Fluorescent antibody test for rapid diagnosis of coronaviral enteritis of turkeys (bluecomb).

Intestinal tissues obtained from coronavirus-infected embryos and turkeys were examined by fluorescent antibody tissue section technique (FAT). Evidence of viral antigen was demonstrated in the cytoplasm of the intestinal epithelial cells covering the villi. Embryo intestines that were examined from 24 to 96 hours after inoculation were positive for immunofluorescence (IF), whereas bursa of Fabricius was negative. Poults hatched from infected embryos were examined at 2 days of age and were positive for IF. Coronaviral antigen was detected by FAT in the cytoplasm of intestinal epithelial cells of the jejunum, ileum, duodenum, and cecum in all turkeys that were examined from 24 hours to 28 days.     

Mise en évidence de l'expression du virus leucémogène bovin (BLV) dans les lymphocytes de mouton par une technique d'immunofluorescence utilisant des anticorps monoclonauxDetection of bovine leukemia virus (BLV) in sheep lymphocytes by immunofluorescencence test using monoclonal antibodies

An indirect immunofluorescence (IF) test was developed to detect bovine leukemia virus (BLV) antigen expression in infected sheep lymphocytes, using monoclonal antibodies anti BLV-major envelope glycoprotein gp51. Peripheral blood lymphocytes were cultivated for 48 h in presence of phytohemagglutinin (PHA) (50 μg/ml), and then fixed with acetone. The cells were assayed for the IF test. All experimentally infected sheep were positive with this test.     

Development of an ELISA for detection of myxoma virus-specific rabbit antibodies: test evaluation for diagnostic applications on vaccinated and wild rabbit sera.

An enzyme-linked immunosorbent assay (ELISA) was developed and compared with 2 reference diagnostic tests (indirect immunofluorescence [IF] and complement fixation) to detect myxoma virus-specific antibodies in sera from 50 rabbits experimentally vaccinated with an attenuated strain of myxoma virus or with a Shope fibroma virus. The ELISA was highly specific (100% specificity) and sensitive (100%, 21 days after homologous vaccination). In a comparison of the ELISA with the IF test in 128 wild rabbits from France, discrepant results were obtained in only 11 (8.6%) animals, which were positive with the ELISA and negative with the IF test. The higher sensitivity and the good specificity of the ELISA was confirmed in a serologic survey of 118 rabbits from 2 Kerguelen (Indian Ocean) islands, where the prevalence of myxomatosis varied considerably. The ELISA is an alternative serologic test for diagnosis, vaccine evaluation, and seroepidemiologic surveys of myxomatosis.     

Anaplastic ependymoma in the cervical spinal cord of a maltese dog

An 8-year and 6-month-old female Maltese dog showed a stoop with rigidity of her cervix and back. Neurologic examination showed loss of proprioception, and deficiency of pain response. Postmortem examination revealed the neoplastic mass replacing the central area in the cervical spinal cord at the level from 4th to 5th segments. Histologically, the mass was composed of neoplastic ependymal cells. The neoplastic cells showed marked atypism, and occasionally formed ependymal rosettes. Based on the morphologic features, the tumor was diagnosed as anaplastic ependymoma. Immunohistochemistry showed that the neoplastic cells were negative for glial fibrillary acid protein, and slightly positive for vimentin and cytokeratin.     

Analysis of a sheep anti-pig T lymphoblast serum with specificity for E rosette-forming lymphocytes

Sheep antibodies against a pig E-rosette-forming lymphoblastic T lymphoma raised by two intravenous injections of 10(10) cells showed little lymphocytotoxic activity which could be absorbed with red cells, alveolar macrophages or kidney or liver cell homogenates. Bone marrow absorption yielded subpopulation specific antibody which binds to E rosette-forming cells (E.RFC) using either complement-mediated cytotoxicity or indirect antiglobulin rosette formation. In 30 blood lymphocyte preparations from 20 pigs with a range of approximately 20-85% E rosettes the mean E% 43.5 +/- 2.7 agreed with the % antigen+ cells by cytotoxicity mean = 42.6 +/- 2.7 and in each individual sample these figures also agreed closely. In samples of blood lymphocytes enriched and depleted for E rosettes, results of %E+ also agreed closely with % antigen+ cells. This relationship also held for thymocytes and the specific antibodies could be completely absorbed with thymocytes. These data show that the antibody identified peripheral and thymic E.RFC. Bound to lymphocytes the antibody inhibited E rosette-formation with sheep red blood cells (SRBC) in saline (S) and dextran (DS) and with pig RBC in dextran and in Ficoll, but did not affect B cells shown by immunofluorescence, direct antiglobulin rosette formation or Fc rosette-formation, either in saline or dextran, (which include T gamma cells). E rosette inhibition was dependent on antibody concentration, showing single and double sigmoid curves for S and DS rosettes respectively, consistent with differing ease of inhibition of the strong and weak rosette formation. The same spectrum of inhibition of rosette formation by antibody binding followed subsequent incubation with C'6-deficient rabbit serum, but with C'-sufficient serum resulted in loss of cells which require dextran for Fc rosette-formation (T gamma). Thus the serum reveals E rosette-forming T cells and their subpopulations, perhaps by binding to the SRBC receptor.     

Detection of chlamydial antibody by fetal serology--an aid to the diagnosis of ovine abortion.

Two serological tests (indirect immunofluorescence and enzyme-linked immunosorbent assay) were developed for the detection of fetal antibody to Chlamydia psittaci. Fetal blood and thoracic fluid from 126 field cases of suspected ovine chlamydial abortion were examined using both tests. Placenta and fetal tissues (lung, liver, and kidney) from the same animals were also examined by the following conventional diagnostic methods: isolation in McCoy cells, detection of chlamydial lipopolysaccharide (LPS), modified Ziehl-Nielsen staining, and direct fluorescent antibody staining of chlamydia in frozen cryostat sections. Seventy cases were positive by fetal serology, and of these, 68 were also positive by isolation and/or LPS detection. The remaining 56 cases had negative fetal serology, and of these, 39 were positive by isolation and/or LPS detection. Results indicate that fetal serology, although less sensitive than either isolation in McCoy cells or detection of chlamydial LPS antigen, may be of particular use when placenta is not available.     

Detection of papillomaviruses in cutaneous fibromas of white-tailed and mule deer

Naturally occurring cutaneous fibromas affecting white-tailed deer (Odocoileus virginianus) and mule deer (O hemionus), and cutaneous fibropapillomas of domestic cattle were tested for papillomavirus using indirect immunofluorescence (IF), peroxidase-antiperoxidase (PAP), and negative-stain electron microscopic techniques. Papillomavirus was consistently detected using rabbit antiserum against papillomavirus group-specific antigen in all mule deer fibromas and bovine fibropapillomas; only 16 of 28 white-tailed deer fibromas tested by IF and 9 of 15 tested by PAP were detected. Normal skin from white-tailed deer or cattle was consistently negative for virus. Similar results were obtained by negative-stain electron microscopic examination of partially purified tumor homogenates. Using deer fibroma virus or bovine papillomavirus type 1-specific antisera, viruses were typed by IF, PAP, and immunoelectron microscopy.     

Antibody directed against Marek's disease-associated tumor surface antigen can be eluted from Marek's disease tumor cells.

Antibody directed against Marek's disease-associated tumor surface antigen (MATSA) was eluted from tumor cells of lymphomas and peripheral blood lymphocytes that were isolated from Marek's disease virus-infected chickens. Feather follicular Marek's disease virus (MDV) antigen could not be demonstrated with this antibody by indirect immunofluorescent (IF) staining. Monoclonal antibody directed against MATSA could completely block the activity of eluted antibody and vice versa. By indirect IF staining using eluted antibody and fluorescein isothiocyanate (FITC) labelled antichicken globulin conjugate. MATSA-bearing cells were detected in MDV infected and herpes virus of turkey (HVT) vaccinated birds. Blocking of immunoglobulin molecules present on B-cells by anti-chicken globulin is critical in this test.     

Prevalence of autoantibodies that bind to kidney tissues in cats and association risk with antibodies to feline viral rhinotracheitis,calicivirus, and panleukopenia

BackgroundThe feline viral rhinotracheitis, calicivirus, and panleukopenia (FVRCP) vaccine, prepared from viruses grown in the Crandell-Rees feline kidney cell line, can induce antibodies to cross-react with feline kidney tissues.ObjectivesThis study surveyed the prevalence of autoantibodies to feline kidney tissues and their association with the frequency of FVRCP vaccination.MethodsSerum samples and kidneys were collected from 156 live and 26 cadaveric cats. Antibodies that bind to kidney tissues and antibodies to the FVRCP antigen were determined by enzyme-linked immunosorbent assay (ELISA), and kidney-bound antibody patterns were investigated by examining immunofluorescence. Proteins recognized by antibodies were identified by Western blot analysis.ResultsThe prevalences of autoantibodies that bind to kidney tissues in cats were 41% and 13% by ELISA and immunofluorescence, respectively. Kidney-bound antibodies were observed at interstitial cells, apical border, and cytoplasm of proximal and distal tubules; the antibodies were bound to proteins with molecular weights of 40, 47, 38, and 20 kDa. There was no direct link between vaccination and anti-kidney antibodies, but positive antibodies to kidney tissues were significantly associated with the anti-FVRCP antibody. The odds ratio or association in finding the autoantibody in cats with the antibody to FVRCP was 2.8 times higher than that in cats without the antibody to FVRCP.ConclusionsThese preliminary results demonstrate an association between anti-FVRCP and anti-cat kidney tissues. However, an increase in the risk of inducing kidney-bound antibodies by repeat vaccinations could not be shown directly. It will be interesting to expand the sample size and follow-up on whether these autoantibodies can lead to kidney function impairment.     

Olfactory neuroblastoma in a horse

An 11-year-old thoroughbred gelding was euthanatized because of right nasal cavity tumor. The tumor consisted of round to oval cells with a scanty cytoplasm and hyperchromatic nuclei. Homer-Wright rosettes and pseudorosettes, as well as microcysts were seen. Neoplastic cells were immunoreactive to vimentin, S-100 protein, and neuron-specific enolase, glial fibrillary acidic protein and microtube-associated protein in varying degrees, indicating neurogenic nature. Based on these findings, this tumor was diagnosed as an olfactory neuroblastoma. Since this type is an uncommon tumor showing histological variety, the nature is discussed.     

Enzyme-linked immunosorbent assays for detecting antibody to Rickettsia australis in sera of various animal species

New endemic areas of spotted fever-like rickettsial disease have been found in south-eastern Australia (Gippsland, Victoria and Flinders Island, Tasmania). The rickettsia responsible is currently unknown although it may be Rickettsia australis. To investigate serological evidence of rickettsial exposure in various wild animal species, a competitive ELISA was developed which detected antibodies to R. australis. It was based on inhibition of an indirect ELISA detecting antibody to R. australis in guinea pig sera. Pre- and post-infection sera from 2 dogs, 2 rabbits, 5 mice and 6 rats, experimentally infected with R. australis, were tested by competitive ELISA. The results showed that all pre-infection sera were negative and all post-infection sera positive for antibody to R. australis. To test the utility of the competitive ELISA for detecting natural rickettsial infection in non-laboratory animals, 51 dog sera, negative for rickettsial antibody by immunofluorescence (IF) and 20 IF positive dog sera (collected from various locations on the east coast of Australia) were tested. Compared to the IF test the competitive ELISA was 90% sensitive and 96% specific. This new test has potential for detecting antibody to R. australis in the sera of different wild animal species.     

A Case of Olfactory Neuroblastoma Induced in A Rat by N-Nitrosobis(2-hydroxypropyl)amine

N-nitrosobis(2-hydroxypropyl)amine (BHP) is a well-known carcinogen and induces tumors in various tissues. In the present paper, a case of olfactory neuroblastoma (ONB) induced in a rat by BHP is described. The tumor was observed in one out of 25 male rats that received 2000 ppm of BHP in drinking water from 6 to 18 weeks of age and were sacrificed at 26 weeks of age. Histologically, the tumor arose in the posterior nasal cavity and consisted of small round cells and elongate cells with scant basophilic cytoplasm. The neoplastic cells proliferated with compartmentalization into the lobules by fibrovascular septa. True rosettes, pseudorosettes and an intercellular fibrillar matrix were occasionally observed. Immunohistochemically, the tumor cells were positive for NF120/200 and β III-tubulin. These results indicate that the present tumor is the first case of ONB induced in a rat by BHP treatment.     

The Fluorescent Antibody Technique in the Diagnosis of Equine Rhinopneumonitis Virus Abortion

Using two known positive equine viral rhinopneumonitis (EVR) sera, conjugates were prepared with fluorescein isothiocyanate and tested for specificity using EVR infected tissue culture cells. The conjugate was then applied to selected tissues from 32 aborted fetuses and foals submitted during a natural outbreak of EVR. Antigen was detected in various tissues by immunofluorescence in 20 cases (62.5%). In 24 cases bovine fetal kidney cell monolayers were inoculated with a pool of lung and liver and EVR virus was isolated from 15 (62.5%). Histological examination of various tissues from 29 cases resulted in the diagnosis of EVR in 19 (65.5%), based upon the presence of focal areas of necrosis and intranuclear inclusion bodies. Correlation of results was not obtained in two cases. One was diagnosed positive histologically and negative on fluorescence, the other was negative histologically and by virus isolation but showed fluorescence. The distribution of fluorescence in various infected fetal tissues indicated that the combined examination of lung and thymus gland was most likely to provide a positive diagnosis.     

Reaction of convalescent bovine antisera with strain-specific antigens of parapoxviruses

Two strains of papular stomatitis (PS) virus, 1 of milker's nodules (MN) virus and 1 of contagious ecthyma (CE) virus possessed 2 distinct external structures when examined by electron microscopy. The innermost, designated coat was closely apposed to the tubular surface, whereas the outer envelope loosely surrounded the virion. When convalescent sera from cattle infected with PS virus were used for immunoelectron microscopy, antibody reacted with coats and envelopes of the PS virus strains, but only with coats of MN and CE viruses. Convalescent sera from cattle infected with PS or MN virus contained complement-dependent antibodies cytolytic to cells infected with the homologous virus. In an indirect immunofluorescence test, the sera reacted with homologous strains to higher titers than with heterologous strains.     

Cultivation and partial characterization of bovine astrovirus

Bovine astrovirus serotype 2 (US2) was adapted to primary neonatal kidney cell (NBK) cultures by the addition of 50 micrograms ml-1 of trypsin in the medium. Infectious virus was released from the cells within 7 days post-infection in early passages and within 3 days in later passages. In the absence of trypsin, neither passage of infected cells nor release of infectious virus occurred. The virus was shown to be similar to the fecal astrovirus by a neutralization test and by ultrastructural studies of infected cells. Primary embryo bovine kidney (EBK) and NBK cell cultures supported infection with both fecal and tissue culture adapted (TCA) astrovirus. The time-related development of infection, as studied by immunofluorescence, was similar for both fecal and TCA astrovirus and for both cell culture types. The first indication of viral infection and expression of viral antigens occurred at 7 h post-infection and was characterized by the appearance of a diffuse faint immunofluorescence (IF) of the cytoplasm. Soon after, two or three brilliant IF granules were observed in the nucleus, which appeared to involve the nucleoli. Subsequently, dense granular IF was seen in the perinuclear region of the cytoplasm, which later extended to involve all the cytoplasmic area. In both EBK and NBK cultures infected with either fecal or tissue culture adapted astrovirus, only a minority of cells became infected, even when the multiplicity of infection exceeded one. Occasionally 10-20% of cells were infected, but in most cultures the proportion did not exceed 2% and in NBK cultures, from 3/9 calves, no infected cells were observed. The virus did not infect bovine cell lines. Infectivity of the virus was not removed by treatment with chloroform, and iododeoxyuridine and actinomycin D when added to the medium, did not block replication. Masses of virions were observed by electron microscopy in discrete areas in the cytoplasm, with similar distributions as the viral antigen foci as seen by IF. The mean diameter of the virions was 34 nm. In conclusion, bovine astrovirus lacks both essential lipids and an envelope, probably has an RNA genome, may have a nuclear phase of replication involving the nucleoli which is not blocked by DNA inhibitors, and has a selective cell tropism.     

Swine reproductive and respiratory syndrome in Québec: Isolation of an enveloped virus serologically-related to Lelystad virus

Sera were collected from convalescent sows and sick piglets from six pig farms in southern Quebec that have experienced outbreaks of the so-called porcine reproductive and respiratory syndrome. By indirect immunoperoxidase, a few of these sera (4 of 14) (28.6%) were found to be positive for antibody to the Lelystad virus, whereas by indirect immunofluorescence 30 of 36 (83.3%) were positive for antibody to the antigenically-related American isolate ATCC-VR2332. Pregnant sows inoculated intranasally with filtered homogenates prepared from the lungs of necropsied piglets obtained from a seropositive farm developed fever, inappetence, and reproductive failure characterized by stillbirths and various stages of mummification. Lesions of interstitial pneumonia were induced in experimentally-infected specific pathogen-free piglets. A virus, having morphological and biological characteristics of viruses assigned to the family Togaviridae, was isolated from lung tissues of experimentally-infected animals; it could only be propagated in primary cultures of porcine alveolar macrophages. Identification of the virus was confirmed by indirect immunofluorescence using a monoclonal antibody directed against the nucleocapsid protein of the ATCC-VR2332 isolate and porcine sera that were found positive for antibody to both the Lelystad and ATCC-VR2332 isolates.     

Immunoferritin and immune electron microscopic study of bovine herpesvirus strain DN-599.

Intracellular antigens of strain DN-599 bovine herpesvirus were detected in the cytoplasm and the nucleus of infected bovine embryonic kidney cells by the indirect immunoferritin (IF) technique. Specific tagging was observed in viral envelope and capsids. Aggregates of viral particles heavily coated with antibody were seen by immune electron microscopy (IEM).     

Présence de la maladie bactérienne du rein chez les salmonidés au Québec

Presence of the bacterial kidney disease in salmonid fish in Quebec During the summers of 1979 and 1980, wild and hatchery fish were analysed for the presence of the bacterial kidney disease (BKD) agent in salmonid fish in Quebec by the indirect immunofluorescence technique. The causative agent of BKD was detected in all hatcheries tested. Ten to 25% of the fish were positive. The presence of this agent was independent of age and species. We were unable to detect the BKD in fish from the rivers in the northern part of Quebec (over the 50th parallel).     

Detection of challenge virus in fetal tissues by nested PCR as a test of the potency of a porcine parvovirus vaccine

To estimate the potency of a porcine parvovirus (PPV) vaccine, three vaccinated and three non-vaccinated pregnant gilts were infected with PPV and the distribution of the virus was studied in the tissues of their 51 fetuses. Virus detection was attempted using haemagglutination (HA) and immunofluorescence (IF) assays, as well as by standard (single) and nested polymerase chain reactions (PCR). None of the detection methods yielded positive results when used to test for the presence of virus in suspensions of organs from the fetuses from the vaccinated gilts. However, the virus was detected in the fetuses from non-vaccinated gilts as follows: HA was positive in 14 cases out of 23 (60.8%), IF in 16/23 (69.5%), standard PCR in 12/20 (60%), and the nested PCR in 19/23 (82.6%). Although the correlation among the results of various methods of virus detection was rather close (r<0.83), the sensitivity of the nested PCR was the highest, both when testing dilutions of PPV and when analysing the fetal organs. The nested PCR therefore provides a reliable approach for studies of virus distribution in fetal organs, with special reference to potency tests on vaccines.     

Distribution of lymphoid leukosis virus and p27 group-specific antigen in tissues from laying hens

In order to gain insight into transmission and pathogenesis of infection, specimens from laying hens that had been naturally exposed to lymphoid leukosis virus (LLV) were tested for group-specific antigen (gsa) of the virus by immunofluorescence (IF), complement fixation (CF), and the enzyme-linked immunosorbant assay (ELISA). Electron microscopic examinations determined the distribution of C-type virus particles in tissues, and the phenotypic-mixing test served as a biological assay for exogenous LLV. The IF gsa was found in all organs tested, and fluorescence was usually found where virus particles were concentrated. In the oviduct and intestine, IF gsa was frequently at the border of the lumina and in the connective tissue associated with basal membranes of glands. In skin, the antigen was detected in smooth muscle, in feather pulp, and in basal epidermal cells of developing feathers. Results of various tests on Ottawa strains of chickens were usually in agreement. For example, among hens that shed gsa into egg albumen, only the viremic hens were consistently positive for IF gsa in both spleens and oviducts. Geometric mean CF titers of antigen were respectively five- and 23-fold higher in spleens and oviducts from viremic hens than in those from nonviremic hens. These findings suggest that the gsa was associated with exogenous virus infection. In Cornell S strain hens that had not been exposed to LLV, gsa was detected in splenic tissue by CF and ELISA but not by the IF test. This gsa was presumed to be of endogenous origin.