inflammatory cell infiltrate. The polymer formulations appeared to be safe, as the animals remained healthy and active throughout the study with no changes in food or water consumption, weight loss, or abnormal behavior observed. Tissue response to both formulations was considered mild and similar to that for other biodegradable polymers, in that the reaction was limited to tissue immediately adjacent to the residual polymer fragments and consisted of a mild fibroplasia with the presence of a few lymphocytes and macrophages. There were no differences between the two formulations in tissue response, and both formulations were considered acceptable for use as injectable implant systems.

A Retrospective Study of Visceral and Nonvisceral Hemangiosarcoma and Hemangiomas in Domestic Animals

Cases of hemangiosarcoma submitted to the Colorado State University Veterinary Diagnostic Laboratory during a 6-year period were reviewed. Visceral hemangiosarcomas represent less than 2% of canine specimens submitted for histologic examination and nonvisceral hemangiosarcoma less than 1%. Most nonvisceral hemangiosarcomas of dogs occur in skin. Hemangiosarcomas are less common in cats and usually occur in skin. They are also rare in other animal species. Animals with nonvisceral hemangiosarcomas are usually mature; dogs and cats average 10 years of age. The tumors develop in many different locations, and there is no sex predilection. A wide variety of dog breeds are affected, but Italian greyhounds, greyhounds, and whippets are overrepresented. Clinical outcomes of 76 cases of nonvisceral hemangiosarcomas in dogs and cats were obtained from submitting veterinarians. Completeness of excision of a tumor is the most important factor that can be used in predicting clinical outcome for an affected dog or cat. In all cases in which the animals were clinically normal for at least 1 year after surgical removal of a nonvisceral hemangiosarcoma, the margins were reported to be free of neoplastic cells. Degree of differentiation, mitotic rate, size of tumor, and presence or absence of epidermal ulceration, mast cells, or solar elastosis did not correlate with clinical outcome.

Specific detection of shedding and latency of bovine herpesvirus 1 and 5 using a nested polymerase chain reaction

A sensitive method for simultaneously detecting and discriminating between bovine herpesviruses types 1 and 5 (BHV-1 and BHV-5) was developed using a nested polymerase chain reaction (PCR) technique. Following amplification using type-common primers derived from gC sequences, amplification using type-specific nesting primers produced different-sized bands specific to the corresponding types, as demonstrated by blot hybridization. Less than 0.1 plaque-forming units (PFU) of each virus and 75 fg or less of viral DNA were routinely detected. The PCR technique amplified correct product from 4 BHV-5 isolates and from 48 BHV-1 isolates, all from the United States, and did not amplify heterologous herpesviruses. The PCR technique was more sensitive than virus isolation in detection of BHV-1 or BHV-5 in nasal secretions from experimentally and naturally infected calves, and it detected BHV-1 or BHV-5 in trigeminal ganglia from these calves.

Encephalitis Induced by Bovine Herpesvirus 5 and Protection by Prior Vaccination or Infection with Bovine Herpesvirus 1

Calves were intranasally challenged with bovine herpesvirus 5 (BHV5) and followed for the development of viral infection, clinical encephalitis, histologic lesions in the brain, and viral sequences in the trigeminal ganglia. Calves that were previously vaccinated with bovine herepesvirus 1 (BHV1, n = 4) or previously infected with BHV1 (n = 5) or that had not been exposed to either virus (n = 4) were compared. No calf developed signs of encephalitis, although all calves developed an infection as indicated by nasal secretion of BHV5 and seroconversion to the virus. Histologic lesions of encephalitis consisting of multifocal gliosis and perivascular cuffs of lymphocytes were observed in calves not previously exposed to BHV1. BHV5 sequences were amplified from the trigeminal ganglia of calves previously vaccinated and from calves not previously exposed to BHV1; calves sequentially challenged with BHV1 and later BHV5 had exclusively BHV1 sequences in their trigeminal ganglia. Administration of dexamethasone 28 days after BHV5 challenge did not influence clinical disease or histologic lesions in either previously unexposed calves (n = 2) or previously immunized calves (n = 2), although it did cause recrudescence of BHV5, as detected by nasal virus secretion.

Malignant Catarrhal Fever in Bison, Acute and Chronic Cases

Acute malignant catarrhal fever (MCF) was diagnosed in 10 bison from 6 herds and ranging from 1 to 6 years of age. The pattern of clinical signs and morphologic lesions differed among bison. Combinations of corneal opacity, lacrimation, nasal discharge, depression, excess salivation, anorexia, diarrhea, melena, and hematuria were observed. Vasculitis characterized by lymphoid infiltrates in the adventia with variable extension into media and intima was found in multiple tissues in each animal. Fibrinoid vascular necrosis was rare. Ulceration in the alimentary tract was found in 9/10 bison, and ulceration or hemorrhage in the urinary bladder was found in 8/10 bison. Lymphoid infiltrates were present in 7 of 9 livers and 9 of 9 kidneys examined histologically. Hyperplasia of lymph nodes was observed in 5 bison. Chronic MCF was diagnosed in 1 bison with an 80-day course of illness that began with lacrimation, corneal opacity, mucoid nasal discharge, depression, and anorexia. These signs ceased after 15 days but circling and blindness developed on day 76. Chronic vascular lesions characterized by endothelial cell hypertrophy, intimal thickening, fragmentation of the internal elastic membrane, smooth muscle hypertrophy, and adventitial infiltrates of lymphocytes and plasma cells were found in many organs. The retinal arteries had chronic inflammation and acute transmural fibrinoid necrosis. The retinas were infarcted. Polymerase chain reaction technique for amplification of ovine herpesvirus 2 sequences was performed on formalin-fixed tissues, and viral sequences were detected in 1–7 tissues from each animal. These viral sequences were not found in tissues of 4 bison not affected by MCF.

Epizootic malignant catarrhal fever in three bison herds: differences from cattle and association with ovine herpesvirus-2

Three bison herds in Colorado experienced high mortality from malignant catarrhal fever (MCF). In comparison with cattle, the bison had a more rapidly progressive disease, fewer clinical signs, and milder inflammatory histologic lesions. There was consistent association with ovine herpesvirus-2 (OHV-2). Contact with sheep was not consistent. Of 17 animals in herd A, 15 died of acute MCF; 1 was slaughtered while healthy; and 1 developed clinical signs of MCF, was treated with corticosteroids and antibiotics, and died of fungal abomasitis and rhinitis after 5 months. In herds B and C, approximately 300 of 900 and 18 of 20 died of MCF following brief clinical disease. The nearest sheep were 1 mile away from herd A, but direct contact with sheep could be documented in herds B and C. Complete gross and histologic examinations were conducted on 34 animals, including all animals in herd A, and MCF was diagnosed in 31. In addition, field necropsies were performed on all dead animals in herd B and most in herd C and MCF was diagnosed on the basis of the gross lesions in most animals. Clinical signs of each animal in herd A were recorded. Illness was brief, usually 8–48 hours. Clinical signs were subtle; separation from the herd was often observed. In all 3 herds, hemorrhagic cystitis and multifocal ulceration of the alimentary tract were consistently found at necropsy. Mild lymphocytic vasculitis was present in multiple organs. Ovine herpesvirus-2 was found by polymerase chain reaction (PCR) in 71 of 105 formalin-fixed tissue specimens from 29 of 31 animals with MCF. In herd A, blood samples from 13 animals were collected at 5 time points and tested by PCR for the presence of OHV-2 viral sequences in peripheral blood leukocytes. Nine bison with a positive PCR test and 4 with negative results prior to clinical illness died of MCF.

Histologic Features and Clinical Outcomes of Melanomas of Lip, Haired Skin, and Nail Bed Locations of Dogs

Outcomes, signalments, and the relationship of histologic features with the outcome of melanomas located in lip, nail bed, and haired skin of dogs were reviewed. These melanomas were diagnosed as benign or malignant based on histologic features. Melanomas of the lip arose from mucous membrane in most cases. 32 dogs with lip melanomas that had histologic features of malignancy, 22 died because of the tumor within 1 year and 10 were tumor free for at least 1 year following removal. Of 10 dogs with melanomas with benign histologic features on the mucous membrane of the lip, 9 were tumor free for at least 1 year. Of 4 dogs with benign appearing tumors of the haired skin of the lip, 3 were tumor free for at least 1 year. Melanomas with histologic features of malignancy occurred in many locations of haired skin, and 11 of 24 dogs were tumor free for at least 1 year. All nail bed melanomas had histologic features of malignancy and all invaded the third phalanx, but 6 of 14 dogs were tumor free for at least 1 year after amputation of the digit. Among these dogs, the 1-year survival rates for tumors classified as malignant by histologic features were 69% for lip, 46% for haired skin, and 43% for nail bed. However, the clinical outcome of an individual malignant tumor could not be predicted accurately by any specific histologic features.

Canine Liver Iron, Copper, and Zinc Concentrations and Association with Histologic Lesions

Concentrations of iron, copper, and zinc were measured in livers of 95 dogs that were suspected of having liver disease. Iron concentrations ranged from 177 to 7,680 ppm (dry weight basis); 54 dogs had iron concentrations greater than the normal concentration of 1,200 ppm. Iron stores were present in Kupffer cells and macrophages but not hepatocytes. The dogs did not have lesions of hemochromatosis. Dogs with high liver iron tended to have high liver copper and inflammatory lesions. High liver copper concentrations usually were associated with hepatocellular necrosis and fibrosis. High liver zinc was found in only 5 animals and was accompanied by histologic inflammatory lesions in one. In humans, increased iron concentration in the liver exacerbates liver damage caused by a variety of insults, and the same may be true for dogs.

Malignant Catarrhal Fever: Polymerase Chain Reaction Survey for Ovine Herpesvirus 2 and Other Persistent Herpesvirus and Retrovirus Infections of Dairy Cattle and Bison

Using a polymerase chain reaction (PCR) test for sequences of ovine herpesvirus 2 (OHV2), this virus was shown to be significantly associated with sheep-associated malignant catarrhal fever (SA-MCF) in terminal cases of disease in 34 cattle and 53 bison. Ovine herpesvirus 2 was not detected in cattle (38) and bison (10) that succumbed to other diseases. Other persistent herpesviruses, retroviruses, and pestivirus, some of which have been previously isolated from cases of SA-MCF, were not associated with the disease. These included bovine herpesvirus 4 (BHV4), bovine lymphotrophic herpesvirus (BLHV), bovine syncytial virus (BSV, also known as bovine spumavirus), bovine immunodeficiency virus (BIV), and bovine viral diarrhea virus (BVDV). A PCR survey for OHV2 in DNA from individual cows peripheral blood lymphocytes in 4 dairies showed that the 1 dairy that was in close contact to sheep had a prevalence of OHV2 of 21.3%, whereas the 3 other dairies had no OHV2. Prevalence of the other herpesviruses and retroviruses in the dairy cows was variable, ranging from 2% to 51% for BHV4, 52% to 78.7% for BLHV, and 10% to 34% for BSV Bovine lymphotrophic herpesvirus and BSV were also found in a few (1–4 of 21 tested) cases of terminal SA-MCF, but BIV and BVDV were not found in either the dairy cows sampled, or in the cases of SA-MCF. No significant correlation was found between the presence of any 2 viruses (OHV2, BHV4, BLHV, BSV) in the dairy cows or terminal cases of SA-MCF.

Use of an Immunoperoxidase Technique to Detect Equine Herpesvirus-1 Antigen in Formalin-Fixed Paraffin-Embedded Equine Fetal Tissues

An indirect immunoperoxidase (IP) procedure using the avidin-biotin-peroxidase complex detection technique was developed to detect viral equine herpesvirus-1 (EHV-1) antigen in formalin-fixed paraffin-embedded tissues from aborted equine fetuses. The procedure was applied to liver, lung, and other tissues from 20 cases of confirmed or suspected EHV-1 -induced abortions. Specific staining was observed in tissue sections from EHV-1-infected fetuses. Positive IP staining was present in tissues of 7 cases that were also positive by fluorescent antibody (FA) and virus isolation (VI) and that had typical histologic lesions. There was no IP staining in 7 cases that had no histologic lesions and negative FA and VI results. Five cases had typical histologic lesions and positive results in only 1 laboratory test; 3 were positive by VI and 2 by FA. Liver of 1 case was positive by IP, but tissues were too autolytic for other tests to be evaluated.