Molecular diagnosis of avian nephritis: preliminary report

Kidney samples from chickens diagnosed with acute nephritis and gout were subjected to histological and electron microscopic examination. The investigations revealed cytoplasmic inclusion bodies in the tubular epithelial cells containing round virions of about 30 nm in diameter. Since avian nephritis virus (ANV) is known as a potential causative agent of the so-called baby chick nephropathy, an RT-PCR assay was developed for the molecular detection of ANV-specific nucleic acid in the specimen. The specificity of the assay was confirmed by direct sequencing of the amplicon obtained in the reaction. The nucleotide sequence of the PCR product showed 92% identity with the reference ANV sequence deposited in the GenBank database. After having been validated on some other suspicious cases of avian nephritis, the PCR method described in this study can be a potential tool for routine diagnostic examination of samples submitted from cases of gout and nephropathy in chickens.     

The indirect immunofluorescence assay using cardiac tissue from chickens, quails and ducks for identification of influenza A virus during an outbreak of highly pathogenic avian influenza virus (H5N1): a rapid and simple screening tool for limited resource settings

Here we describe the diagnostic utility of the indirect immunofluorescence assay (IFA) during a recent outbreak of highly pathogenic avian influenza (HPAI) subtype H5N1 virus in southern Thailand and demonstrate the usefulness of the cardiac tissue from infected chickens, quail, and ducks for diagnosis. The most reliable sample for IFA diagnosis of influenza A virus was cardiac tissue (83.0%; 44/53) which when divided by species (chicken, quail and duck cardiac tissues) gave respective positivity rates of 88% (22/25), 88.9% (16/18) and 60.0% (6/10). Cardiac tissue also gave the highest IFA intensity for the three species. We believe that the IFA method has wide applicability in developing countries or remote settings where clinically similar avian diseases with high morbidity and mortality such as Newcastle disease and fowl cholera are common and could be rapidly excluded thereby conserving valuable reference laboratory capacity for true HPAI outbreaks.     

三种鉴别诊断PRRSV美洲型经典毒株和变异株的方法比较

我国流行的猪繁殖与呼吸综合征病毒（Porcine reproductive and respiratory syndrome virus, PRRSV）包括美洲型经典毒株和变异株。本研究应用RT—PCR、直接免疫荧光（ direct immunofluorescence asay, DFA ）及病毒分离3种检测方法对99份不同类型的猪繁殖与呼吸综合征临床疑似病料和人工感染病料进行检测。结果显示,RT—PCR检测试剂盒敏感性最高,DFA鉴别诊断试剂盒次之,病毒分离最差。DFA与病毒分离、RT—PCR的总符合率分别为92％和85．5％。RT—PCR灵敏性高,可作为PRRSV鉴别诊断的首选方法;DFA鉴别诊断法所需时间较短、特异性好,但需要一定的操作经验;病毒分离敏感性低、操作繁琐,阳性分离需要其它方法验证,不适合于PRRSV的鉴别诊断。     

Demonstration of the agent of bovine viral diarrhea-mucosal disease (BVD/MD) in comparison between organ fluoresence freezing method and the direct cultivation in tissue culture]

Organs of 100 calves whose clinical symptoms indicated a BVD/MD viral infection have been examined in the laboratory by different methods on BVD/MD virus in order to compare the effectivity of the single test-systems. By inoculation of organ material into tissue culture from foetal calf kidneys and additional staining with swine-fever conjugate in the CCSC-system 59 calves were detected as BVD/MD virus carriers. Taking this result for comparative purposes equal to 100% there could be stated an effectivity of 73% by inoculation of tissue cultures and judging cpe instead of staining with fluorescent antibody as above. Only 34% reactions could be demonstrated by means of heterotypic immunofluorescence in organ tissues. For diagnostic purposes a combination of the easy and quick method of heterotypic immunofluorescence in organ tissues and a cultural virus isolation from organ material with additional immunofluorescence is recommended.     

Development of an enzyme-linked immunosorbent assay for detecting infectious bursal disease virus (IBDV) infection based on the VP3 structural protein

Infectious bursal disease virus (IBDV) causes a contagious immunosuppressive disease in chickens. The aim of the present study is to develop an enzyme-linked immunosorbent assay (ELISA) using the expressed VP2 or VP3 protein of IBDV as the coating antigen for detecting antibodies to IBDV. Experimental results were compared with virus neutralization assay and a commercial-available ELISA. These assays were used to examine the sera from farm chickens and chickens vaccinated experimentally. The VP3-based ELISA had a higher correlation coefficient (R(2)) of 0.812 with a commercial ELISA kit at a serum dilution of 1:500 than that of VP2-based ELISA (R(2)) of 0.671. The relative sensitivity between virus neutralization and VP2-ELISA and VP3-ELISA was 96% (251/262) and 100% (262/262), respectively, and that between virus neutralization and a commercial ELISA was 99% (257/261). Additionally, compared with virus neutralization assay, the reference technique for diagnosing IBDV, VP3-based ELISA had an agreement value of 99%, superior to that of VP2-based ELISA (95%) or the commercial kit (89%). These results revealed that the capability of either VP2-ELISA or VP3-ELISA in detecting the field chicken sera was comparable to the commercial one, which is generally used to replace the virus neutralization assay. However, the preparation of VP3 is derived from an Escherichia coli expression system with a high yield and purification efficiency by Ni(2+)-NTA gels, which is more favorable to the insect cell-derived particles formed by VP2. Therefore, VP3-ELISA could be developed as an efficient and low cost diagnostic method for IBDV infection in field chickens.     

Percutaneous fine-needle biopsy of deep thoracic and abdominal masses in dogs and cats

Percutaneous fine-needle biopsy was used to investigate thoracic and abdominal masses in the dog and cat. One hundred and thirty-two cases were included in the study; 20 cases were excluded from the comparative study due to poor cellularity or blood contamination (retrieval rate 86.8 per cent). One hundred samples (56 dogs and 44 cats) were classified by cytology as neoplastic. All the cytological diagnoses of neoplasia were confirmed by histological samples obtained either by non-surgical methods, at surgery or during postmortem examination. No false positive diagnoses of neoplasia were made. Thirty-two samples were cytologically classified as 'negative for neoplasia'. Subsequent histological examination revealed 18 true negative and 14 false negative results. The procedure had an overall 89.4 per cent (118 cases out of 132) agreement between the diagnosis of inflammatory disease versus neoplasia, with a sensitivity of 87.8 per cent, a specificity of 100 per cent, a predictive value of a positive test of 100 per cent and a predictive value of a negative test of 56.3 per cent.     

Evaluation of brush cytology in the diagnosis of chronic intranasal disease in cats

Brush cytology was used as a diagnostic aid in 85 cats affected with chronic intranasal disease. Fifty-three of these cases, sampled over a five-year period, were included in this study, while the other cases were excluded due to poor cellularity of the cytological samples (nine cases) or a lack of histological or follow-up data (23 cases). Thirty-six brush samples were classified by cytology as inflammatory. Subsequent histological examination revealed a false negative diagnosis of neoplasia in six cats, two of which had malignant tumours (one adenocarcinoma and one lymphoma), the remaining four having benign tumours (two adenomas and two osteochondromas). Seventeen samples were classified by brush cytology as neoplastic. This was confirmed in 16 of these cases by histology or follow-up (nine epithelial malignant tumours, six lymphomas and one osteosarcoma). In the remaining case, a false positive diagnosis of lymphoma was made. The procedure had an overall 86.8 per cent (46/53) agreement between the diagnosis of inflammatory conditions versus neoplasia, with a sensitivity of 72.7 per cent, a specificity of 96.8 per cent, a predictive value of a positive test of 94.1 per cent and a predictive value of a negative test of 83.3 per cent.     

The diagnosis of Neospora abortions in cattle

An indirect immunofluorescent antibody test (IFAT) and an enzyme-linked immunosorbent assay (ELISA) for specific anti-Neospora antibodies in bovine sera and foetal fluids were compared with histological examination results on aborted foetal material. The agreement between serological and histological examination results was poor, while the two serological tests showed a high degree of agreement. Serological testing of diagnostic serum samples and foetal fluids suggests that the prevalence of anti-Neospora antibodies in cattle which recently aborted is around 40%, in line with previous estimates of the number of abortions in dairy cattle caused by Neospora sp. A sero-epidemiological approach to the diagnosis of Neospora abortions in cattle may be suggested from these data.     

Concurrent malignant catarrhal fever and bovine virus diarrhoea virus infection in a dairy herd

An account is given of an outbreak of malignant catarrhal fever which occurred in a 98-cow dairy herd. Ten animals died or were slaughtered and the disease was confirmed by clinical and histological examination. Serological tests for malignant catarrhal fever virus were positive in three of four animals. The diagnosis of malignant catarrhal fever was complicated by the presence of bovine virus diarrhoea virus infection in three of the early cases. The initial cases of malignant catarrhal fever occurred in a group of nine-month-old calves which were housed in an old milking parlour with 19 pedigree Suffolk ewes at lambing time. Later cases occurred in two adult cows and in two heifers. Investigations of the remainder of the herd for evidence of bovine virus diarrhoea virus did not reveal the presence of any persistently infected cattle. Serological examinations for antibody to malignant catarrhal fever and bovine virus diarrhoea virus were carried out on the 19 Suffolk ewes. Six of them had neutralising antibody titres to malignant catarrhal fever virus and three were positive in the indirect immunofluorescence test. The possible roles of bovine virus     

The Dominant white mutation in the PMEL17 gene does not cause visual impairment in chickens

Objective  To examine whether the Dominant white mutation (causing a hypopigmented phenotype in chicken) affects the visual ability and gives rise to ocular abnormalities in chickens ( Gallus gallus ).
Procedure  Chickens homozygous for either the Dominant white mutation or the wild-type alleles were tested in a visual contrast behavioral test and subjected to histological and ophthalmologic examination.
Results  There were no differences between the genotypes in the visual contrast behavioral test, and there were no abnormal structures among the Dominant white chickens in the ophthalmic examination. The histological sections from the Dominant white chickens did not differ from the wild-type chicken in structure, photoreceptor density, or RPE pigmentation.
Conclusions  The results indicate that the Dominant white mutation in PMEL17 does not seem to affect the visual ability or eye structures in chickens.     

Development of enzyme-linked immunosorbent assay with nucleoprotein as antigen for detection of antibodies to avian influenza virus

During the avian influenza outbreak of 2003-04 in Southeast Asia, two avian influenza viruses (AIV), one of H5N1 subtype and the other H9N2 subtype, were isolated and identified from local farms. The nudeoprotein (NP) gene of the H5N1 AI isolate was cloned, and the segment encoding amino acid 47-384, which covers its major antigenic domains, was subcloned and expressed in E. coli. Subsequently, the NP (47-384) expression product was purified and used as the diagnostic antigen to develop a NP-based type-specific indirect enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to AI from chicken sera. The ELISA is shown to be specific for AIV and does not cross-react with chicken sera that has antibodies to other avian viruses. The NP(47-384)-ELISA was compared with a hemagglutination inhibition test and a commercial AIV ELISA kit in evaluating 150 sera samples from experimentally AIV-infected or vaccinated specific-pathogen-free (SPF) chickens. Our NP(47-384)-ELISA was more sensitive than the two tests and showed an 82% agreement ratio with the HI test and an 80.67% agreement ratio with the commercial kit. The NP(47-384)-ELISA and the commercial AIV ELISA were used to evaluate 448 field sera samples from diseased chickens or vaccinated chickens during the 2003-04 AI outbreak in China. The two ELISA tests had a 95% agreement ratio. We conclude that the NP(47-384)-ELISA developed in our laboratory was specific and sensitive and it has great application potential in China's long-term prevention and control of AI.     

Laboratory decision-making during the classical swine fever epidemic of 1997-1998 in The Netherlands

The National Reference Laboratory for classical swine fever (CSF) virus in the Netherlands examined more than two million samples for CSF virus or serum antibody during the CSF epizootic of 1997–1998. The immense amount of samples and the prevalence of border disease (BD) virus and bovine viral diarrhoea (BVD) virus infections in Dutch pig herds necessitated the diagnostic efforts of the laboratory to be focused on generating CSF specific test results throughout the eradication campaign.

Detection of 82% of the 429 outbreaks was achieved through the combined use of a direct immunofluorescence and peroxidase assay (FAT/IPA) with samples (tonsils) collected from clinically-suspected pigs. This suggests that in the majority of the outbreaks, the pigs had clinical signs that were recognised by the farmer and/or veterinarians, indicating the presence of CSF virus in a pig herd. A positive diagnosis of 74% of all the tissue samples (tonsils) collected at infected pig holdings was established by FAT. More than 140,000 heparinised blood samples were examined by virus isolation, resulting in the detection of 4.5% of the infected herds. CSF virus was isolated in approximately 29% of all the blood samples collected from pigs at infected or suspected farms.

Several serological surveys — each done within a different framework — led to the detection of 13.5% of the total number of outbreaks. The detection of CSF virus antibody in serum was carried out by semi-automated blocking ELISA. Approximately 28.5% of the sera which reacted in the ELISA were classified as CSF virus-neutralising antibody positive and 26.5% as positive for other pestiviruses following the virus neutralisation test (VNT).

We concluded that two of the CSF laboratory diagnostic methods described were determinative in the eradication campaign: first, the FAT for the screening of diseased pigs; and second, the ELISA and VNT when millions of predominantly healthy pigs needed to be screened for the presence of CSF serum antibody. Decision-making on the basis of results generated by either method can, however, be seriously hindered when samples are examined from pig herds with a high prevalence of non-CSF pestiviruses.     

A latex agglutination test for the rapid detection of avian influenza virus subtype H5N1 and its clinical application.

A rapid and simple latex agglutination test (LAT) for the detection of avian influenza virus (AIV) subtype H5N1 in chicken allantoic fluids, tracheal swabs, and tissues was developed. Monoclonal antibodies against the hemagglutinin glycoprotein of H5N1 were covalently coupled onto the surface of carboxylated latex bead using a water-soluble carbodiimide to obtain sensitized latex particles (SLP). These SLPs strongly agglutinated in the presence of allantoic fluid containing H5N1, but not fluids containing other AIV sub-types such as HIN1, H3N2, H4N6, and H9N2. Using this LAT, the virus was detectable in tracheal swabs 24 hours to 30 days after inoculating chickens with H5N1, with detection rates ranging from 45.5 to 79.2%. Much higher rates of detection were obtained from tissues collected postmortem from H5N1 experimentally infected chickens; lung tissue yielded the highest detection rate (96.7%), followed by kidney, spleen, brain, and liver tissues (90%). Lower detection rates were achieved with heart (41.7%) and cloacal tissues (26.8%). When the LAT was compared with other detection methods, the agreement with the viral isolation, H5 antigen immunochromatographic test,and H5 real-time RT-PCR test was 93.97, 95.18, and 87.95%, respectively. The test was highly specific for H5N1 in chickens and water fowls and had sensitivity comparable to other diagnostic tests evaluated.     

Laboratory experience during the classical swine fever virus epizootic in the Netherlands in 1997-1998

From February 1997 till May 1998 the national reference laboratory for classical swine fever (CSF) in the Netherlands was confronted with millions of samples taken from pigs during an outbreak of CSF in a pig dense region. In a limited period major logistic problems needed to be solved regarding the processing of samples and information at the laboratory facilities.In total over 2.3 million samples were examined by different CSF diagnostic methods. The majority (approximately 2.1 million) of these samples were blood samples which were tested for CSF serum antibody in a semi-automated ELISA. Approximately 166,000 samples were examined for the presence of CSF virus or viral antigen. Automated preparation and testing of blood samples for CSF serum antibody, the obligatory identification and registration system of pig holdings and the computerised laboratory management system made it possible to process the huge amount of samples and information presented in a limited period. The majority of the test results was sent to the veterinary authorities via e-mail or a computerised fax system.Of the 429 outbreaks 82% were detected via a direct immunofluorescence technique performed on cryostat sections of the tonsil. The sampling of clinically suspected pigs ('guided' sampling) for this diagnostic method provided rapid positive and negative results and thus played a paramount role during the eradication campaign. Serological surveys identified 13.5% of the infected pig holdings: such surveys proved very effective in the screening of holdings which were subjected to restrictions (protection or surveillance zones) for many months. Virus isolation performed on different types of samples detected 4. 5% of the infected pig holdings.In conclusion, analysis of data collected in the laboratory and epidemiological analysis should result in an improved eradication plan for the future control of outbreaks of CSF in the Netherlands supported by optimised CSF diagnostic methods.     

Non-exfoliative canine cytology: the value of fine needle aspiration and scraping cytology

The results of the cytological and histological examination of 348 canine lesions, localised in various organs, were compared with respect to the tumourous or non-tumour nature of the lesions and the malignancy or benignancy of tumours. The retrieval rate was 92.5%. Regarding the distinction between tumourous and non-tumourous lesions, the cytological examination showed a diagnostic accuracy of 83.9%, a sensitivity of 95.6%, a specificity of 65.4% and a predictive value for the presence of tumour of 93.5%. The diagnostic accuracy of cytology concerning the differentiation in malignancy and benignancy of the neoplasms was 83.7%, with a sensitivity of 86.8%, a specificity of 79.4% and a predictive value for the presence of malignant tumour of 85.6%. These results confirm the value of non-exfoliative cytology as a diagnostic method, providing rapid and valuable information with regard to diagnosis and prognosis and, consequently, for therapeutic handling. An eventual histological diagnosis remains indicated, especially in case of soft-tissue and mammary lesions.     

A comparison of gel diffusion, fluorescent antibody and virus isolation methods in experimental and natural cases of infectious bursal disease.

In studies with chicks inoculated with the Sk-1 strain of infectious bursal agent the bursa of Fabricius was found to be the tissue of choice for virus isolation as well as for use in the fluorescent antibody test and the agar gel diffusion test. In separate experiments positive results were obtained until postinoculation days 3 or 4 by the agar gel diffusion test, 5 or 6 by the fluorescent antibody test and 14 by the virus isolation method, respectively. Bursas from chickens involved in seven natural outbreaks of infectious bursal disease were then examined by these three methods. Virus was isolated from six outbreaks and infectious bursal agent antigen was demonstrated in three by the agar gel diffusion test method and seven (three by direct examination and four after one passage in chicks) by the fluorescent antibody test method. Passage in chicks was required when nonspecific fluorescence complicated the interpretation of fluorescent antibody test results.     

Field trial of six serological tests for bovine brucellosis

Serum agglutination (SAT), complement fixation (CFT), indirect ELISA (iELISA), competitive ELISA (cELISA), Rose Bengal (RBT) and EDTA-modified agglutination (EDTA) tests were used in parallel on serological samples from 19,935 cattle in 301 herds. The study herds were selected according to putative exposure to Brucellaabortus with cases defined by bacteriological culture or test agreement. No single test identified all infected cattle and, at diagnostic thresholds, relative sensitivity was highest in the iELISA (67.9%) or RBT (78.1%), using bacteriological culture or test agreement, respectively, to define cases. As screening tests, the relative sensitivity of the SAT was highest (75.9% by culture or 84.9% by test agreement), with an optimal threshold of 31 IU. The relative specificity of the diagnostic tests ranged from 99.6% (SAT 31IU) to 100% (iELISA, RBT and CFT). The trial confirmed the value of the SAT as a screening test and the value of parallel testing.     

Comparative study of serological methods for the detection of antibodies to porcine reproductive and respiratory syndrome virus.

A comparison was made of serological diagnostic methods used for the detection of antibodies against porcine reproductive and respiratory syndrome (PRRS) virus. In the "phase I" PRRS test panel comparison, a panel of sera collected from 135 pigs of various ages, from North American herds with and without PRRS histories, were sent to 4 different laboratories and tested by an indirect immunofluorescent assay (IFA), an immunoperoxidase monolayer assay (IPMA) and an indirect enzyme-linked immunosorbent assay (iELISA). In the "phase II" PRRS test panel comparison, a panel of 382 sera collected from pigs of various ages, PRRS histories, and from various locations in North America and France, were divided into 2 panels (A & B) and sent to 3 Canadian laboratories and tested by the IFA and iELISA. In the phase I comparison, agreement between the IFA of laboratory 4 and the iELISA and IPMA of laboratory 3 was excellent (kappa values of 95% and 98%, respectively). This contrasted with the poor agreement between these laboratories and the IFA results of laboratories 1 and 2 in the phase I trial. In the phase II comparison, the results demonstrated good agreement between various tests both within and between laboratories. The overall performance of the iELISA was superior in the combination of sensitivity (96.1%) and specificity (100%) relative to the reference classification of the serum samples and repeatability (kappa value 98%). The iELISA is technically superior to IFA and IPMA, time efficient, cost effective and suitable for testing of a large number of samples over a short period of time. Thus, the iELISA may be a better alternative to IFA or IPMA for routine detection of PRRS viral antibodies in swine sera.