Restriction endonuclease analysis of alcelaphine herpesvirus 1 DNA and molecular cloning of virus genomic DNA for potential diagnostic use.

Alcelaphine herpesvirus 1 (AHV-1) genomic DNA was analyzed using restriction enzymes having recognition sequences both low in guanine-cytosine content (BamHI, KpnI, HindIII) and high in guanine-cytosine content (SmaI, AvaI, ApaI). The results from the restriction enzyme analyses along with exonuclease treatment demonstrated that the termini of AHV-1 DNA are likely composed of polyrepetitive sequences high in guanine-cytosine content similar to those found in Herpesvirus saimiri DNA. Cleaving AHV-1 DNA with the restriction enzyme SmaI produced polyrepetitive sequences that appeared as low molecular weight supermolar bands less than 1 kilobase pairs (kb) in size. Analysis of AHV-1 DNA cleaved with HindIII indicated that the polyrepetitive sequences are approximately 29.5 kb in size. The total molecular weight for AHV-1 DNA was determined to be approximately 118 kb. Seventy-five percent of the AHV-1 genome was cloned into the plasmids pAT153 and pBR322. Cloned DNA fragments representing unique sequences of the AHV-1 genome hybridized with AHV-1 genomic DNA but showed no appreciable hybridization with AHV-2 DNA or bovine herpesvirus 1, 2, or 4 DNA.

Application of polymerase chain reaction to detect animals latently infected with agents of malignant catarrhal fever.

Oligonucleotide primers derived from alcelaphine herpesvirus 1 (AHV-1) isolate WC11 DNA, the first identified agent of malignant catarrhal fever (MCF), were used to assay blood lymphocyte DNA using the polymerase chain reaction (PCR). Multiple species of exotic ruminants were examined to determine the suitability of this technique for detecting animals that may be latently infected. To correlate the PCR results with those of serology, serum samples were obtained from each animal concurrently with lymphocyte collection and subjected to an AHV-1 virus-neutralization assay (VNA). A total of 86 MCF-susceptible animals were tested, and the results of the VNA and PCR assays were compared. PCR results were positive for 44 animals. Of these, 13 were positive by VNA. Animals positive by both VNA and PCR were all wildebeest, the asymptomatic carriers of AHV-1, confirming the ability of the primers to amplify AHV-1 sequence. Positive PCR results from species other than wildebeest may represent sequence amplified from viruses related to AHV-1, which may not induce antibodies capable of neutralizing the WC11 isolate used in the VNA. This study demonstrates that PCR is capable of detecting the presence of MCF agents in various populations of captive ruminants prior to the appearance of clinical MCF so that the sources of infection can be more reliably ascertained.

Use of disinfectants in zoos and game parks.

Disinfection is used in the animal quarters of zoos and game parks as an adjunct to physical cleaning and the removal of potentially contaminated materials. Disinfection is particularly useful in reducing infection risks in young animal nursery facilities, and in routine cleaning operations of animal quarters and feeding utensils. Specific disinfectants may be selected for certain known microbial contaminants following an infectious disease outbreak. For example, premises contaminated by foot and mouth disease virus are usually disinfected with 2% sodium hydroxide (lye). The disinfectants most commonly used in zoo operations usually have a broad spectrum of microbicidal activities, such as o-phenylphenol salts, especially sodium o-phenylphenol. Equally popular for routine cleaning and sanitizing operations in zoos are quaternary ammonium compounds and chlorine bleach (sodium hypochlorite). It is important to remember that many disinfectants are protoplasmic poisons, or may be caustic or corrosive. Animals must usually be excluded from facilities being disinfected and premises should be rinsed thoroughly, after a suitable environmental exposure time to such disinfectants, before animals are allowed to return.

Fatal respiratory disease in Nilgiri tahr: possibly malignant catarrhal fever.

Nilgiri tahr (Hemitragus hylocrius) are native to India and are a rare zoo exhibit. This report describes an acute respiratory disease in tahr that caused the death of 15 of 16 animals in an extensive exhibit of about 35 acres where they were housed together with a variety of other exotic species of ruminants. The deaths occurred in two separate outbreaks and were associated with losses from malignant catarrhal fever in other ruminants in the exhibit. The most prominent clinical sign was severe dyspnea, and death occurred within five days. The principal lesions were an acute nonsuppurative inflammation of the respiratory tract and pulmonary vessels, lymphadenopathy and lymphoid cell infiltration in the organs of some animals. It was conjectured that the tahr died of a unique pneumonic form of malignant catarrhal fever. Attempts at viral isolation were negative.

Prevalence of antibodies to alcelaphine herpesvirus-1 and nucleic acid hybridization analysis of viruses isolated from captive exotic ruminants.

A serologic survey was conducted to determine the prevalence of antibodies to alcelaphine herpesvirus-1 (AHV-1) in captive exotic ruminants within the United States. Forty-six percent of the members of the subfamily Alcelaphinae (wildebeest, topi, hartebeest) in the family Bovidae had virus-neutralizing antibody to AHV-1. Other subfamilies of Bovidae with high prevalence of virus-neutralizing antibodies to AHV-1 included Hippotraginae (oryx and addax) and Caprinae (sheep and goats), with prevalence of 45% and 29%, respectively. Herpesviruses that have been isolated from captive exotic ruminant species, including healthy animals and those with clinical malignant catarrhal fever at the Oklahoma City Zoo and the San Diego Zoo/Wild Animal Park, were analyzed by DNA restriction enzyme analysis and blot hybridization. Variation has been detected among the genomes of several malignant catarrhal fever virus isolates obtained from various exotic species of ruminants, using the DNA restriction enzymes BamHI and HindIII. The DNA of these virus isolates is distinct from that of bovine herpesviruses 1, 2, and 4, as demonstrated by restriction enzyme analysis and nucleic acid hybridization. On the basis of restriction enzyme analysis and nucleic acid hybridization data, the DNA from each of the putative alcelaphine herpesvirus isolates examined, except for the topi virus isolate, had a high degree of DNA sequence similarity with the original AHV-1 isolate, WC-11, from a blue wildebeest.

Evaluation of a bovine viral diarrhea vaccine produced in a porcine kidney cell line.

An attenuated bovine viral diarrhea (BVD) live-virus vaccine, produced in a continuous porcine cell line, evoked an immune response which produced a protective level of serum antibodies in vaccinated cattle. Post-vaccinal reactions to the vaccinal virus were not observed in cattle vaccinated at 5 feedlots or in cattle vaccinated in experimental tests. The vaccinal virus did not produce demonstrable viremia or detectable excretion of virus from the respiratory or digestive tracts. Leukopenia or abnormality in differential leukocyte values did not occur as with virulent BVD viral infection. Transmission of vaccinal virus from vaccinated cattle to susceptible controls did not occur under conditions in which vaccinated and nonvaccinated cattle were in constant contact for 28 days.

Reservoir of St. Louis encephalitis virus in Ohio bats.

Inoculation of the big brown bat (Eptesicus fuscus) with a small dose of a St. Louis encephalitis (SLE) virus strain isolated in Ohio indicated that the big brown bat was susceptible to infection. The virus was maintained in the bats through hibernation (70 days), and the bats developed a viremia within 4 days of arousal from hibernation (105 days after inoculation). A field survey of 390 big brown bats and little brown bats (Myotis lucifugus) conducted in 5 regions of Ohio during 1979 to 1981 revealed a SLE virus-neutralizing antibody prevalence of 9%. Cohabitation of natural caves and abandoned mineshafts in Ohio by Culex pipiens mosquitoes, big brown bats, and little brown bats was also documented. Demonstration of a 9% prevalence rate of neutralizing antibody to SLE virus in big brown bats and little brown bats in Ohio during a nonepizootic period indicated that the bat may be involved in the maintenance of SLE virus in enzootic foci and could have a role in dissemination of SLE virus to epizootic foci.

Dexamethasone-induced recrudescence of malignant catarrhal fever and associated lymphosarcoma and granulomatous disease in a Formosan sika deer (Cervus nippon taiouanus).

Malignant catarrhal fever (MCF) was diagnosed in a 2-week-old Formosan sika deer. The fawn had been previously exposed to a clinically normal neonatal wildebeest calf from which alcelaphine herpesvirus-1 was isolated. Alcelaphine herpesvirus-1 was isolated from buffy coat leukocytes and nasal and ocular secretions of the fawn during the acute illness. The fawn clinically recovered after 3 weeks. Virus was not recovered from blood at this time. Dexamethasone, given 4 months after clinical recovery, resulted in reisolation of MCF virus from blood and recrudescence of clinical MCF. The deer was euthanatized. At necropsy, pathognomonic lesions of MCF, granulomatous disease, and malignant lymphoma were observed. Antibodies to bovine leukosis viral antigens were not detected in the serum. The epidemiologic and pathogenetic importance of the findings are discussed.

Enzyme-linked immunosorbent assay for the detection of antibodies to the alcelaphine herpesvirus of malignant catarrhal fever in exotic ruminants.

An ELISA for antibodies to the alcelaphine herpesvirus-1 of malignant catarrhal fever was developed. Of sera that represented 42 exotic ruminant species, 216 were evaluated by the ELISA and a virus-neutralization test. A significant correlation (r = 0.564, P less than 0.001, n = 216) between the ELISA and virus-neutralization test results was found. Of the sera having positive test results by virus neutralization, 86.1% also had positive results by the ELISA, and of the sera having negative test results by virus neutralization, 83.9% also had negative results by the ELISA. The presence of antibody, as measured by the ELISA, correlated with clinical signs of malignant catarrhal fever and the isolation of herpesvirus.

Ultrastructure of cellular changes in the replication of the alcelaphine herpesvirus-1 of malignant catarrhal fever.

Cell cultures inoculated with 5 different viral isolates from 4 species of ruminants with clinical signs of malignant catarrhal fever (from the San Diego Wild Animal Park) were examined by electron microscopy. Each had the morphology of a herpesvirus (118 to 220 nm) and was icosahedral, and the nucleocapsid matured in the nucleus of the infected cell. Envelopment of budding occurred with each viral isolate at the nuclear and the plasma membranes. The virions egressed from the cell by budding from the plasma membrane or through channels of the Golgi apparatus or the endoplasmic reticulum. A proposed scheme for the morphogenesis of the herpesvirus of malignant catarrhal fever is presented.

Diagnosis of malignant catarrhal fever by polymerase chain reaction amplification of alcelaphine herpesvirus 1 sequence.

We derived sequence information from cloned HindIII fragment "D" of alcelaphine herpesvirus 1 strain WC11, an agent of malignant catarrhal fever (MCF). Based on this sequence, oligonucleotide primers were selected and synthesized for use in a polymerase chain reaction amplification assay. These primers were used to test samples of total nucleic acids isolated from multiple tissues taken from an Indian gaur (Bos guarus gaurus) at the San Diego Wild Animal Park in San Diego, California (USA) which had clinical signs of a natural infection of MCF. Six of eight tissue samples examined had amplifiable sequences present. A nucleic acid probe complementary to the sequence of the original clone between the primer sites also was synthesized and used to confirm the identity of the amplified viral sequences, thus providing a diagnosis of MCF at the molecular level.

Epidemiologic and pathologic aspects of an epizootic of malignant catarrhal fever in exotic hoofstock.

An epizootic of malignant catarrhal fever (MCF) occurred at the Los Angeles Zoological Park which resulted in the deaths of four exotic ungulates. The source of infection was considered to be a newly purchased wildebeest bull (Connochaetes taurinus taurinus) that had been negative for antibody to MCF virus by an indirect immunofluorescent test. The need to re-evaluate regulations for the transportation and housing of young wildebeest is emphasized by this MCF outbreak. The diagnostic technology now available for identifying asymptomatic carriers of MCF virus and the present understanding of the behavior and pathogenesis of this highly cell-associated herpesvirus in exotic ruminants should provide a basis for the prevention and control of MCF in zoological parks.

Cryptosporidial infections in captive wild animals.

Neonatal diarrhea was an important cause of morbidity and mortality in a hand-rearing facility for exotic ruminants at the San Diego Wild Animal Park. Studies undertaken to determine the causes of the problem revealed that oocysts of Cryptosporidium sp. were demonstrable in auramine O stained fecal smears from 52 of 183 (28.4%) animals examined. Cryptosporidial infection was identified in 21 of 40 species of exotic ruminants with diarrhea. In addition, cryptosporidia were associated with gastroenteric disease in two primates and two reptiles. It was observed also that auramine O stained coccidial oocysts of the genus Eimeria, which were present in five of 183 (2.7%) of the specimens examined.

Bovine viral diarrhea virus infection in captive exotic ruminants.

Antibody to bovine viral diarrhea (BVD) virus was found in 174 of 1,905 (9.1%) exotic ruminants. Of the seropositive animals, 129 had titers of at least 1:8. When serums from zoos that had vaccinated against BVD were excluded from analysis, 60 of 1,390 samples (4.3%) were seropositive for BVD. Noncytopathogenic strains of BVD virus were isolated from 5 exotic ruminants.

Prevalence of antibody to bovine herpesvirus 1 in wild ruminants captive in United States zoos.

Serum samples (n = 1,146) representing 100 species of exotic ruminants now captive in United States zoos were assayed for neutralizing antibody to infectious bovine rhinotracheitis (IBR) virus (bovine herpesvirus 1). Thirty-four animals (3%) of 11 species had antibody to IBR virus. Because of the low prevalence of IBR antibody found, it was concluded that vaccination against IBR virus probably is not necessary for captive wild ruminants in United States zoos.

Lymphoproliferation in captive wild ruminants affected with malignant catarrhal fever: 25 cases (1977-1985).

The severity of lymphoproliferative disease associated with malignant catarrhal fever was extremely variable among 25 animals at the San Diego Wild Animal Park. Severe lymphoproliferative disease was seen in 3 of 10 Formosan Sika deer (Cervus nippon taiouanus), 3 of 6 Indian Axis deer (Cervus a axis), 3 of 6 Barasingha deer (Cervus d duvauceli), and 1 of 3 Nilgai (Boselaphus tragocamelus). Two Sika deer and 2 Barasingha deer had lesions morphologically indistinguishable from lymphosarcoma. Our findings were consistent with the hypothesis that alcelaphine herpesvirus-1 has oncogenic potential.

Rabies and other viral diseases.

This article presents discussions of the primary viral diseases with neurologic manifestations that affect cattle: rabies, pseudorabies, malignant catarrhal fever, infectious bovine rhinotracheitis, and sporadic bovine meningo-encephalomyelitis. Characteristics of the etiologic agents, and descriptions of clinical signs, pathology, epidemiology, diagnosis, prevention and control are presented.