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.     

Investigation of an outbreak of mucosal disease in a beef cattle herd in southwestern Saskatchewan.

This study describes the epidemiological investigation of an outbreak of mucosal disease that occurred on a ranch in southwestern Saskatchewan. Over a six-month period during the fall and winter of 1991-1992, in a herd of 515 beef cattle and 96 bison, 20 yearling cattle from a group of 105 housed in one feedlot pen died from mucosal disease. A further eight yearlings were slaughtered for salvage because they were at risk of dying from mucosal disease. Mucosal disease mortalities were the first observed evidence of fetal infections with bovine viral diarrhea virus in this herd. Animals that died from mucosal disease exhibited signs of ill thrift prior to death. Deaths from mucosal disease were confined to the progeny of one herd of beef cows. Following an outbreak of fetal infection with bovine viral diarrhea virus during 1989-1990, at least 28 (22%) of the 128 calves born from this herd of cows in the spring of 1990 were persistently infected with bovine viral diarrhea virus. However, only one calf born from this herd in 1991, and five calves born from all herds in 1992 were persistently infected. Of the five persistently infected calves born in 1992, three were born to persistently infected replacement heifers born in 1990. These heifers calved without assistance in 1992, but only one of their calves survived past three days of age, and it was persistently infected. In January 1992, 82% of the total herd had reciprocal antibody titers to bovine viral diarrhea virus of > or = 1024 which suggested a high level of herd immunity to bovine viral diarrhea virus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal and polyclonal antibodies for immunohistochemical detection of bovine parainfluenza type 3 virus in frozen and formalin-fixed paraffin-embedded tissues.

Accurate identification of bovine parainfluenza type 3 virus in bovine respiratory disease requires dependable, sensitive, and specific techniques for detection in affected animals. Immunohistochemical testing can be a rapid and reliable means of demonstration of virus in tissues from suspect cases; however, this procedure is dependent upon the quality of the antisera directed against the viral antigens. The production of rabbit polyclonal and murine monoclonal antibodies directed against bovine parainfluenza type 3 virus and techniques for their use in fresh-frozen and formalin-fixed paraffin-embedded tissues in immunofluorescence and immunoperoxidase-based immunohistochemical tests are described.     

Diagnosis of bovine viral diarrhea virus infection using monoclonal antibodies

The monoclonal antibody (MAb) D89 against bovine viral diarrhea virus (BVDV) was used in conjunction with fluorescein-conjugated anti-mouse immunoglobulin in an indirect fluorescent antibody (IFA) procedure on frozen tissue sections and cell culture. During the 2-year study, BVDV was isolated from specimens submitted in 460 cases. The D89 Mab detected all but 2 BVDV isolates, both cytopathic. In 316 of the cases in which BVD virus was detected by IFA, specimens were inoculated on bovine turbinate cells and examined for BVDV antigens at 3-5, 10, and 20 days postinoculation. The BVDV was detected in 238/316 cases (75%) after 3-5 days incubation. The remainder were not detected until 10 or 20 days postinoculation. Virus isolation was enhanced in the early test if plates were centrifuged at the time of inoculation. Results suggest that D89 monoclonal antibody is a suitable diagnostic reagent for the detection of BVDV isolated from diagnostic specimens. The D89 MAb can be used for the detection of BVDV in both cell culture and tissues. Combination of D89 with another BVDV MAb (C17) did not improve the ability to detect BVDV in tissues compared to using D89 only, and the combined Mab's resulted in an increase in nonspecific fluorescence when used on tissues. Although pooling of different BVDV monoclonal antibodies may be necessary to detect all strains of BVDV in cell culture, pooling should be used with caution on tissues. Early detection of BVDV in cell culture by this IFA procedure permits faster confirmation of BVDV diagnosis when compared to the usual routine testing for noncytopathic BVDV at termination of first passage in cell culture.     

Detection of Infectious Bovine Rhinotracheitis and Bovine Viral Diarrhea Viruses in the Nasal Epithelial Cells by the Direct Immunofluorescence Technique

Nasal epithelial cells were collected by cotton swabs for the diagnosis in experimental and field cases of infectious bovine rhinotracheitis and field cases of bovine viral diarrhea in calves. A portion of the cells was washed twice in phosphate buffered saline and a 25 µL drop was placed on microscope slides. The cells were dried, fixed and stained according to the direct fluorescent antibody technique. Another portion of the same specimen was inoculated onto primary bovine skin cell cultures for virus isolation. In the experimental studies for infectious bovine rhinotracheitis, 29/35 specimens were positive by fluorescent antibody technique and 32/35 by cell culture and in the field cases, 22/119 were positive by fluorescent antibody technique and 19/119 by cell culture. In the field cases of bovine viral diarrhea, 28/69 samples were positive by fluorescent antibody technique and 14/69 by cell culture. When fluorescent antibody technique was performed on inoculated cell cultures a total of 24/69 specimens were positive for bovine viral diarrhea. The sensitivity of fluorescent antibody technique was thus comparable to that of cell culture method for infectious bovine rhinotracheitis and bovine viral diarrhea.     

Lesions and localization of viral antigen in tissues of cattle with experimentally induced or naturally acquired mucosal disease, or with naturally acquired chronic bovine viral diarrhea.

Tissues from cattle that died of experimentally induced mucosal disease (n = 3), naturally acquired mucosal disease (n = 6), or naturally acquired chronic bovine viral diarrhea (n = 4) were examined. Consistent findings were lymphocytic depletion of lymphoid tissues, degeneration of myenteric ganglion cells, and mild adrenalitis. Intracytoplasmic viral antigen was detected in myenteric ganglia and in endocrine glandular cells. Noncytopathic virus was isolated from all cattle, and cytopathic virus was isolated from 12 of 13 cattle.     

Coinfection with bovine viral diarrhea virus and Mycoplasma bovis in feedlot cattle with chronic pneumonia

Chronic, antibiotic-resistant pneumonia, sometimes with concurrent polyarthritis, occurs in feedlot cattle in western Canada. The prevalence of Mycoplasma bovis, bovine viral diarrhea virus, and Haemophilus somnus was determined by using immunohistochemical staining of lung and heart tissue from 2 groups of animals with this history. Mycoplasma bovis antigen was present in 44/48 cases submitted between 1995 and 1998 (retrospective group) and 15/16 of cases from 1999 (prospective group), and was associated with pulmonary necrosis. Bovine viral diarrhea virus antigen was present in association with microscopic vascular lesions in 31/48 retrospective and 9/16 of prospective cases. Types Ib and II bovine viral diarrhea virus were isolated from 4/16 prospective cases. Haemophilus somnus antigen was present in heart, lung, or both of 15/48 retrospective and 8/16 prospective cases. The results suggest that there may be synergism between bovine viral diarrhea virus and M. bovis in this pneumonia with arthritis syndrome.     

Surveillance for persistent bovine viral diarrhea virus infection in four artificial insemination centers

Four large bovine artificial insemination centers implemented a program of surveillance of resident and newly acquired bulls for persistent bovine viral diarrhea virus infection. Infection was identified in 12 of 1,538 bulls. Several clinical abnormalities, including acute and chronic mucosal disease, were evident among the persistently infected bulls. Semen produced by such bulls consistently contained bovine viral diarrhea virus, and such contamination was not always accompanied by diminished seminal quality. Infected bulls were detected by means of virus isolation tests performed on blood specimens, but not by use of serologic testing. Ten of the 12 persistently infected bulls were results of embryo transfer. Virologic surveillance of breeding herds, artificial insemination and embryo transfer centers, and the cattle trade is necessary to prevent spread of this virus by modern cattle breeding practices. Attention is also necessary to prevent contamination by this virus of the fluids used for recovery, in vitro manipulation, and transfer of bovine embryos.     

Immunologic and virologic findings in a bull chronically infected with noncytopathic bovine viral diarrhea virus

Depressed lymphocyte blastogenesis in response to mitogen stimulation, depressed iodination of protein by neutrophils, and enhanced ingestion of Staphylococcus aureus by neutrophils were detected in a bull with chronic bovine viral diarrhea (BVD). Before developing chronic BVD, the bull was vaccinated with a killed cytopathic BVD virus. Neutralizing antibodies specific for the vaccine virus were detected in serum specimens obtained from the bull immediately before death. A noncytopathic BVD virus was isolated from the spleen after death. The immunologic and virologic findings in this bull supported reported research findings on the pathogenetic mechanisms involved in chronic BVD and mucosal disease.     

Expression of a 50 kDa putative receptor for bovine viral diarrhea virus in bovine fetal tissues.

The expression of a 50 kDa bovine viral diarrhea virus putative receptor in different bovine fetal tissues from 3-month old fetuses was studied. The receptor expression was examined by immunocytochemical staining and by immunoblotting using antiidiotypic probe (anti-D89). Intense specific staining in enterocytes of the small and large intestines, cortical tubular epithelial cells of kidneys, respiratory epithelial cells of the trachea and esophageal mucosal epithelial cells was observed, demonstrating the strong expression of bovine viral diarrhea virus receptor in the tissues. Weak staining was found in cerebellum, thymus, spleen, liver, cerebrum, and lung tissues; however, heart tissues were negative. Immunoblotting results correlated with the immunoperoxidase staining assays. Thus, the expression levels of the receptor are variable in different tissues. This pattern of expression may provide clues to the pathogenic potential of bovine viral diarrhea virus in the bovine fetus.     

Evaluation of an antigen-capture ELISA for the detection of bovine herpesvirus type 1 shedding from feedlot cattle

A total of 457 nasal swab specimens from cases of respiratory disease in 2 feed lots were evaluated for the detection of bovine herpesvirus Type 1 (BHV-1) by ELISA. Thirty-three were found to be positive for BHV-1 by the recovery of infectious virus and 21 of these were positive by ELISA, yielding a sensitivity of 64%. Fifteen other virus isolations were made and included bovine viral diarrhea viruses, rhinoviruses and parainfluenza Type 3 viruses; none of these cases were positive with the BHV-1 ELISA. Specificity of the ELISA was 100%. Eighty percent of the specimens with BHV-1 titers greater than 10(5) TCID50 were detected by ELISA; the median amount of virus in positive specimens that were detected by ELISA was 7 X 10(5) TCID50 and the median amount of virus in specimens not detected was 1.5 X 10(4) TCID50. BHV-1 infection was most frequently diagnosed in feedlot cattle that had been in the feedlot for 40-80 days. Approximately half of the infected cattle were carrying virus-neutralizing antibodies in their serum.     

Bovine viral diarrhea virus: biotypes and disease.

Bovine viral diarrhea virus continues to produce significant economic losses for the cattle industry and challenges investigators with the complexity of diseases it produces and the mechanisms by which it causes disease. This paper updates and attempts to clarify information regarding the roles of noncytopathic and cytopathic bovine viral diarrhea viruses in persistent infections and mucosal disease. It also covers, in brief, what is known of the new diseases: thrombocytopenia and hemorrhagic disease, and a disease resembling mucosal disease that is apparently caused solely by noncytopathic virus. Although a good understanding of the roles of the 2 biotypes in the production of persistent infections and the precipitation of mucosal disease has been obtained, there are still unanswered questions regarding the origin of cytopathic viruses and the mechanism by which they cause pathological changes in cells. It is apparent, however, that cytopathic bovine viral diarrhea viruses arise by mutation of noncytopathic viruses, and it is known that p80 is the marker protein for cytopathic viruses. The previous distinction between mild bovine viral diarrhea and fatal mucosal disease has been eroded with the emergence of new virulent bovine viral diarrhea viruses. The new diseases pose a threat to the cattle industry and present a new challenge for investigators. Index Veterinarius (1984-1994) and Medline (1985-1994) databases and personal files updated since 1987 from BIOSIS Previews and Biosciences Information Services were used to search the literature.     

牛病毒性腹泻/粘膜病研究进展

牛病毒性腹泻(BVD)是由牛病毒性腹泻病毒(BVDV)引起的一种传染病。被感染后,会导致牛群腹泻、发热、黏膜糜烂溃疡、先天性免疫等症状。本文通过对牛病毒性腹泻流行情况、检测方法、临床类型以及预防与防控等进行论述,为我国的牛病毒性腹泻防治研究提供参考。     

Tween 80: a marker for differentiation of hog cholera and bovine viral diarrhea viruses.

Markers for differentiating hog cholera and bovine viral diarrhea viruses were studied using Tween 80, chloroform, trichlorotrifluoroethane and tri (n-butyl) phosphate. Attenuated A and virulent Ames strains of hog cholera virus were employed. Moreover, the NADL PK-15 cell culture adopted strain and low cell culture passaged Purdue strain of bovine viral diarrhea virus were used. These viruses were reacted with 2,500 micrograms/ml of Tween 80 for one hour at 37 degrees C. When attenuated A and virulent Ames strains of hog cholera virus with titers greater than 10(6) and 10(5) plaque forming units respectively, were reacted with Tween 80 the titer of each strains was reduced by approximately 10(4) plaque forming units of virus. When either strain of bovine viral diarrhea virus was reacted with Tween 80, virus was not detected.     

Bovine viral diarrhea virus in New World camelids.

A virus known to cause multiple problems in cattle, bovine viral diarrhea virus, was isolated from 3 different cases in New World camelids. Virus isolation, immunoperoxidase staining, and fluorescent antibody staining were used to detect the virus. The herds involved were screened for antibody titers to bovine viral diarrhea and virus isolation from the buffy coat. Bovine viral diarrhea virus should be considered as a cause of death in young and old New World camelids.     

Natural infection of pigs with bovine viral diarrhea virus and its differential diagnosis from hog cholera.

Natural infection of pigs with bovine viral diarrhea virus (BVDV) through contact with infected cattle has caused problems in diagnosing hog cholera (HC). Low cross-reacting serum antibody titers against HC caused by BVDV infection were found in clinically normal pigs as well as those suspected of having HC. Bovine viral diarrhea virus was isolated from specimen tissues and initially identified as HC virus (HCV), using the fluorescent antibody cell culture technique. Additional cell cultures, as well as pig and calf trials, were necessary to identify it as BVDV. The isolate caused clinical signs of illness in the calves, whereas the pigs remained healthy. Bovine viral diarrhea virus may be detected in tissue sections or isolated in cell cultures and confirmed as HCV, using the HC fluorescent antibody conjugate. Laboratories performing the neutralization test for HC should use discretion when interpreting HC titers unless BVD titers are determined on the same serums.     

Experimental primary postnatal bovine viral diarrhea viral infections in six-month-old calves

Eight clinically healthy calves were inoculated intranasally, four with either noncytopathic or four with cytopathic bovine viral diarrhea virus, and were necropsied 5 or 12 days post-inoculation. The most frequent gross lesion associated with noncytopathic or cytopathic viral infection was proximal colonic mural edema. Consistent microscopic findings were acute to subacute tracheitis, mild enterocolitis with edema, petechial hemorrhages of mesenteric lymph nodes with mild follicular lymphocytic depletion, and paracortical lymphocytic hyperplasia. At necropsy, cytopathic virus was recovered from 4/4 calves and noncytopathic virus was isolated from 2/4 calves. Neutralizing antibodies to noncytopathic bovine viral diarrhea virus were detected in the two calves from which noncytopathic virus was not recovered. Immunohistochemical analysis of lymphoid tissues demonstrated a small, randomly distributed population of mononuclear cells that contained bovine viral diarrhea viral antigen in 7/8 calves.     

Comparative pathogenicity of selected bovine viral diarrhea virus isolates in gnotobiotic lambs

The infectivity and pathogenicity of selected bovine viral diarrhea virus (BVDV) isolates were determined in gnotobiotic, colostrum-deprived neonatal lambs. Five-day-old cesarean-derived gnotobiotic lambs were exposed to 1 of 10 BVDV isolates via aerosol suspension. These isolates were from tissues or secretions of calves or lambs affected with respiratory tract disease, weak neonatal calves, aborted bovine fetuses, or reference Singer or Draper BVDV. The pathogenicity of each isolate, relative to the others, was evaluated in lambs by measurement of the neutralizing antibody response, virus isolation from nasal secretions or tissues, and postmortem lesions. The BVDV isolates varied in their infectivity and pathogenicity. Singer, the cytopathic reference strain, was the most lymphotrophic isolate and stimulated the greatest neutralizing antibody response. Encephalitis was the most consistent lesion observed and was used as the final determinant of relative pathogenicity of the viruses. The most neuropathogenic isolates were the 2 viruses originating from lambs affected with respiratory tract disease, the 2 weak neonatal calf isolates, and 1 isolate from an aborted bovine fetus. The least pathogenic isolates were the 2 reference isolates, Draper and Singer; the 2 mucosal disease isolates; and 1 isolate originating from an aborted bovine fetus.     

Serologic examination of aborted ovine and bovine fetal fluids for the diagnosis of border disease, bluetongue, bovine viral diarrhea, and leptospiral infections

Fetal serum from most of 994 bovine and 553 ovine aborted fetuses was tested serologically for antibodies to border disease (BD), bovine viral diarrhea (BVD), and bluetongue (BT) viruses, and to Leptospira sp., and the results were compared with the results of isolation procedures, fluorescent antibody tests (FAT), and histologic examinations of the same fetuses. Antibodies to BT virus were not found in any of the 994 bovine and 553 ovine fetuses. Antibody titers to BVD virus were present in 39 of 966 bovine fetuses tested, and BVD virus was detected in 4 of the 39. Four of 74 fetuses in which the BVD virus was detected by FAT or isolation had titers to BVD virus. Microagglutination (MAT) titers to 1 or more of 5 serovars of leptospires were present in 52 of 773 bovine fetal sera tested. Leptospires were not detected by FAT in any bovine fetuses that had leptospiral antibody titers. Leptospires were detected by FAT in 15 aborted calves, and none of these had MAT titers. Antibody titers to BD virus were present in 80 of 486 fetal lamb sera tested, and the virus was detected by FAT or isolation in 3 of the 80 fetuses. Border disease virus was detected in 14 of 486 fetal lambs tested. Twelve of the 14 were tested serologically and 3 had titers to BD virus. Leptospiral antibody titers were present in 27 of 326 ovine fetal sera tested. Leptospires were not detected in any of the 326 ovine fetuses tested by FAT.     

Mechanism of protection from primary bovine viral diarrhea virus infection. I. The effects of dexamethasone.

A series of investigations was designed to study the role of cellular immunity and passive antibody in protecting neonatal calves from primary bovine viral diarrhea virus infection. Administration of corticosteroids (dexamethasone) in doses capable of suppressing cellular immunity markedly potentiated systemic bovine viral diarrhea virus infection in calves which lacked bovine viral diarrhea passive neutralizing antibody. Immunosuppressed calves did not form neutralizing antibody to bovine viral diarrhea virus and developed a fatal viremia. Calves with high levels of passive bovine viral diarrhea neutralizing antibodies were protected from the effect of corticosteroids. The results suggest an essential role for humoral passive antibody, but not for cellular immunity, in protection from primary systemic bovine viral diarrhea virus infection in calves.