Detection of bovine coronavirus and type A rotavirus in neonatal calf diarrhea and winter dysentery of cattle in Quebec: evaluation of three diagnostic methods.

The use of direct electron microscopy, enzyme-linked immunosorbent assay, and protein A-gold immunoelectron microscopy for the identification of bovine coronavirus and type A rotavirus were examined. Two hundred and forty-nine samples from diarrheic calves and winter dysenteric cattle from seven geographic areas in Quebec were examined for the presence of viruses by direct electron microscopy of negatively stained preparations. In addition, all the samples were analyzed by enzyme-linked immunosorbent assay, and a random selection of 47 samples were also analyzed by protein A-gold immunoelectron microscopy. Thirty-nine percent of samples examined by direct electron microscopy contained viral particles; bovine coronavirus and type A rotavirus were the most common viruses involved. Overall agreement between any two of the methods used compared favorably with results obtained by others using similar methods. The presence of coronavirus and rotavirus in fecal samples obtained from neonatal calves and the presence of coronavirus in samples from winter dysenteric adult cattle suggested their etiological roles in the respective diseases. Furthermore, results from protein A-gold immunoelectron microscopy of coronavirus-like particles implied that a different coronavirus or some other viruses might be involved in these diseases. Finally, the efficiency of direct electron microscopy, enzyme-linked immunosorbent assay and protein A-gold immunoelectron microscopy as diagnostic tools is discussed.     

犬瘟热的微生物学诊断研究

对临床诊断为犬瘟热的１４例病犬，取其脑组织，运用细胞培养、合胞体检查、包涵体检查、免疫荧光试验、电镜观察等５种检测方法，就犬瘟热的微生物学诊断进行了系统的研究。结果表明，犬脑组织块与猫胚（ＦＥ）细胞或非洲绿猴肾（Ｖｅｒｏ）细胞共同培养，盲传的培养物出现不稳定的细胞病变，通过对不同代次的培养物检查包涵体，并作超薄切片或负染，电镜观察犬瘟热病毒（ＣＤＶ）粒子，证实分离到ＣＤＶ野毒。病犬脑组织切片经ＨＥ染色检查包涵体及用ＣＤＶ荧光抗体直接法检测脑抹片及接种犬脑的细胞培养物，均获得一定的阳性结果。建立的改良离子捕获电镜法，用于检测犬脑匀浆和犬脑接种细胞培养物，与离子捕获电镜法、免疫电镜法及直接电镜法相比，效果更为理想。１４例临床诊断为犬瘟热的病犬，综合运用上述微生物学手段检测，结果为１２例阳性，２例疑似。     

Lesions of spontaneous canine viral enteritis

Spontaneous enteric disease characterized by hemorrhagic diarrhea and high mortality occurred in puppies from commercial kennels in three midwestern states. Microscopic lesions resembling those of panleukopenia in cats were seen in the intestines. The predominant features were necrosis of crypt epithelium, collapse or dilation of crypt lumina and villous atrophy. Viral particles morphologically resembling parvovirus were found in the feces by direct electron microscopy. The canine virus reacted with antibody to feline panleukopenia virus by immunoelectron microscopy and fluorescent antibody technique. Fluorescent antibody was used to detect virus in the crypt epithelium of affected dogs. Feline kidney cells inoculated with fecal preparations had cytopathic effect and positive fluorescence by fluorescent antibody technique.     

Viral agents associated with outbreaks of diarrhea in turkey flocks in Quebec.

The relative importance of various enteric viruses associated with diarrhea of turkey poults was investigated by an evaluation of specimens received since 1982. Specimens originated from one to eight week old turkey poults, with mild to severe diarrhea, from 114 flocks in 42 commercial operations located in southern Quebec. The acute phase of enteritis occurred usually in poults between two and four weeks of age. Clarified intestinal contents were examined by direct electron microscopy and enzyme immunoassays. Enzyme-linked immunosorbent assays were performed with antisera to bovine rotavirus group antigen, avian reovirus types 1 to 5, and the prototype strain of the turkey enteric coronavirus. The presence of viruses could be demonstrated by electron microscopy in 55.3% of the specimens, and at least five different viruses were incriminated either alone or in combination. The coronavirus was by far the most common enteric virus with a prevalence of 47.5%. By enzyme-linked immunosorbent assay, rotavirus, reovirus and turkey coronavirus were detected in 14.5%, 18.1% and 61.4% of the specimens, respectively. By electron microscopy, 56.6% of these cases were positive for at least one virus.     

Scanning electron, light, and immunofluorescent microscopy of coronaviral enteritis of turkeys (Bluecomb).

Intestinal sections from both experimental and field cases of turkey coronaviral enteritis (TCE) were examined by scanning electron microscopy and light microscopy through 10 days after inoculation and by a direct fluorescent antibody test for TCE through 12 days. Serums were collected for an indirect fluorescent antibody test for TCE through 160 days after inoculation. Lesions observed with the scanning electron microscopy were catarrhal enteritis with hemorrhage per diapedesis, epithelial desquamation, and villous atrophy which developed and regressed within 6 days after inoculation. Light microscopy demonstrated similar lesions, except that villus-to-crypt ratios remained depressed 10 days. The direct fluorescent antibody test demonstrated the presence of coronaviral antigen throughout the sampling period, and serum antibodies to TCE were present until at least 160 days, when the experiment was terminated.     

Comparison of results using electron microscope, immunodiffusion and fluorescent antibody analyses to detect rotavirus in diarrheic fecal samples of calves.

Seventy-nine diarrheic calf fecal samples were examined by electron microscopy, immunodiffusion and the fluorescent antibody technique for the presence of rotavirus (reovirus-like agent). Thirty-eight (48%) of the samples were positive by electron microscopy, 59% by immunodiffusion and 20% positive by fluorescent antibody technique analyses. Another 9% were suspect-positive by fluorescent antibody technique. Chymotrypsin treatment of the fecal samples increased the ease of observing the viral particles by electron microscopy and also intensified the immunodiffusion arcs obtained. Immunodiffusion analyses using specific antisera to the virus would appear to be a practical method of detecting rotavirus in diarrheic fecal samples.     

家兔黏膜免疫器官肥大细胞在巴氏杆菌感染中的特征——组织化学与电镜观察

对5例巴氏杆菌感染家兔的空肠与圆小囊肥大细胞（mast cell，MC）进行了组织化学与电镜的观察，发现在细菌感染导致的病变组织边缘、病变组织内血管附近结缔组织中及变性细胞周围组织的上皮下与黏膜下层都出现了大量甲苯胺蓝（Toluidine Blue，TB）阳性肥大细胞。电镜下，MC有的直接与变性细胞密切接触，有的则和血管内皮紧密相触，在此部位的血管内可见有淋巴细胞贴附填充。MC胞浆内充盈大量的特征性颗粒，有时这些颗粒向细胞表面突出形成边缘空隙，即形成所谓的脱颗粒管道，将颗粒内容物逐渐排除细胞外，遗留空腔。     

用离子捕获电镜技术快速检测犬瘟热病毒

应用离子捕获电镜术与直接负染电镜术比较，检测了临床上疑似为犬瘟热的犬病料。结果表明，离子捕捉电镜技术具有快速、简便、直观、较感度高等优点，可作为快速检测病毒的常规方法推广应用。     

Comparative studies of the detection of rotavirus in fecal samples of calves with diarrhea with the latex test "Slidex Rota-Kit 2" and electron microscopy

Of 68 fecal samples from calves with diarrhoea which were tested for rotavirus with the latex agglutination test "Slidex Rota-Kit2" and by electron microscopy 33 samples were positive and 33 were negative with both tests respectively. Divergent results (latex test positive/EM negative and vice versa) were observed in one specimen only, respectively. Cross reactions with other viruses diagnosed by electron microscopy were not observed with the latex agglutination test. The "Slidex Rota-Kit2" is another suitable test for the diagnostic laboratory as well as for the veterinary practitioner for the detection of rotavirus in fecal samples of calves.     

The laboratory diagnosis of Orthopoxvirus infection in the domestic cat

Twenty-eight cases of naturally occurring feline Orthopoxvirus infection were diagnosed by combinations of virus isolation, electron microscopy, agar gel precipitation, histopathology and serology. Twenty-five of 28 cases were positive by virus isolation on the chorioallantoic membrane of hens' eggs and in tissue culture, 22 of 28 by electron microscopy, three of five by histopathology, none by agar gel precipitation, and 17 of 17 by serology. The techniques used are described, and the results compared and discussed.     

Latex test for rapid rotavirus diagnosis in calves

A latex agglutination test (LA) was compared with direct electron microscopy (EM) for detection of rotavirus infection in calves. A total of 375 samples from 62 calves were collected. Samples were taken when the calves were 1, 3, 5, 7, 10 and 20 days old and some scours samples were collected as well. Altogether 45/375 (12%) specimens were positive in LA and 10/375 (2.7%) were positive in EM. Samples positive in EM were also positive in LA. Out of the 62 calves studied 26/62 (42%) were positive in LA and 8/62 (13%) in EM. We found the LA very easy to perform, to be more sensitive than the EM method and probably a rather specific method for detection of rotavirus infection.     

Alkaline phosphatase reaction of canine mammary mixed tumours: a light and electron microscopic study

Alkaline phosphatase (ALK-P) reactions at the light and electron microscopic levels were performed on eight canine mammary mixed tumours. In the morphologically normal gland next to the tumours, the myoepithelial cells and the stromal tissues near the acinar basement membranes both showed a positive ALK-P reaction by light microscopy. At the electron microscopic level, those parts of the myoepithelial cell membrane that were in contact with other cells--myoepithelial or lumenal epithelial--showed an ALK-P-positive reaction, as did the adjacent stromal tissue. The characteristic tumour cells, at sites of early proliferation, were ALK-P-positive, while in the mucoid and chondroid areas of the mixed tumours they were largely ALK-P-negative by light microscopy. By electron microscopy, those neoplastic cells which were in contact with other cells typically showed a positive ALK-P reaction, while those cells which were in contact with mucoid or chondroid matrix were largely negative. Although the cytoplasmic filaments of the neoplastic cells were 10 nm thick, and those of normal myoepithelial cells were 6 to 8 nm thick, the observations reported provide further evidence for the view that the essential cells of the canine mammary mixed tumour are derived from myoepithelial cells.     

Herpes viral hepatitis in a toucan

Herpesvirus infection was diagnosed in a toucan. The herpesvirus was isolated from the liver and identified by electron microscopy in the liver and in cell culture. A negative immunofluorescent reaction was obtained when virus-infected cell cultures were reacted with a conjugate to the herpesvirus of Pacheco's disease. The main pathologic finding in the toucan consisted of a severe necrotizing hepatitis with intranuclear inclusions in the liver and spleen. A presumptive diagnosis of chlamydiosis was also made, based on a positive direct fluorescent monoclonal antibody reaction to chlamydial antigens in impression smears of liver and spleen. Chlamydial isolation attempts were unsuccessful. The toucan had been in contact with two macaws that had died 5 days before the toucan died and were diagnosed by histology as having herpesvirus hepatitis.     

Comparison of the diagnostic methods for studying parvovirus and rotavirus infections of dogs

79 feces samples of dogs between 7 weeks till 13.5 years of age, showing clinical signs of a hemorrhagic gastroenteritis, were tested by a commercial ELISA (DiaSystems Canine Parvo, Tech-America) for Canine Parvovirus (CPV) and by two Latex-Agglutination tests for Rotavirus (30 probes were tested by Rota Screen, 49 samples by Slidex Rota-Kit 2, both tests from BioMerieux). All samples were also examined by electron microscopy. The results of the simultaneous investigations showed 28 times positive and 28 times negative for CPV (70.9%). In 93.7% the investigations for Rotavirus-infection showed identical results by the Latex-Agglutination and electron microscopy: 73 samples were negative, one case showed a positive reaction. In 4 feces samples Rotavirus could only be detected by the Latex-test. In one sample a double-infection (CPV/Rotavirus) could be observed by all methods, in two cases the double-infection was only found by using the Latex-Agglutination. No other viruses could be found by the electron microscope than those described above. The results and the performance of the methods are discussed and compared with the data of other authors.     

1株猪源牛病毒性腹泻病毒的分离与鉴定

从BVDV阳性仔猪病料中分离病毒,为开展猪源BVDV病原学研究奠定基础。将处理后BVDV阳性仔猪组织样品接种MDBK细胞,分离到1株猪源BVDV,命名为SD0803株。通过细胞培养、直接免疫荧光、5′-UTR与Npro PCR扩增、电镜观察、TCID50测定及对其分子进化特征加以分析。结果表明,该毒株在MDBK细胞上盲传至13代未出现细胞病变。在直接免疫荧光试验中呈阳性荧光信号。PCR扩增分别获得5′-UTR与Npro预期大小DNA片段。电镜观察,病毒粒子略呈圆形,有囊膜,直径约50nm。病毒滴度为10-6.5 TCID50.0.2 mL-1。SD0803 5′-UTR、Npro序列进化分析显示,该分离株属于BVDV-1,与已知BVDV-1各亚型之间同源性较低,单独成一分支。结果表明,成功分离鉴定1株猪源BVDV SD0803,该毒株为非致病变型BVDV-1,极有可能为BVDV-1新的亚型。     

Rapid coagglutination test for detection of rotaviruses in turkeys

Avian rotaviruses present in fecal samples were readily detected using a staphylococcal protein-A coagglutination test on a white porcelain plate. Staphylococci, which produced large amounts of protein-A, were coated with rabbit anti-avian rotavirus serum. The antibody-coated staphylococci were agglutinated specifically by rotavirus present in the fecal sample. The macroscopic agglutination reaction occurred within a few minutes. A total of 40 fecal samples were tested by the coagglutination test. The sensitivity and specificity of the coagglutination test were compared with those of electron microscopy, enzyme-linked immunosorbent assay, and tissue-culture virus-isolation methods. Of the 31 fecal samples positive for rotavirus on electron microscopy, 27 (87%) were positive on coagglutination test. Of the nine electron-microscopy-negative samples, seven (78%) were also negative on coagglutination test. It was concluded that the staphylococcal protein-A coagglutination test can be used as a simple, rapid screening test for avian rotavirus.     

Evaluation of ELISA and electron microscopy for the detection of coronavirus and rotavirus in bovine faeces

Enzyme-linked immunosorbent assay (ELISA) was compared with electron microscopy in the examination of faeces from experimental calves and showed 100 per cent agreement in the detection of 19 bovine coronavirus and 15 bovine rotavirus electron microscope positive samples. In a limited field survey of calf diarrhoea 75 selected faeces were examined independently by ELISA and electron microscopy and the agreement between the two tests was 95 per cent for coronavirus and 84 per cent for rotavirus. A further comparison was made with 74 samples submitted for routine diagnosis and this yielded agreements of 82 per cent (coronavirus) and 89 per cent (rotavirus). Factors contributing to discrepant results were examined and the relative advantages and disadvantages of the two tests for routine detection of these enteric viruses are discussed.     

Routine isolation and cultivation of bovine rotaviruses in cell culture

Using the bovine embryonic kidney cell line, Aubek, bovine rotaviruses were routinely isolated from fecal samples of calves with diarrhea. Of 125 fecal samples positive for rotavirus by immune electron microscopy and the enzyme-linked immunosorbent assay, 61 isolates were recovered and cultivated continuously.     

Detection of mink enteritis virus in mink feces, using enzyme-linked immunosorbent assay, hemagglutination, and electron microscopy

Twenty-five mink were inoculated with mink enteritis virus (MEV). Fecal specimens were collected daily and were simultaneously evaluated for MEV antigen by use of a direct enzyme-linked immunosorbent assay (ELISA), hemagglutination (HA), and electron microscopy. Results of the evaluations indicated that MEV was shed in the feces on postinoculation days 5 and 6. The virus was not detectable by ELISA or HA after postinoculation day 6, although viruses were found in reduced numbers by use of electron microscopy. The ELISA was specific for MEV, and the sensitivity of the ELISA for MEV was comparable with that of HA.     

Natural infection with an attaching and effacing Escherichia coli in a diarrheic puppy.

Enteric infection with an attaching and effacing Escherichia coli was diagnosed in a puppy with protracted diarrhea. Extensive colonization of the small intestinal mucosa was observed by light and scanning electron microscopy and characteristic lesions of bacterial attachment of the brush border of the enterocytes were demonstrated by transmission electron microscopy. The E. coli strain isolated from the small intestine belonged to serotype O49:H10, did not produce any known E. coli enterotoxin or cytotoxin, was not invasive, and was negative for the known fimbrial colonization factors produced by animal and human enterotoxigenic E. coli. A positive immunoperoxidase reaction was obtained on the bacteria attached to the enterocytes with an anti-E. coli O49 antiserum.