Donal O'Toole

Comparison of ear notch immunohistochemistry, ear notch antigen-capture ELISA, and buffy coat virus isolation for detection of calves persistently infected with bovine viral diarrhea virus

Two techniques performed on skin biopsy samples (ear notches), immunohistochemistry (IHC) and antigen-capture ELISA (AgELISA), were compared for detection of bovine viral diarrhea virus (BVDV) persistent infection (PI) in 559 Angus calves between the ages of 1 and 5 months. The calves also were tested for BVDV infection using virus isolation (VI) and reverse transcription (RT)-PCR on buffy coat samples and for antibodies to BVDV types 1a and 2 by serum neutralization (SN). Sixty-seven of 559 (12.0%) calves tested positive at initial screening by IHC, AgELISA, or VI, and all 67 were kept for a minimum of 3 months and retested monthly by IHC, AgELISA, VI, RT-PCR, and SN. Of the calves positive at initial screening, 59/67 (88.1%) were determined PI and 8/67 (11.9%) were determined acutely infected. Both IHC and AgELISA detected 100% of PI calves; however, IHC and AgELISA also detected 6 and 8 acutely infected calves, respectively, at initial screening. Furthermore, IHC and AgELISA continued to detect 3 and 4 acutely infected calves, respectively, 3 months after initial screening. Three acutely infected calves had IHC staining indistinguishable from PI calves at initial screening. Both IHC and AgELISA are accurate at detecting BVDV-infected calves, but veterinarians and producers should be advised that both tests detect some calves acutely infected with BVDV in addition to PI animals. Repeat testing using VI or RT-PCR on buffy coat samples should be performed at 30 days after initial screening to conclusively discriminate between acute and PI.

Chronic and recovered cases of sheep-associated malignant catarrhal fever in cattle

Malignant catarrhal fever (MCF) is traditionally regarded as a disease with a short clinical course, low morbidity and high case fatality rate. Owing to the limitations of the assays used for laboratory diagnosis, it was difficult to characterise the clinical spectrum of sheep-associated MCF, particularly when the cattle recovered from an MCF-like clinical syndrome. Over a period of three years, 11 cattle that survived MCF for up to two-and-a-half years were identified on four premises. A clinical diagnosis of MCF was confirmed by the detection of ovine herpesvirus-2 DNA in peripheral blood leucocytes using a polymerase chain reaction (PCR) assay that detects a specific 238 base-pair fragment of viral genomic DNA. Of the 11 cattle examined, six recovered clinically with the exception of bilateral corneal oedema with stromal keratitis (four animals) and unilateral perforating keratitis (one animal). The 10 animals available for postmortem examination had disseminated subacute to chronic arteriopathy. Recovery was associated with the resolution of the acute lymphoid panarteritis that characterises the acute phase of MCF, and with the development of generalised chronic obliterative arteriosclerosis. Bilateral leucomata were due in part to the focal destruction of corneal endothelium secondary to acute endothelialitis. Formalin-fixed tissues and/or unfixed lymphoid cells from all 11 cattle were positive for sheep-associated MCF by PCR. These observations indicate that recovery and chronic disease are a significant part of the clinical spectrum of MCF and that such cases occur with some frequency in the area studied. The affected cattle remain persistently infected by the putative sheep-associated MCF gammaherpesvirus.

Pathology of experimentally induced chronic selenosis (alkali disease) in yearling cattle

Prolonged oral exposure of cattle to elevated dietary selenium (Se) in forage and seleniferous plants in seleniferous areas of the western United States is associated historically with 2 clinical syndromes: alkali disease and “blind staggers.” The potential for Se-induced disease in cattle and other species is considerable in areas with seleniferous shales, Se-accumulating plants, arid climates, and alkaline soils. These 2 Se-associated conditions were defined in the 1930s and 1940s, and the nosology of blind staggers is questionable. Seventeen yearling steers fed 0.15, 0.28, and 0.8 mg Se/kg body weight as selenomethionine or selenite for 120 days were euthanized and examined postmortem. Significant lesions were confined to 4 steers in the medium- and high-dose selenomethionine group and to 1 steer in the high-dose selenite group. Grossly, dystrophic hoof lesions developed in 2 steers, 1 of which had extensive separation of horn from lamellar and coronary epidermis and also lost hair from the tail switch. Histologically, tubules in the stratum medium of hooves from these 5 steers were replaced by islands of parakeratotic cellular debris, separated by more normal hoof matrix. Two of the 5 steers also had hyperplasia, acanthosis, parakeratosis, and disorganized germinal epithelium of varying severity in hoof epithelium, particularly at the tips of epidermal lamellae. These changes may distinguish the hoof lesions of chronic selenosis from those of chronic laminitis in cattle, in which dermal (chorial) changes predominate. In skin from the distal part of the tail of the animal that lost its switch, most follicles were atrophic and devoid of hairshafts and displayed dyskeratosis and mild superficial follicular keratosis. No significant lesions developed in tissues other than integument. Autometallographic staining for catalytic Se bonds in various tissues, including skin, liver, and kidney, revealed no positive staining of hair shafts; the correlation between stain intensity and dose group was poor. These findings indicate that dietary exposure for 4 months to 0.28 and 0.8 mg Se/kg in the form of selenomethionine and to 0.8 mg Se/kg in the form of sodium selenite reproduces in some cattle mild (subclinical) to severe (clinical) forms of alkali disease. No significant neurological, renal, or hepatic lesions developed, supporting the contention that blind staggers is caused by factors other than excessive dietary selenium.

A deletion mutation in bovine SLC4A2 is associated with osteopetrosis in Red Angus cattle

BackgroundOsteopetrosis is a skeletal disorder of humans and animals characterized by the formation of overly dense bones, resulting from a deficiency in the number and/or function of bone-resorbing osteoclast cells. In cattle, osteopetrosis can either be induced during gestation by viral infection of the dam, or inherited as a recessive defect. Genetically affected calves are typically aborted late in gestation, display skull deformities and exhibit a marked reduction of osteoclasts. Although mutations in several genes are associated with osteopetrosis in humans and mice, the genetic basis of the cattle disorder was previously unknown.ResultsWe have conducted a whole-genome association analysis to identify the mutation responsible for inherited osteopetrosis in Red Angus cattle. Analysis of >54,000 SNP genotypes for each of seven affected calves and nine control animals localized the defective gene to the telomeric end of bovine chromosome 4 (BTA4). Homozygosity analysis refined the interval to a 3.4-Mb region containing the SLC4A2 gene, encoding an anion exchanger protein necessary for proper osteoclast function. Examination of SLC4A2 from normal and affected animals revealed a ~2.8-kb deletion mutation in affected calves that encompasses exon 2 and nearly half of exon 3, predicted to prevent normal protein function. Analysis of RNA from a proven heterozygous individual confirmed the presence of transcripts lacking exons 2 and 3, in addition to normal transcripts. Genotyping of additional animals demonstrated complete concordance of the homozygous deletion genotype with the osteopetrosis phenotype. Histological examination of affected tissues revealed scarce, morphologically abnormal osteoclasts displaying evidence of apoptosis.ConclusionsThese results indicate that a deletion mutation within bovine SLC4A2 is associated with osteopetrosis in Red Angus cattle. Loss of SLC4A2 function appears to induce premature cell death, and likely results in cytoplasmic alkalinization of osteoclasts which, in turn, may disrupt acidification of resorption lacunae.

PREVALENCE OF ANTIBODY TO MALIGNANT CATARRHAL FEVER VIRUS IN WILD AND DOMESTIC RUMINANTS BY COMPETITIVE-INHIBITION ELISA

A competitive-inhibition ELISA (CI-ELISA), based on a monoclonal antibody to an epitope conserved among malignant catarrhal fever virus (MCFV) strains of both wildebeest and sheep origin, was used to determine the prevalence of antibody to MCFV in selected domestic and wild ruminants, both free-ranging and captive, from the USA. We evaluated 2528 sera from 14 species between 1990 and 1995, including 80 pronghorn antelope (Antilocapra americana), 339 bighorn sheep (Ovis canadensis), 103 bison (Bison bison), 17 black-tailed deer (Odocoileus hemionus columbianus), 395 domestic cattle (Bos taunts), 291 domestic goats (Capra hircus), 680 domestic sheep (Ovis ammon), 323 elk (Cervus elaphus), 41 llamas (Lama glama), 21 mouflon sheep (Ovis musimon), 54 mountain goats (Oreamnos americanus), 101 mule deer (Odocoileus hemionus), 20 muskox (Ovibos moschatus), and 63 white-tailed deer (Odocoileus virginianus). A high seroprevalence (37 to 62%) was observed in domestic sheep, domestic goats, muskox, and some bighorn sheep populations. Seroprevalence in these species was generally age-related: a very low seroprevalence was present in these animals under one year of age. A low seroprevalence (2% to 13%) was found in clinically-susceptible species such as domestic cattle, deer, elk and bison, supporting the concept that significant numbers of non-lethal infections occur among clinically susceptible ruminants.

Malignant Catarrhal Fever in a Bison (Bison Bison) Feedlot, 1993–2000

A fatal enteric syndrome was identified in American bison (Bison bison) at a large feedlot in the American Midwest in early 1998. An estimated 150 bison died of the syndrome between January 1998 and December 1999. The syndrome was identified as malignant catarrhal fever (MCF), primarily the alimentary form. Clinical onset was acute, and most affected bison died within 1–3 days; none recovered. Consistent lesions were hemorrhagic cystitis, ulcerative enterotyphlocolitis, and arteritis-phlebitis. Vasculitis was milder and more localized than that in cattle with MCF, and in contrast to the situation in cattle, lymphadenomegaly was minimal. Virtually all affected bison examined were positive for ovine herpesvirus 2 (OvHV-2) by polymerase chain reaction (PCR) assay. A retrospective study of archived tissues established that MCF occurred in the yard as early as 1993. A prospective study was undertaken to establish the importance of MCF relative to other fatal diseases at the feedlot. The fate of a group of 300 healthy male bison in a consignment of 1,101 animals was followed for up to 7 months to slaughter. At entry, 23% (71/300) of bison were seropositive for MCF viruses, and 11% (8/71) of these seropositive bison were PCR positive for OvHV-2. Forty seronegative bison were selected at random from the group, and all were PCR negative for OvHV-2. There was no change in seroprevalence in the group during the investigation. The minimum infection rate for MCF virus was 36.3% (93/256). Twenty-two (7.3%) of the 300 bison in the feedlot died. Of these, 15 had MCF, 4 had acute or chronic pneumonia, and 3 were unexamined. Losses in the entire consignment were higher (98/1,101; 8.8% death loss); 76% of deaths were attributable to MCF. The study failed to reveal a relationship between subclinical infection and development of clinical disease.

Chronic generalized obliterative arteriopathy in cattle: a sequel to sheep-associated malignant catarrhal fever.

Malignant catarrhal fever (MCF) in cattle is generally associated with a short clinical course and a high case fatality rate (90–95%). The lesions in cattle that survive acute MCF for a prolonged period or appear to recover have not been documented. In a naturally occurring outbreak of MCF in a herd of beef cattle in Wyoming, 7 of 84 yearling heifers (8.3% of replacement herd) and 2 of 230 cows (0.9% of cow herd) developed clinical signs of pyrexia, mucopurulent discharge, bilateral keratitis, and weight loss following contact with ewes that had lambed 34–62 days earlier. Six of 9 affected cattle were examined postmortem following clinical signs (CS) that developed 2–150 days earlier. Three cattle with CS for ≤39 days had lesions of regional lymphadenopathy and widespread severe segmental lymphoid arteritis-phlebitis that were typical of acute MCF, and proliferative intimal lesions were present in a small proportion of arteries at days 20 and 39 of CS. By contrast, 3 cattle that survived to 90, 105, and 150 days after clinical onset had distinctive arterial lesions in multiple organs, characterized by proliferative concentric fibrointimal plaques, disrupted inner elastic lamina, focally atrophic tunica media, and vasculitis of variable severity. Immunohistochemical and ultrastructural examination of intimal plaques identified the predominant cellular component to be smooth muscle cells with a contractile phenotype. No viral structures were seen. Serologic studies, using a competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) that detects antibody to an epitope broadly conserved among isolates of the MCF virus, found that 2 chronically affected cattle were serologically positive between days 42 and 100 of CS, with seroconversion in 1 animal between days 52 and 73 of CS. Seroprevalence was 7.9% in the 76 remaining healthy animals of the replacement heifer herd and 40% (75% in adult sheep and 4% in lambs) in the in-contact sheep flock 77 days after onset of CS in the index case. This episode suggests that, in addition to the common and well recognized acute form of MCF in cattle, this viral infection encompasses a disease spectrum that includes chronic disease and partial to “complete” clinical recovery, and in recovered animals chronic obliterative arteriopathy is the preeminent lesion. The MCF CI-ELISA may be helpful in identifying such cattle and in epidemiologic studies of MCF outbreaks.

Diagnosis of malignant catarrhal fever by PCR using formalin-fixed, paraffin-embedded tissues.

A previously described polymerase chain reaction (PCR) assay (amplification of a 238-bp fragment of ovine herpesvirus 2 [OHV-2] genomic DNA) for diagnosis of sheep-associated malignant catarrhal fever (MCF) was adapted for use on formalin-fixed, paraffin-embedded tissues. Variables affecting its use were examined. Archived tissues from cattle, white-tailed deer (Odocoileus virginianus), and bison (Bison bison) diagnosed with MCF by clinical signs or histologic lesions were obtained from 2 veterinary diagnostic laboratories. Tissues from healthy animals and from animals diagnosed with other common bovine viral diseases were examined as controls. A total of 86 blocks from 37 suspect MCF cases were examined. Forty-one blocks from healthy animals and animals with unrelated viral diseases were examined as controls. The assay was specific for sheep-associated MCF and did not yield false-positive signals from healthy animals or from cases of infectious bovine rhinotracheitis, bovine virus diarrhea, mucosal disease, or parainfluenza-3 virus infection. A wide variety of tissues were suitable substrates, including spleen, lymph node, intestine, brain, lung, and kidney. Extracted DNA provided a more suitable target than did unextracted tissue lysate. The highest levels of viral DNA were present in lymphoid organs and intestine, but the data indicate that in acute clinical cases, most organs contain sufficient viral DNA to serve as a suitable diagnostic specimen. Fixation of 0.5-cm3 blocks of tissue in 10% neutral buffered formalin was deleterious to the target DNA, and PCR signals progressively diminished after fixation for >45 days. Detection of genomic DNA of OHV-2 by PCR was successful for archived tissues that were 15 years old.

Malignant Catarrhal Fever-like Disease in Sheep after Intranasal Inoculation with Ovine Herpesvirus-2

A malignant catarrhal fever (MCF)–like disease was induced experimentally in 3 sheep after aerosol inoculation with ovine herpesvirus-2 (OvHV-2). Each of 3 OvHV-2–negative sheep was nebulized with 2 ml of nasal secretions containing approximately 3.07 − 109 OvHV-2 DNA copies from a sheep experiencing an intensive viral-shedding episode. Ovine herpesvirus-2 DNA became detectable by polymerase chain reaction in the peripheral blood leukocytes of all 3 sheep within 3 days, and all 3 seroconverted between 6 and 8 days postinfection (PI). The sheep developed clinical signs, with copious mucopurulent nasal discharge and fever around 14 days PI. One of the 3 clinically affected sheep was euthanized at 18 days PI. Major lesions at necropsy were multifocal linear erosions and ulcers in mucosa of the cheeks, tongue, pharynx, and proximal esophagus and mild disseminated pneumonia. Microscopically, there was extensive moderate superficial histiocytic–lymphocytic rhinitis with epithelial dissociation and degeneration. Moderate multifocal histiocytic bronchointerstitial pneumonia was associated with loss of terminal bronchiolar epithelium. Lymphocytic vasculitis was present only in the lung. The remaining 2 sheep recovered clinically, approximately 25 days PI. The study revealed that clinical signs and lesions resembling MCF can develop when uninfected sheep are exposed to a high dose of aerosolized OvHV-2.

TOXOPLASMA GONDII INFECTIONS IN CAPTIVE BLACK-FOOTED FERRETS (MUSTELA NIGRIPES), 1992–1998: CLINICAL SIGNS, SEROLOGY, PATHOLOGY, AND PREVENTION

An epizootic of toxoplasmosis occurred among 22 adult and 30 kit black-footed ferrets (Mustela nigripes) maintained under quarantine conditions at the Louisville Zoological Garden (Louisville, Kentucky, USA) in June, 1992. Black-footed ferrets appear to be highly susceptible to acute and chronic toxoplasmosis. Clinical signs were observed in 19 adults and six kits and included anorexia, lethargy, corneal edema, and ataxia. Two adults and six kits died with acute disease. High antibody titers to Toxoplasma gondii were detected by latex agglutination and modified agglutination assay in 10 black-footed ferrets. One adult and six kits that died with acute clinical signs were necropsied and T. gondii-like organisms were found microscopically in multiple organs. Diagnosis of toxoplasmosis was confirmed by immunohistochemical staining with anti-T. gondii antibodies and by ultrastructural examination. Although the source of T. gondii for black-footed ferrets was not identified, frozen uncooked rabbit was the most likely source. Chronic toxoplasmosis resulted in the death of an additional 13 black-footed ferrets that were adults during the epizootic. Affected animals developed chronic progressive posterior weakness and posterior ataxia 6–69 mo after the epizootic began. Meningoencephalitis or meningoencephalomyelitis associated with chronic toxoplasmosis were identified at necropsy in all 13 ferrets. Precautions to prevent introduction of pathogens into the colony were insufficient to exclude T. gondii. Although toxoplasmosis may cause significant mortality in mustelids, the high mortality of black-footed ferrets in this epizootic was of concern due to their endangered status. This is the first detailed report of toxoplasmosis in black-footed ferrets.