Pathologic Findings and Viral Antigen Distribution During Natural Infection of Ring-Necked Pheasants With H5N2 Highly Pathogenic Avian Influenza Virus A.

Highly pathogenic avian influenza (HPAI) is a major viral disease of poultry characterized by acute onset, systemic infection, and rapid death. In January 2015, H5N2 HPAI was identified by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and gene sequencing as the cause of rapid death in 40 of 390 ring-necked pheasants (approximately 10% mortality), raised in a game bird farm in Washington State. We report clinicopathologic findings and viral antigen distribution in pheasants that died during the outbreak. Affected birds were depressed with reluctance to move, ruffled feathers, and drooping heads. Congestion of the cerebellar meningeal blood vessels was the only consistent gross pathologic finding. Meningoencephalitis with vasculitis and necrosis in the spleen and heart were the major microscopic lesions in the birds. Viral antigen was consistently detected in the brain, heart, and ovary with variable presence in other organs.

Clinicopathologic features of a systemic coronavirus-associated disease resembling feline infectious peritonitis in the domestic ferret (Mustela putorius).

From 2002 to 2007, 23 ferrets from Europe and the United States were diagnosed with systemic pyogranulomatous inflammation resembling feline infectious peritonitis (FIP). The average age at the time of diagnosis was 11 months. The disease was progressive in all cases, and average duration of clinical illness was 67 days. Common clinical findings were anorexia, weight loss, diarrhea, and large, palpable intra-abdominal masses; less frequent findings included hind limb paresis, central nervous system signs, vomiting, and dyspnea. Frequent hematologic findings were mild anemia, thrombocytopenia, and hypergammaglobulinemia. Grossly, whitish nodules were found in numerous tissues, most frequently the mesenteric adipose tissue and lymph nodes, visceral peritoneum, liver, kidneys, spleen, and lungs. One ferret had a serous abdominal effusion. Microscopically, pyogranulomatous inflammation involved especially the visceral peritoneum, mesenteric adipose tissue, liver, lungs, kidneys, lymph nodes, spleen, pancreas, adrenal glands, and/or blood vessels. Immunohistochemically, all cases were positive for coronavirus antigen using monoclonal antibody FIPV3-70. Electron microscopic examination of inflammatory lesions identified particles with coronavirus morphology in the cytoplasm of macrophages. Partial sequencing of the coronavirus spike gene obtained from frozen tissue indicates that the virus is related to ferret enteric coronavirus.

Comparison of two automated immunohistochemical procedures for the diagnosis of scrapie in domestic sheep and chronic wasting disease in North American white-tailed deer (Odocoileus virginianus) and mule deer (Odocoileus hemionus).

Two commercially available automated immunohistochemistry platforms, Ventana NexES and DakoCytomation Autostainer Universal Staining System, were compared for diagnosing sheep scrapie and cervid chronic wasting disease. Both automated platforms used the same antiprion protein monoclonal primary antibodies, but different platform-specific linker and amplification reagents and procedures. Duplicate sections of brainstem (at the level of the obex) and lymphoid tissue (retropharyngeal lymph node or tonsil) from the same tissue block were immunostained for the comparison. Examination of 1,020 tissues from 796 sheep revealed 100% concordance of results between the Ventana NexES and DakoCytomation platforms for diagnosing sheep scrapie from lymphoid tissue (103/103 positive; 405/405 negative) and brainstem (120/120 positive; 392/392 negative). Similarly, examination of 1,008 tissues from 504 white-tailed deer revealed 100% concordance between the Ventana NexES and DakoCytomation platforms for diagnosing chronic wasting disease from lymphoid tissue (104/104 positive; 400/400 negative) and brainstem (104/104 positive; 400/400 negative). Examination of 1,152 tissues from 482 mule deer revealed a concordance of 98.6% in lymphoid tissue and 99.9% in brainstem between the Ventana NexES and DakoCytomation platforms for diagnosing chronic wasting disease. The results indicate equivalence or near equivalence between the DakoCytomation and Ventana NexES autostainer platforms for identification of the disease-associated prion protein (PrPd)-positive and PrPd-negative brain and lymphoid tissues in sheep, white-tailed deer, and mule deer.

Immunization with native surface protein NcSRS2 induces a Th2 immune response and reduces congenital Neospora caninum transmission in mice.

NcSRS2, a tachyzoite surface protein of Neospora caninum, is an immunodominant protein with respect to induction of antibody production and has a role in attachment and invasion of host cells. Native NcSRS2 was isolated from whole tachyzoite lysate antigen by affinity chromatography using NcSRS2 specific monoclonal antibody and used to immunize BALB/c mice in a congenital transmission study. NcSRS2 was a highly conserved protein as indicated by comparison of deduced amino acid sequence obtained from NcSRS2 gene sequences of 10 geographically distinct N. caninum isolates. Mice immunized with purified native NcSRS2 produced antigen-specific antibody, primarily of IgG 1 subtype. Following challenge during gestation with 10(7) tachyzoites, immunized mice had a statistically significant decreased frequency of congenital transmission compared to non-immunized mice (P

Leukoencephalitis associated with selective viral replication in the brain of a pony with experimental chronic equine infectious anemia virus infection.

Neurologic disease occurs sporadically in horses infected with the equine infectious anemia virus (EIAV). This report describes a case of clinically severe neurologic disease in a pony experimentally infected with EIAV. This pony did not have fever or anemia, which are the characteristic clinical signs of disease. The histopathologic changes were characterized as lymphohistiocytic periventricular leukoencephalitis. Polymerase chain reaction and in situ hybridization data showed that the brain lesions were directly associated with viral replication and that high-level viral replication occurred selectively within the lesion and not in other tissues. These findings suggest that EIAV-associated neurologic disease is the direct result of viral replication.

Resistance to scrapie in PrP ARR/ARQ heterozygous sheep is not caused by preferential allelic use.

BACKGROUND: In sheep, susceptibility to scrapie, which is similar to human prion diseases such as Kuru and variant Creutzfeldt-Jakob disease (vCJD), is determined by prion protein (PrP) gene (Prnp) polymorphisms. Sheep with genotype ARQ/ARQ, denoting polymorphisms at codons 136, 154, and 171, are susceptible, whereas those with genotypes ARR/ARQ and ARR/ARR are resistant, indicating dominance of ARR over the ARQ allele. AIMS: Based on familial CJD E200K, 129V, where preferential use of the 200E allele in EK heterozygous individuals confers resistance, heterozygous ARR/ARQ sheep were used to test the hypothesis that resistance is caused by preferential use of the ARR allele. METHODS: After assessment of equivalent PrP expression across genotypes, allele use was analysed by sequencing reverse transcription polymerase chain reaction derived DNA clones containing the Prnp gene coding sequence. RESULTS: The ARR to ARQ ratio was 1.1 in 133 clones, representing Prnp mRNA from three ARR/ARQ sheep, indicating equal use of both alleles. CONCLUSIONS: Dominance of the resistant associated allele in sheep scrapie involves mechanisms other than the absence of PrP derived from the disease associated ARQ allele.

Validation of a commercially available monoclonal antibody-based competitive-inhibition enzyme-linked immunosorbent assay for detection of serum antibodies to Neospora caninum in cattle.

A previously described monoclonal antibody (MAb)-based competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was modified to optimize performance, and the assay was validated in various defined cattle populations for detection of serum antibody to Neospora caninum, a major cause of bovine abortion. Modifications to the cELISA included capturing native N. caninum antigen with a parasite-specific MAb (MAb 5B6-25) and directly conjugating the competitor MAb (MAb 4A4-2), with both MAbs binding different epitopes of a conserved, immunodominant 65-kDa tachyzoite surface antigen. The assay was validated using three serum sets, a "gold standard" set of 184 cow sera defined by fetal histopathology and N. caninum immunohistochemistry and by maternal N. caninum indirect fluorescence assay (IFA) at a 1:200 serum dilution, a relative standard set of 330 cow sera defined by IFA alone, and a set of 4,323 cow sera of unknown N. caninum status. A test cutoff of 30% inhibition was identified. The diagnostic sensitivity was 97.6%, and diagnostic specificity was 98.6% for the gold standard abortion-defined sera. The diagnostic sensitivity was 96.4%, and diagnostic specificity was 96.8% for the relative standard IFA-defined sera. Testing of the 4,323 bovine sera of unknown N. caninum status revealed a distinct bimodal distribution and steep sigmoid frequency curve with only 1.8% of samples within 5% of the test cutoff, indicating a sharp discrimination between test-positive and test-negative samples. In summary, the modified N. caninum cELISA provided a simple, rapid, and versatile method to accurately identify N. caninum infection status in cattle using a single cutoff value.

Characterization of caprine interleukin-4.

Caprine interleukin-4 (IL-4) cDNA was cloned from RNA of mitogen stimulated goat peripheral blood mononuclear cells utilizing reverse transcriptase-polymerase chain reaction. The sequence of caprine IL-4 cDNA corresponds to a 535 nucleotide mRNA with 5'- and 3'-untranslated regions and a 405 nucleotide open reading frame, the first 66 nucleotides of which encode a putative signal peptide. Mature IL-4 is a 12.8kDa protein containing six cysteine residues and two potential N-linked glycosylation sites and is highly homologous with other ruminant IL-4. The predicted molecular mass of mature unglycosylated IL-4 was confirmed by western blot of recombinant caprine IL-4 expressed in bacteria with a monoclonal antibody against a carboxyterminal peptide derived from the predicted amino acid sequence of bovine IL-4. Eukaryotic expression plasmids containing caprine IL-4 cDNA were used to characterize recombinant IL-4. Transcription of IL-4 mRNA was confirmed by transfection of COS-7 and goat synovial membrane cells, and recombinant IL-4 produced by stably transfected L929 cells inhibited inducible nitric oxide synthase in macrophages. Genetic immunization of mice with a caprine IL-4 cDNA expression plasmid induced antibodies against recombinant caprine IL-4 produced in bacteria.

Immunohistochemical detection of Prion protein (PrP-Sc) and epidemiological study of BSE in Korea.

Though the aetiology of transmissible spongiform encephalopathies (TSEs) remains uncertain, proteinase resistant prion protein (PrP-Sc), a converted form of the normal cellular prion protein (PrP-C), accumulates in the lysosome of cells of the nervous systems of animals with TSEs. In this study, clinical and epidemiological examinations of bovine spongiform encephalopathy (BSE) were conducted in Korea. During the investigated period, none of the cattle exhibited typical clinical signs of BSE, such as behavioral disturbances, high sensitivity, and abnormal locomotion. Immunohistochemical analysis and western immunoblotting were established to detect PrP-Sc in the brain tissue using monoclonal antibody (MAb) F89/160.1.5, produced by immunizing mice with a synthetic peptide which corresponds to bovine PrP residues 146-159, NH2-SRPLIHFGSDYEDRC-COOH. Although some BSE-like spongiform changes were observed in bovine brains randomly collected from Korean slaughterhouses from 1996 to 1999, no PrP-Sc was detected in those brains with the established immunohistochemistry and western immunoblotting assay. Also, no positive reaction was observed in bovine brains infected with rabies. These immunohistochemical and western immunoblotting methods using MAbs, specifically reactive with conserved epitopes on ruminant PrP, can be used for postmortem diagnosis of BSE. Further, the method can be applied to antemortem and the preclinical diagnosis of ovine scrapie by detecting PrP-Sc in lymphoid tissues, such as the tonsils, third eyelid or peripheral lymph nodes.

Immunization of BALB/c mice with killed Neospora caninum tachyzoite antigen induces a type 2 immune response and exacerbates encephalitis and neurological disease.

BALB/c mice were immunized subcutaneously with soluble Neospora caninum tachyzoite antigen (NSO) entrapped in nonionic surfactant vesicles (NISVs) or administered with Freund's complete adjuvant (FCA). Following virulent parasite challenge, groups of mice immunized with NSO and either NISVs or FCA had clinical neurological disease and increased numbers of brain lesions compared to groups of mice inoculated with FCA, NISVs, or phosphate-buffered saline (PBS) alone. Increased numbers of brain lesions were statistically significant only between mice immunized with NISV-NSO and NISV- or PBS-treated mice. Following parasite challenge, brain inflammatory infiltrates in all experimental and control groups of mice were relatively similar and consisted of compact infiltrates of macrophages admixed with various numbers of lymphoid cells. Increased brain lesions in NSO-immunized mice were associated with increased antigen-specific interleukin 4 (IL-4) secretion and increased IL-4:gamma interferon secretion ratios from splenocytes in vitro and increased antigen-specific immunoglobulin G1 (IgG1):IgG2a ratios in vivo. Thus, immunization with whole killed N. caninum antigen and either liposoidal or Freund's adjuvant induced a type 2 immune response that was associated with worsened disease. The present studies emphasize the need to identify specific N. caninum antigens or other delivery systems that will elicit protective immune responses to neosporosis.

Toxoplasmic encephalitis in a free-ranging Rocky Mountain bighorn sheep from Washington.

A 4-mo-old free-ranging Rocky Mountain bighorn sheep (Ovis canadensis canadensis) from the Hells Canyon area (Washington, USA) was diagnosed with encephalitis associated with Toxoplasma gondii infection. The sheep had concurrent pneumonic pasteurellosis and resided in a geographic area with endemic Pasteurella-associated pneumonia and mortality in bighorn sheep. The brain had multifocal necrotizing and nonsuppurative encephalitis with intralesional protozoa. The protozoa were identified as T. gondii by immunohistochemistry. To our knowledge, this is the first report of T. gondii infection in a Rocky Mountain bighorn sheep.

PrP(c) mRNA, but not PrP(Sc) is found in the salivary glands of scrapie-infected sheep.

Transmission studies in transmissible spongiform encephalopathies (TSEs) have become increasingly important due to the possible transmission of bovine spongiform encephalopathy to humans resulting in new variant Creutzfeldt-Jacob disease. The horizontal transmission of scrapie, a TSE of sheep, is poorly understood. Possible sources of horizontal transmission are the submandibular and parotid salivary glands. TSEs like natural sheep scrapie are characterized by the conversion of a normal protease sensitive prion protein, PrP(c), to an abnormal protease resistant prion protein, PrP(Sc). Since the presence of PrP(Sc) is an indicator of disease, the salivary glands of scrapie-infected sheep were examined for the presence of PrP(Sc). Although PrP(c) mRNA was detected in the salivary glands, PrP(Sc) was not found in the salivary glands of scrapie-infected sheep. These data suggest that the salivary glands are unlikely sources of horizontal transmission of natural sheep scrapie.

Preclinical diagnosis of scrapie by immunohistochemistry of third eyelid lymphoid tissue.

Ovine scrapie is a member of the transmissible spongiform encephalopathies (TSEs), a heterogeneous family of fatal neurologic disorders characterized by deposition of an abnormal isoform (prion protein [PrP] PrP-Sc) of a cellular sialoglycoprotein in neural tissue. PrP-Sc is detectable in some lymphoid tissues of infected sheep months or years before development of clinical disease. Detection of PrP-Sc in these tissues is the basis for live-animal testing. In this study, we characterize the performance of a preclinical diagnostic test for ovine scrapie based on a monoclonal antibody (MAb)-based immunohistochemistry assay of nictitating membrane ("third eyelid")-associated lymphoid tissue. The results of third eyelid immunohistochemistry assay agreed with the scrapie status of the sheep for 41 of 42 clinical suspects with confirmed scrapie and 174 of 175 sheep without scrapie. Third eyelid sampling agreed with the scrapie status for 36 of 41 clinically normal sheep positive for PrP-Sc immunostaining of brain tissue, including 27 sheep with positive biopsy specimens that progressed to clinical disease with confirmed scrapie 3 to 20 months after biopsy. The assay used MAb F89/160.1.5, which binds to residues 142 to 145 of ovine PrP. This antibody can be used in combination with MAb F99/97. 6.1, which binds to residues 220 to 225. One or both MAbs in this cocktail recognize PrP sequences conserved in most mammalian species in which natural TSEs have been reported. Immunohistochemistry assay of routinely formalin-fixed lymphoid tissues with a cocktail of pan-specific MAbs is a practical, readily standardized live-animal and preclinical test for ovine scrapie.

Neospora caninum seroprevalence and associated risk factors in beef cattle in the northwestern United States.

A Neospora caninum seroprevalence and risk factor survey of 2585 cows was conducted in 55 beef cow-calf herds located in five northwestern states of the USA. Blood samples were collected by private veterinary practitioners and management practices were surveyed using a mail questionnaire. Producers were randomly selected from those that employed these veterinarians to perform annual herd pregnancy examinations. Questions were asked about animal management, grazing and feeding, immunization and record keeping practices. Blood was collected from a systematically selected sample of cows in each herd, and age, origin, and pregnancy status were recorded. Blood samples were analyzed for antibodies against N. caninum antigen using a monoclonal antibody-based competitive inhibition (CI) ELISA. Overall seroprevalence was 24% and within herd seroprevalence ranged from 3 to 67% with a median of 19%. Within herd seroprevalence and mean inhibition percentage were different between the five states. Herds that managed their cows on range for summer grazing had lower seroprevalence than those that did not, while increased seroprevalence was associated with higher winter stocking density. Cows less than 3 years of age had higher CI ELISA inhibition percent values than cows greater than 6 years of age. No relationship was noted between serologic status and individual cow origin (purchased or raised), or pregnancy status at the time of sampling.

Neutralization of maternal IL-4 modulates congenital protozoal transmission: comparison of innate versus acquired immune responses.

IL-4 levels were modulated in mice to test the hypothesis that induction of a maternal type 1 response would decrease the frequency of congenital Neospora caninum transmission. This hypothesis tested the relationship between IL-4 and both innate and adaptive immunity utilizing two basic experimental designs. In the first, maternal IL-4 was neutralized with mAb during pregnancy in naive mice concomitant with initial, virulent infection. In the second, maternal IL-4 was neutralized before pregnancy concomitant with a priming inoculation consisting of live, avirulent N. caninum tachyzoites followed by virulent challenge during subsequent gestation. In mice that were naive before pregnancy, neutralization of IL-4 during gestational challenge did not result in decreased congenital transmission as measured by PCR performed on 1-day-old neonatal mice. In mice that were primed and modulated before pregnancy, congenital transmission from gestational challenge was significantly decreased compared with control mice. Reduction in transmission constituted a decrease in the numbers of mice transmitting N. caninum and a lower frequency of transmission by individual dams (p < 0.05). Decreased congenital transmission was associated with significantly lower levels of maternal splenocyte IL-4 secretion, lower IL-4 mRNA levels, and higher levels of IFN-gamma secretion. Protected mice had significantly decreased Neospora-specific IgG1 compared with nonmodulated mice. These studies define a relationship between maternal Ag-specific immunity and the frequency of congenital transmission and demonstrate that modulation of type 2 cytokine responses can change the frequency of congenital protozoal transmission.

Interferon-gamma and interleukin-12 mediate protection to acute Neospora caninum infection in BALB/c mice.

The type of immune response required to protect mice against clinical disease during acute Neospora caninum challenge was investigated in BALB/c mice. Groups of female BALB/c mice were infected i.p. with N. caninum tachyzoites concomitant with either: (1) antibody to interferon-gamma; (2) recombinant murine interleukin-12; or (3) recombinant murine interleukin-12 plus antibody to interferon-gamma. Mice treated with anti-interferon-gamma alone had increased morbidity/mortality, decreased body weight, increased foci of liver necrosis and increased numbers of N. caninum tachyzoites in the lung by 7 days p.i. compared with controls. Increased disease and parasite load in the anti-interferon-gamma-treated mice was associated with antigen-specific antibody IgG1 > IgG2a and a three-fold decreased ratio of antigen-specific interferon-gamma:interleukin-4. Mice treated with recombinant murine interleukin-12 had decreased encephalitis and brain parasite load at 3 weeks p.i. compared with control mice treated with PBS. In recombinant murine interleukin-12-treated mice, decreased brain lesions and parasite load were associated with antigen-specific antibody IgG2a > IgG1 and a three-fold increased ratio of antigen-specific interferon-gamma:interleukin-4 from splenocytes; the interleukin-12 effect was dependent upon interferon-gamma, as indicated by concomitant in vivo interferon-gamma neutralisation. By 6 weeks p.i. with N. caninum, there were no differences in brain lesions and parasite load between interleukin-12- and PBS-treated groups, indicating that the effects of interleukin-12 on driving a protective type 1 response were transient. These data indicate a role for interferon-gamma, interleukin-12 and type 1 immune responses in control of acute neosporosis in mice.

Detection by PCR of Neospora caninum in fetal tissues from spontaneous bovine abortions.

The routine diagnosis of Neospora caninum abortion is based upon histopathologic changes in fetal tissues and identification of tissue parasites by immunohistochemistry. Confirmation of N. caninum infection by immunohistochemistry has low sensitivity. In the present study, we examined the utility of PCR in detecting N. caninum infection in fetal tissues from spontaneous bovine abortion. DNA was obtained from fresh and formalin-fixed tissues from 61 bovine fetuses submitted for abortion diagnosis. Histopathology and immunohistochemistry determined the true status of N. caninum infection in each fetus. In formalin-fixed paraffin-embedded tissues, PCR detected N. caninum DNA in 13 of 13 true-positive fetuses (100%) and in 1 of 16 true-negative fetuses (6%). In fresh or frozen tissues, PCR detected N. caninum DNA in 10 of 13 true-positive fetuses (77%) and 0 of 11 true-negative fetuses (0%). PCR also detected N. caninum DNA in 6 of 8 fetuses that had typical lesions of N. caninum but were immunohistochemistry negative, indicating a higher sensitivity of PCR in comparison to that of immunohistochemistry. N. caninum DNA was amplified most consistently from brain tissue. PCR detection of N. caninum DNA in formalin-fixed, paraffin-embedded tissues was superior to that in fresh tissues, presumably because of the increased accuracy of sample selection inherent in histologic specimens.

Detection of equine antibodies to babesia caballi by recombinant B. caballi rhoptry-associated protein 1 in a competitive-inhibition enzyme-linked immunosorbent assay.

A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was developed for detection of equine antibodies specific for Babesia caballi. The assay used recombinant B. caballi rhoptry-associated protein 1 (RAP-1) and monoclonal antibody (MAb) 79/17.18.5, which is reactive with a peptide epitope of a native 60-kDa B. caballi antigen. The gene encoding the recombinant antigen was sequenced, and database analysis revealed that the gene product is a rhoptry-associated protein. Cloning and expression of a truncated copy of the gene demonstrated that MAb 79/17.18.5 reacts with the C-terminal repeat region of the protein. The cELISA was used to evaluate 302 equine serum samples previously tested for antibodies to B. caballi by a standardized complement fixation test (CFT). The results of cELISA and CFT were 73% concordant. Seventy-two of the 77 serum samples with discordant results were CFT negative and cELISA positive. Further evaluation of the serum samples with discordant results by indirect immunofluorescence assay (IFA) demonstrated that at a serum dilution of 1:200, 48 of the CFT-negative and cELISA-positive serum samples contained antibodies reactive with B. caballi RAP-1. Four of five CFT-positive and cELISA-negative serum samples contained antibodies reactive with B. caballi when they were tested by IFA. These data indicate that following infection with B. caballi, horses consistently produce antibody to the RAP-1 epitope defined by MAb 79/17.18.5, and when used in the cELISA format, recombinant RAP-1 is a useful antigen for the serologic detection of anti-B. caballi antibodies.

Monoclonal antibody F89/160.1.5 defines a conserved epitope on the ruminant prion protein.

The transmissible spongiform encephalopathies are a heterogeneous group of fatal neurodegenerative disorders occurring in humans, mink, cats, and ruminant herbivores. The occurrence of novel transmissible spongiform encephalopathies in cattle in the United Kingdom and Europe and in mule deer and elk in parts of the United States has emphasized the need for reliable diagnostic tests with standardized reagents. Postmortem diagnosis is performed by histologic examination of brain sections from affected animals. The histopathological criteria for transmissible spongiform encephalopathies include gliosis, astrocytosis, neuronal degeneration, and spongiform change. These lesions vary in intensity and anatomic location depending on the host species and genetics, stage of disease, and infectious agent source. Diagnosis by histopathology alone may be ambiguous in hosts with early cases of disease and impossible if the tissue is autolyzed. Deposition of the prion protein (an abnormal isoform of a native cellular sialoglycoprotein) in the central nervous system is a reliable marker for infection, and immunohistochemical detection of this marker is a useful adjunct to histopathology. In the present paper we describe monoclonal antibody (MAb) F89/160.1.5, which reacts with prion protein in tissues from sheep, cattle, mule deer, and elk with naturally occurring transmissible spongiform encephalopathies. This MAb recognizes a conserved epitope on the prion protein in formalin-fixed, paraffin-embedded sections after hydrated autoclaving. MAb F89/160.1.5 will be useful in diagnostic and pathogenesis studies of the transmissible spongiform encephalopathies in these ruminant species.