Lesions and retroviruses associated with naturally occurring ovine pulmonary carcinoma (sheep pulmonary adenomatosis)

Five sheep with ovine pulmonary carcinoma were markedly dyspneic and had sporadic coughing; two had copious watery nasal exudate. In four, lesions consisted of multifocal nodules of neoplastic cuboidal epithelial cells in acinar or papillary patterns. Electron microscopically, cells had microvilli, tight junctions, and cytoplasmic lamellar bodies typical of alveolar type II cells. One sheep had a single lung tumor of nonciliated bronchiolar epithelial cells. Vacuolated alveolar macrophages surrounded adenomatous foci. One sheep had a metastatic lesion in the caudal mediastinal lymph node. All sheep had histologic lesions of lymphoid interstitial pneumonia (LIP, ovine progressive pneumonia) consisting of peribronchiolar and interstitial lymphoid hyperplasia, and fibromuscular proliferation; all had serum precipitating antibodies to ovine lentivirus. Lung fluids or tumor homogenates contained a 26-kd peptide that crossreacted with a primate-derived type D retrovirus as detected by immunoblotting or interspecies competition radioimmunoassay. Ovine lentivirus was isolated from concentrated lung fluids or tumor tissues of four sheep tested and from tumor cell DNA of one animal transfected into ovine muscle cells. These studies document the presence of type D-related retrovirus antigen in ovine pulmonary carcinoma (OPC) in the United States and indicate that lentivirus-induced LIP is a lesion frequently associated with this disease.     

The etiology and pathogenesis of ovine pulmonary carcinoma (sheep pulmonary adenomatosis)

Ovine pulmonary carcinoma (OPC, sheep pulmonary adenomatosis, jaagsiekte) occurs naturally as a contagious bronchioloalveolar carcinoma of sheep in the Americas, Europe, Africa and Asia. The disease is endemic and economically important in Peru and apparently more common than previously suspected in the U.S.A. The tumor is a result of transformation of type II alveolar epithelial cells or non-ciliated bronchiolar cells of the lung. Clinically affected sheep develop dyspnea, tachypnea and often a watery nasal discharge that originates from tumor secretions. The course is progressive and death usually occurs within a few weeks. To study the viral etiology and pathogenesis of OPC in the U.S.A., the disease was experimentally transmitted to neonatal or young lambs with a success rate of 69%. Ovine lentivirus (OvLV), present in the inocula, was concurrently transmitted and induced lymphoid interstitial pneumonia in most animals. While morphological, immunological and other studies implicate a type D or type B retrovirus as the etiologic agent of OPC, this virus has not yet been cultured and the role of ovine lentivirus in the disease remains unknown.     

Alveolar type II cells expressing jaagsiekte sheep retrovirus capsid protein and surfactant proteins are the predominant neoplastic cell type in ovine pulmonary adenocarcinoma

Ovine pulmonary adenocarcinoma is caused by jaagsiekte sheep retrovirus. To gain insight into the histogenesis and viral pathogenesis of this neoplasm, the tumor cell phenotypes and differentiation state were correlated with the distribution of jaagsiekte sheep retrovirus capsid protein in neoplastic and normal cells of the lung in nine naturally occurring and 12 experimentally induced cases of ovine pulmonary adenocarcinoma. Overall, 82% of tumor cells had ultrastructural features consistent with alveolar type II cells, 7% of tumor cells had features of Clara cells, and 11% of tumor cells were insufficiently differentiated to classify. The proportion of the neoplastic cell phenotypes varied within tumors, and no tumor consisted of a morphologically uniform cell population. To further characterize the neoplastic cell population, sections of tumors were immunostained with antibodies to surfactant protein A, surfactant protein C, and Clara cell 10-kd protein. Overall, surfactant proteins A and C were expressed in 70% and 80% of tumor cells, respectively, whereas Clara cell 10-kd protein was expressed in 17% of tumor cells. Jaagsiekte sheep retrovirus capsid protein was detected in 71% of tumor cells and in macrophages (5/21 tumors examined) and in nonneoplastic alveolar and bronchiolar cells (6/14 tumors). Expression of this viral protein in neoplastic cells, classified morphologically and by immunophenotyping primarily as of the alveolar type II lineage, implies an important role for specific virus-cell interactions in the pathogenesis of ovine pulmonary adenocarcinoma.     

Retrovirus-associated ovine pulmonary carcinoma (sheep pulmonary adenomatosis) and lymphoid interstitial pneumonia. I. Lesion development and age susceptibility

To determine the lesion development of retrovirus-induced ovine pulmonary carcinoma (OPC), ten neonatal lambs were inoculated intratracheally with either 1) lung fluid preparations derived from a sheep with Type D retrovirus-associated OPC and concurrent ovine lentivirus (OvLV)-associated lymphoid interstitial pneumonia (LIP) (n = 8); or 2) lung fluid from a sheep with only OvLV-LIP (n = 2). Seven of eight neonates that received Type D retrovirus-associated OPC/OvLV-LIP lung fluid developed both OPC and LIP lesions between 9 and 32 weeks after inoculation. Mild OPC lesions consisted of foci of type II alveolar epithelial cells lining alveoli surrounded by minimal alveolar macrophage infiltrates. More severe OPC lesions consisted of multifocal aggregates of cuboidal to columnar neoplastic cells forming acini or masses associated with abundant alveolar macrophage infiltrates. Lesions of LIP consisted of peribronchiolar and perivascular lymphoid hyperplasia and heterogeneous interstitial leukocytic infiltrates. The two neonates that received OvLV-LIP lung fluid developed rapid and severe LIP, but not OPC lesions. Two lambs (inoculated as neonates with virus-free lung fluid) and three lambs (uninoculated contacts) served as controls and did not develop OPC. To investigate age susceptibility for development of OPC, 20 additional lambs within defined age groups (neonates, 2 weeks old, 5 weeks old, and 10 weeks old) received ultracentrifuged tumor homogenate. Neonatal to 5-week-old lambs inoculated with Type D retrovirus-associated OPC/OvLV-LIP tumor homogenate were equally likely to develop OPC, but lambs inoculated at 10 weeks of age were more refractory to tumor development.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunomorphologic and morphometric changes in pulmonary lymph nodes of sheep with progressive pneumonia

Morphologic, immunohistochemical, and morphometric studies were conducted on the posterior mediastinal lymph nodes of eleven sheep with naturally occurring ovine progressive pneumonia and four apparently healthy sheep with no pulmonary lesions (three seropositive, one seronegative for antibody to ovine progressive pneumonia virus). Compared with lesion-free sheep, sheep with ovine progressive pneumonia had a seven-fold increase in B lymphocyte areas and a 21/2-fold increase in T lymphocyte areas of these lymph nodes. Immunochemistry revealed cytoplasmic immunoglobulin G in scattered cells of germinal centers, medullary cords and interfollicular areas and membrane-associated immunoglobulin G in dendritic cells of germinal centers. Immunoglobulin M staining cells were widely scattered in germinal centers and medullary cords. Although B cell hyperplasia seemed to be the predominant process in lymph nodes of sheep with ovine progressive pneumonia, this was not accompanied by the expected degree of plasmacytosis, morphologically and immunohistochemically. These findings may represent an aberrancy of immunoregulation in ovine progressive pneumonia.     

Evidence of decreased concanavalin A induced suppressor cell activity in the peripheral blood and pulmonary lymph nodes of sheep with ovine progressive pneumonia

Concanavalin A (Con A) induced suppressor cell activity was evaluated in a group of ovine progressive pneumonia virus-antibody positive sheep (OPPV+). Decreased levels of suppressor activity were observed in the peripheral blood and regional pulmonary lymph nodes of animals with clinical and pathological evidence of ovine progressive pneumonia (OPP) when compared to animals with no lesions and animals with caseous lymphadenitis involving the lungs and pulmonary lymph nodes. Decreased levels of Con A stimulated lymphocyte proliferation was also observed in the peripheral blood and iliac lymph nodes of sheep with OPP. Sheep with OPP were found to be hypogammaglobulinemic. Sera from OPPV+ sheep had no effect on T and B cell mitogen stimulated responses of lymphocytes from sheep seronegative for antibodies to ovine progressive pneumonia virus (OPPV-) when compared to normal sheep serum.     

A PCR technique for the detection of Jaagsiekte sheep retrovirus in the blood suitable for the screening of ovine pulmonary adenocarcinoma in field conditions

Ovine pulmonary adenocarcinoma (OPA) is a naturally occurring contagious lung neoplasia caused by jaagsiekte sheep retrovirus (JSRV). Although no specific circulating antibodies against the virus can be detected in infected sheep, JSRV proviral DNA sequences can be found in peripheral blood leukocytes (PBLs) in clinically affected and in a proportion of in contact animals. In this study, existing hemi-nested PCR procedure is compared with a new one-step PCR technique that was developed to minimise potential DNA contamination and reduce sample and reagent handling. Different blood preparations were assessed and the best results were achieved on DNA prepared from buffy coat. The sensitivity of this PCR was lower in JSRV infected sheep without lesions of OPA than in clinically affected sheep, which indicate that this PCR may not be not fully appropriate for screening of individual sheep, but rather to provide results at flock level. This PCR is the only currently available blood test for detection of JSRV infected sheep and may be useful in epidemiological studies and in control programmes of OPA.     

Ultrastructure and frequency of mastitis caused by ovine progressive pneumonia virus infection in sheep

Twenty-five sheep, experimentally (n = 15) or naturally (n = 6) infected with ovine progressive pneumonia virus and noninfected controls (n = 4), were evaluated for histological and ultrastructural lesions of mastitis. Histologically, nine of 15 experimentally infected sheep and all six naturally infected sheep had lympho-plasmacytic mastitis. Severity of the lesion increased with length of time after infection. Periductal lymphatic nodules were seen in five sheep experimentally infected for 2.8 years or longer and in five naturally infected sheep that were 3.7 years old or older. Ultrastructurally, responses to ovine progressive pneumonia virus were diffuse lympho-plasmacytic infiltrates in glandular interstitium, lymphocytic and occasional plasmacytic infiltrates in ductal walls and lumens, lymphoblasts surrounded by small lymphocytes in glandular interstitium, and degeneration of epithelium releasing cells and cellular debris into the lumen. Based on the prevalence of lesions, the mammary tissue was more susceptible to ovine progressive pneumonia virus than other target organs: lung, brain, and synovium. Lesions did not differ between breeds of sheep. Ovine progressive pneumonia virus was not seen in the mammary tissue but was isolated from 15 of 17 mammary glands.     

Jaagsiekte sheep retrovirus found in milk macrophages but not in milk lymphocytes or mammary gland epithelia of naturally infected sheep

Jaagsiekte sheep retrovirus (JSRV) causes ovine pulmonary adenocarcinoma. JSRV can be transmitted via infected colostrum or milk, which contain somatic cells (SCs) harboring JSRV provirus. Nevertheless, the cell types involved in this form of transmission and the involvement of the mammary gland remain unknown. We separated adherent cells (macrophages and monocytes) by plastic adherence, and lymphocytes (CD4+ and CD8+ T cells, and B cells) by flow cytometry, from SCs in milk samples from 12 naturally infected, PCR blood test JSRV–positive, subclinical ewes. These cell populations were tested by PCR to detect JSRV provirus. The ewes were euthanized, and mammary gland samples were analyzed immunohistochemically to detect JSRV surface protein. We did not detect JSRV provirus in any milk lymphocyte population, but milk adherent cells were positive in 3 of 12 sheep, suggesting a potential major role of this population in the lactogenic transmission of JSRV. Immunohistochemistry did not reveal positive results in mammary epithelial cells, pointing to a lack of participation of the mammary gland in the biological cycle of JSRV and reducing the probability of excretion of free viral particles in colostrum or milk.     

Jaagsiekte sheep retrovirus is present at high concentration in lung fluid produced by ovine pulmonary adenocarcinoma-affected sheep and can survive for several weeks at ambient temperatures

Jaagsiekte sheep retrovirus (JSRV) causes a fatal lung cancer of sheep known as ovine pulmonary adenocarcinoma (OPA). OPA is a significant disease in many sheep-rearing countries and there is no effective method of control. A unique feature of OPA is the overproduction of fluid in the lung of affected animals. This lung fluid contains JSRV and provides a means of transmission through the inhalation of virus. In this study we demonstrated that lung fluid from different OPA cases contained between 10⁷ and 10¹⁰ copies of JSRV RNA per ml. Examination of JSRV RNA survival under conditions that mimic natural conditions suggested that intact JSRV virions may persist for several weeks in the environment. These are the first quantitative data on JSRV in lung fluid and provide valuable information for implementing appropriate biosecurity measures to control the spread of JSRV in the field.     

Serum antibody responses in sheep after natural infection with Bacteroides nodosus

Soluble outer membrane protein of Bacteroides nodosus extracted with potassium thiocyanate (KSCN) was employed as antigen in an enzyme linked immunosorbent assay (ELISA) to detect serum antibody in sheep naturally infected with a heterologous serogroup. Serum antibody responses in 55 sheep were monitored for 2 years and maximum levels were directly related to the severity of clinical foot lesions. Serum antibody levels rose 2 weeks after foot lesions developed and declined within several months of resolution of lesions. After the first footrot transmission period, antibody levels persisted significantly (P less than 0.001) longer in sheep that did not become affected in the next transmission period compared with sheep in which footrot recurred. Antibody response did not appear to result in resolution of foot lesions. ELISA using KSCN antigen gave similar results to whole cell ELISA where cells prepared from an homologous serogroup were used as antigen. Both these assays were more sensitive than ELISA in which heterologous whole cell antigen was used. Proteins extracted from the outer membrane of B. nodosus, which are known to be immunogenic in natural infection and common to different serogroups of B. nodosus, appear to be useful antigens for serological investigations of ovine footrot.     

Comparisons of reactivity of pulmonary vascular and airway smooth muscle of sheep and calf to tryptamine analogues, histamine, and antigen.

Serotonin (5-hydroxytryptamine) contracted the pulmonary artery, trachea, and bronchus in both cattle and sheep. Serotonin contracted the calf pulmonary vein, but it relaxed the pulmonary vein of sheep. Histamine constricted the 2 pulmonary blood vessels and trachea in calves and sheep. In contrast, histamine relaxed the ovine bronchus, but not the bronchus of the calf. Antigen (equine serum) constricted pulmonary veins from sensitized calves, but relaxed pulmonary veins from similarly sensitized sheep. Apparently, sheep are naturally more resistant than calves to both histamine and serotonin which contribute to the pulmonary inflammatory reaction.     

ras p21 expression in ovine pulmonary carcinoma

We have adapted an enzyme-linked immunoblot assay (ELIBA) for the detection of a c-ras proto-oncogene and oncogene protein products in human cell lines and tumors of 21,000 daltons molecular weight (p21ras) to studies of tissues derived from sheep. In the ELIBA, a double antibody system is used in which p21ras proteins are initially immunoprecipitated from protein extracts with monoclonal antibodies, and subsequently identified using additional anti-ras antibodies. Binding is identified with a non-radioactive enzyme-linked colorimetric detection system. In the present study, the ELIBA system was used to study twenty-seven ovine lung specimens, representing normal lung, inflammatory, and neoplastic lesions. We detected p21ras protein expression in every tissue examined, but the nature and amount of the protein product varied significantly among the tissues examined. Some tissues expressed multiple ras species. Broncho-alveolar carcinoma specimens were most likely to express c-Ki-ras proteins. Mutant proteins of c-N-ras and c-Ki-ras were detected in several bronchoalveolar carcinoma specimens, based on migrational differences between mutant and normal proteins in 15% polyacrylamide gels. The results of this study demonstrate the utility of the ELIBA system for detection of c-ras expression in ovine lung tissues, and demonstrate the ability of the system to discriminate specific ras protein species. The prognostic significance of ras expression in sheep pulmonary carcinoma has yet to be determined.     

Pulmonary dendritic cells isolated from neonatal and adult ovine lung tissue

Lung dendritic cells (DCs) are potent antigen presenting cells (APCs) that initiate and modulate the adaptive immune response upon microbial infection within the pulmonary environment. For the first time, neonatal and adult lung DCs in a large animal model were compared in these studies. Here, we isolated and identified lung DCs in both neonatal and adult sheep, a valuable experimental animal utilized in pulmonary studies of naturally occurring respiratory diseases. Neonatal lung DCs exhibited characteristic dendrites and morphology when observed by transmission electron microscopy and expressed low to moderate DEC-205, CD80/86, MHC class II and CD 14. Regardless of age, lung DCs were functionally able to endocytose FITC conjugated ovalbumin but to a lesser degree than monocyte-derived DCs. In addition, neonatal lung DCs were demonstrated to be potent stimulators of allogeneic T cell proliferation. Together, these results demonstrate that neonatal and adult lung DCs are functionally similar. It is apparent from the data presented that neonatal pulmonary DCs do not exhibit an intrinsic functional defect that would impair their ability to take up antigen and stimulate na?ve T cells. These data support growing evidence that neonatal immune responses may differ from adults due to different microenvironmental influences rather than differences in dendritic cell maturation states.     

Immunohistochemical and histopathological findings of ovine pulmonary adenocarcinoma (Jaagsiekte) in Egyptian sheep

Ovine pulmonary adenocarcinoma (OPA) is a naturally occurring retrovirus-induced transmissible lung cancer in sheep. Lungs and associated (bronchial and mediastinal) lymph nodes of seven sheep with OPA were examined. Lungs had few multifocal consolidated slightly elevated gray to white masses ranging from 0.5 to 3 cm in diameter. Histopathologically, these masses appeared as well-differentiated acinar adenocarcinoma with little evidence of anaplasia. The acini composed of well-differentiated cuboidal to low columnar epithelium with clear or vacuolated cytoplasm and low mitotic index. No metastases were observed in the bronchial and mediastinal lymph nodes of any animal. The presence of Jaagsiekte sheep retrovirus (JSRV) was demonstrated in the lungs by immunohistochemistry. JSRV protein was detected in all tumor epithelial cells, histologically normal alveolar type II cells, and few bronchiolar epithelial cells, alveolar macrophages, lymphocytes, and plasma cells. This study is the first to confirm the presence of natural OPA in Egypt.     

Recovery of transmissible gastroenteritis virus from chronically infected experimental pigs.

Transmissible gastroenteritis (TGE) virus was reisolated from pulmonary and intestinal tissues from 6 of 9 chronically infected experimental pigs (principals) necropsied 30 to 104 days after inoculation. Tissue homogenates (lung and small intestine) from the principals were prepared and inoculated into 3- to 5-day-old gnotobiotic pigs. The virus reisolated from the tissue homogenates produced a milder disease on 1st passage and a more severe disease on 2nd passage. The chronically infected experimental pigs (principals) developed serum-neutralization titers to TGE of 1:30 to 1:525. There appeared to be no relationship between serum titers and reisolation of TGE virus from the 9 principals. The persistence of virus in lung or intestine to 104 days indicates the recovered (or carrier) pig may be considered the primary source of TGE virus infection.     

Protective effects of vaccination with bovine leukemia virus (BLV) Tax DNA against BLV infection in sheep

A DNA vaccination trial was performed on sheep to determine whether vaccination with bovine leukemia virus (BLV) transactivator Tax DNA is effective against BLV infection. Immunization was carried out with cationic liposomes containing the Tax-expressing plasmid DNA and subsequently all sheep were challenged with BLV. BLV titers in peripheral blood mononuclear cell (PBMC) determined by syncytium formation assay and BLV provirus load detected by genomic PCR analysis showed higher levels of virus titers in control sheep than those in Tax-vaccinated sheep. Higher levels of IFN-gamma mRNA expression have been demonstrated in vaccinated sheep after the challenge. These results suggested that Th1 type immune response induced by Tax DNA vaccine inhibited BLV propagation in vaccinated sheep at the early phase of infection.     

Ovine Lentivirus is Aetiologically Associated with Chronic Respiratory Disease of Sheep on the Laikipia Plateau in Kenya

A study was undertaken to investigate the occurrence of ovine lentivirus (OvLV) infection in sheep with chronic respiratory disease on the Laikipia Plateau, Kenya. All seven Merino crossbred sheep with chronic dyspnoea and emaciation examined for gross and microscopic lesions had lymphoid interstitial pneumonia (LIP), and one also had pulmonary abscesses. Two of the sheep with LIP also had lesions of ovine pulmonary carcinoma (OPC, jaagsiekte). Using in situ hybridization, OvLV DNA localized to a high proportion of pulmonary macrophages in lungs with lesions of LIP. Lung tissue samples from six of these sheep were positive for a syncytium-inducing virus in cultures of lamb testis cells. Thin-section electron microscopy of infected cells showed virions with morphogenesis typical of lentiviruses. In a western blotting assay, monoclonal antibodies to the OvLV capsid (CA, p27) and matrix (MA, p15) proteins of a North American OvLV isolate reacted with similar-sized bands of the virus, and serum from six of the sheep were reactive with CA from the Kenyan viral isolate. Using an OvLV agar gel immunodiffusion (AGID) test, all seven sheep were positive for serum antiviral antibody, as were 29% of 63 clinically normal sheep from Laikipia District. However, when sera from the healthy sheep were tested in a western blot assay, only 52% had IgG reactive to the OvLV CA, indicating a high rate of false negative reactions with the AGID test. Serum samples from 87 Red Maasai or Dorper crossbred sheep from two farms in other parts of Kenya were OvLV seronegative by both the AGID test and the western blot assay. These results document the first identification of OvLV as a cause of chronic respiratory disease in sheep in Kenya and show a high rate of infection in sheep flocks, with a high prevalence of chronic respiratory disease.     

Detection of Mycobacterium avium subsp. paratuberculosis in ovine faeces by direct quantitative PCR has similar or greater sensitivity compared to radiometric culture

The aims of this study were to develop a new real-time quantitative PCR (QPCR) assay based on IS900 for detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP) DNA in faeces, and to use this to detect infected sheep. Both the C and S strains of MAP were detected by the QPCR assay, and no cross reactions were detected with 51 other species of mycobacteria including 10 which contained IS900-like sequences. One copy of IS900 fragment cloned into plasmid pCR2.1 and 1 fg of MAP genomic DNA were consistently detected, while in spiked faecal samples the detection limit was 10 viable MAP per gram of ovine faeces. A total of 506 individual ovine faecal samples and 27 pooled ovine faecal samples with known culture results were tested. The QPCR assay detected 68 of 69 BACTEC culture positive individual faeces and there was a strong relation between time to detection in culture and DNA quantity measured by QPCR (r= -0.70). In pooled faecal samples, QPCR also agreed with culture (kappa=0.59). MAP DNA was detected from some culture negative faecal samples from sheep exposed to MAP, suggesting that the QPCR has very high analytical sensitivity for MAP in faecal samples and detects non-viable MAP in ovine faeces. None of the faecal samples from 176 sheep that were not exposed to MAP were positive in QPCR. This is the first report of a direct faecal QPCR assay that has similar sensitivity to a gold standard radiometric culture assay.     

Comparison of four serologic tests for the detection of antibodies to bovine leukemia virus

Four tests for detection of antibodies to bovine leukemia virus (BLV) were compared. The sera that were tested came from cattle in naturally infected commercial dairy herds, cattle that were infected under experimental conditions, and cattle in an isolated BLV-free herd. The tests that were compared included a radioimmunoprecipitation assay (RIA) with p24 antigen, a RIA with glycoprotein (gp) antigen, an agar-gel immunodiffusion (AGID) test with gp antigen, and a virus-neutralization (VN) test that was based on inhibition of BLV-induced syncytia in cell culture. Results of the 4 serologic tests agreed for 96.8% of the sera from cattle in commercial herds. The gp RIA detected the greatest number of positive sera (188); it was followed in turn by the p24 RIA (187), the VN test (183), and the AGID test (176). The gpd RIA titers of the 12 sera that gave negative AGID results were 175 or less. In RIA, the percentage of precipitation of labeled antigen by positive sera was almost always higher with gp antigen than with p24 antigen. Satisfactory sensitivity in the p24 RIA required the acceptance of a low level of antigen precipitation, 15%, as a positive test. In the gp RIA, however, almost all positive sera precipitated at least 50% of the labeled antigen. Nonspecific precipitation of antigen in the RIA by sera from BLV-free cattle ranged from 4% to 10%. Examination of sequential serum samples from 17 experimentally infected cattle showed that BLV antibody was first detected 2 to 8 weeks after inoculation. In 9 cattle, seroconversion was detected simultaneously by all of the tests. Results from the other 8 cattle indicated that seroconversion could be detected first by p24 RIA, followed by the gp RIA and the VN test. The longest interval between RIA seroconversion and AGID seroconversion was 10 days. Monthly tests of sera from 10 laboratory cattle that were infected by contact exposure showed that 7 animals seroconverted in all tests at the same time. Two cattle were positive first in RIA, but the next month they were also positive in the VN and AGID tests. One animal was positive in the RIA and the VN test for 2 months before antibody was detected by AGID.