Detection of chicken anaemia virus DNA from formalin-fixed tissues by polymerase chain reaction

Chicken anaemia virus was detectable from various samples such as cell-free virus, infected cells, unfixed liver homogenates, formalin-fixed liver homogenate or formalin-fixed paraffin-embedded ( ) tissues from experimental or field infected chicks using assay. The detection limit of the first PCR assay was 1 infected cell or 10^−1·5, ₅₀ of cell-free virus (strain A2). The nested assay increased the sensitivity 10- or 100-fold. was detectable in the other 14 Japanese strains isolated from 1976 to 1994 by the assay. All the amplified products were digested with BglII, HindIII, PstI and SacI. These results suggest that the region amplified was highly conserved among the strains. The nested assay was very sensitive. However, was detectable in most field samples using the first assay. Therefore, the nested assay may not always be necessary. In contrast, the nested PCR assay was necessary to detect in tissues or formalin-fixed material. Use of the assay in detection from tissues may be most valuable in diagnosis of diseases caused by or associated with , because it allows detection of both microscopic lesions and .     

Feline leukemia virus detection by immunohistochemistry and polymerase chain reaction in formalin-fixed, paraffin-embedded tumor tissue from cats with lymphosarcoma.

The prevalence of feline leukemia virus (FeLV) antigen and DNA was assessed in formalin-fixed, paraffin-embedded tumor tissues from 70 cats with lymphosarcoma (LSA). Tissue sections were tested for FeLV gp70 antigen using avidinbiotin complex (ABC) immunohistochemistry (IHC); DNA was extracted and purified from the same tissue blocks for polymerase chain reaction (PCR) amplification of a 166 base pair region of the FeLV long terminal repeat (LTR). Results were related to antemortem FeLV enzyme-linked immunosorbent assay (ELISA) for serum p27 antigen, anatomic site of LSA, and patient age. Viral DNA was detected by PCR in 80% of cases and viral antigen by IHC in 57% of cases. Seventeen cases were PCR-positive and IHC-negative; one case was PCR-negative and IHC-positive. Clinical records included FeLV ELISA results for 30 of 70 cats. All 19 ELISA-positive cats were positive by PCR and IHC; of the 11 ELISA-negative cats that were negative by IHC, seven were positive by PCR. When evaluated according to anatomic site, FeLV DNA and antigen were detected less frequently in intestinal LSAs than in multicentric and mediastinal tumors. Lymphosarcoma tissues from cats < 7 yr were several fold more likely to be positive for FeLV antigen by IHC than were tumors from cats > or = 7 yr. However, there was no significant difference in PCR detection of FeLV provirus between LSAs from cats < 7 yr and those > or = 7 yr.(ABSTRACT TRUNCATED AT 250 WORDS)     

An atypical case of lymphosarcoma (sporadic bovine leukosis) in a heifer.

An 18-month-old Friesian heifer had several unusual, raised, black, cutaneous plaques, some of which were up to 20 cm in diameter, on its head and neck, limbs, thorax and perineum. There was also generalised lymphadenopathy. A clinical diagnosis of lymphosarcoma (sporadic bovine leukosis) was derived from a fine needle aspiration of a skin lesion. Post mortem and histological examinations confirmed a multicentric lymphosarcoma with widespread infiltration into many of the tissues recognised as predilection sites for this type of tumour. However, in the authors' experience, the presence of tumour masses in the trachea and the right mainstem bronchus was atypical.     

Atypical sporadic lymphosarcoma in a 7-month-old Holstein heifer.

A 7-month-old calf with a firm, diffuse infiltration of the left hind limb with sciatic nerve motor deficit was presented. The cytology indicated a malignant, round cell tumor and at necropsy, tissues were positive to a Kappa-lambda immunohistochemistry test. The final diagnosis was sporadic bovine leukosis, juvenile form.     

Detection of leptospires in bovine semen by polymerase chain reaction

OBJECTIVE: In view of the considerable importance of venereal transmission of bovine leptospirosis, the objective of the present study was to compare the polymerase chain reaction (PCR), culture/isolation and serology to detect leptospire infection in bovine semen. DESIGN: Blood for serologic examination and semen for bacterial culture and PCR were collected from 20 bulls at artificial insemination centres in Brazil. Each animal was sampled twice for serology. RESULT: Forty-five percent (9/20) of the serum samples collected showed agglutinin titers to serovar hardjo in the first sample and 25% (5/20) had agglutinin titers to serovar hardjo in the second sample. Eighty percent (16/20) of semen samples were positive by PCR. Leptospires could not be isolated from any of the semen samples examined. CONCLUSION: Polymerase chain reaction can be a method of great potential for the detection of leptospires at artificial insemination centres.     

Detection of bovine leukemia virus in blood and milk by nested and real-time polymerase chain reactions.

Concerns about retroviruses in livestock and products derived from them have necessitated the development of tests to detect the bovine leukemia virus (BLV) in blood and milk from cattle. Dairy cattle (n = 101) from 5 different geographical areas were used for this study. A nested polymerase chain reaction (PCR) identified 98% of BLV seropositive cattle (n = 80) from blood and 65% from milk, whereas real-time PCR detected 94% of BLV seropositive cattle from blood and 59% from milk. Bovine leukemia virus was also detected by PCR in approximately 10% of seronegative cattle (n = 21), most likely because of early detection before seroconversion.     

Direct detection of bovine leukemia virus infection: practical applicability of a double polymerase chain reaction.

A double polymerase chain reaction (PCR) assay has been devised for the direct detection of bovine leukemia virus (BLV). The assay was directly performed on blood leukocytes, avoiding the DNA-purification procedures. The PCR products were identified by gel-electrophoresis and the specificity of the test was confirmed by hybridization with a biotinylated oligonucleotide probe. When testing the sensitivity of PCR, less than eight genome copies of the provirus were detected in the background of two million negative lymphocytes. In a BLV infected herd 22 animals of various age groups were examined by the indirect (serological) diagnostic tests of agar-gel immunodiffusion and indirect ELISA as well as by the direct detection method of PCR. The tests were repeated at monthly intervals on five occasions. When examining the specimens from cows and heifers, a close agreement was found between the results of the various methods. The newborn calves, which were the offspring of BLV infected mothers, were consequently negative in PCR throughout the experimental period. However, in the indirect tests the calves were positive during the first samplings and became negative only around four months of age. Since the indirect tests can not discriminate infection from colostral immunity, PCR proved to be a useful complementary assay for the safe diagnosis of BLV infection in young calves.     

Detection of infectious laryngotracheitis virus in formalin-fixed, paraffin-embedded tissues by nested polymerase chain reaction

Infectious laryngotracheitis virus (ILTV) is routinely diagnosed by histopathologic examination of trachea, eyelid, and lung tissues. Lesions consistent with infectious laryngotracheitis (ILT) infection include syncytial cell formation with intranuclear inclusion bodies. These changes are present during the acute phase of infection. To increase the sensitivity of detecting ILT, a nested polymerase chain reaction (PCR) was developed for detection of ILTV DNA. Nested PCR assay was specific for the amplification of ILTV DNA and did not amplify a variety of other avian pathogens. To further validate the ability of this assay to detect ILT, nested PCR was performed in formalin-fixed, paraffin-embedded tissues from 35 cases of respiratory disease. Of the 35 cases, 12 were considered ILT suspects on the basis of initial clinical observation. Eleven of the 12 ILT-suspect cases were diagnosed as ILT, and the remaining 24 were diagnosed as nonspecific tracheitis (NST) by histopathologic examination. Histopathologically positive samples were confirmed by direct fluorescent antibody test and virus isolation. Of the 11 ILT-positive cases, 10 were positive by nested PCR. In addition, ILTV DNA was detected in 7 of the 24 samples diagnosed as NST upon histopathologic examination. Therefore, by nested PCR, ILTV DNA was detected in tissues independently of the presence of syncytial cells, intranuclear inclusions, or both. ILT nested PCR is a specific and sensitive assay capable of detecting ILT at different stages of infection and can be utilized in combination with histopathological examination to accelerate the diagnosis of ILT infection.     

Detection of Mycobacterium paratuberculosis in formalin-fixed paraffin-embedded tissues by the polymerase chain reaction.

The polymerase chain reaction (PCR) utilizing primers specific for the IS900 sequence of Mycobacterium paratuberculosis was applied to tissue sections of formalin-fixed, paraffin-embedded ileum from cattle with Johne's disease and the results compared to those obtained with acid-fast (Ziehl-Neelsen) and immunohistochemical staining. The PCR was positive in 19/21 tissues (90%) while Ziehl-Neelsen staining was positive in 18/21 (86%) and immunohistochemical staining in 21/21 (100%). The Ziehl-Neelsen and immunohistochemical stains are not, however, specific for M. paratuberculosis. The PCR for detection of M. paratuberculosis using the IS900 sequence is a specific and relatively sensitive method for confirmation of Johne's disease and its application to formalin-fixed, paraffin-embedded tissues may be useful for confirmation of dubious cases, for retrospective studies and for epidemiological analyses.     

Detection and identification of Mycoplasma from bovine mastitis infections using a nested polymerase chain reaction.

A polymerase chain reaction (PCR) test was compared with culture for the detection and diagnosis of bovine Mycoplasma intramammary infection. The PCR test was applied to 24-hour Mycoplasma enrichment cultures of milk from cows with suspected mastitis and from bulk tank milk. In comparison to culture, the sensitivity and specificity of the PCR method were 96.2% and 99.1% for individual cow milk and 100% and 99.8% for the bulk tank milk, respectively. However, in discrepant cases where PCR was positive and culture was negative, the PCR test was correct; subsequent PCR tests and culturing of the individual cow's milk yielded positive results. The PCR test simultaneously detected and differentiated among 11 bovine Mycoplasma species.     

套式RT-PCR检测牛呼吸道合胞体病毒的研究

为了检测牛呼吸道合胞体病毒(BRSV),根据已发表的融合蛋白(F)基因序列,设计了套式RT-PCR引物,初步建立了BRSV的套式RT-PCR检测方法。对138份牛鼻腔棉拭子进行了检测,结果检测到了BRSV阳性样品54份,总的阳性检出率为39.1%。对大部分BRSV阳性样品的F基因扩增产物进行了序列测定与分析。用该套式RT-PCR对牛传染性鼻气管炎病毒、牛副流感病毒3型、牛病毒性腹泻病毒等进行了检测,结果无交叉反应,表明该检测方法具有良好的特异性。本研究首次用套式RT-PCR技术证实了我国部分省的牛群中存在BRSV感染。     

Detection of bovine viral diarrhea virus, using degenerate oligonucleotide primers and the polymerase chain reaction.

A technique for detection of bovine viral diarrhea virus (BVDV) from circulating blood leukocytes, using the polymerase chain reaction, is described. The published nucleotide sequences of 2 strains of BVDV and that of hog cholera virus were aligned and the information was used to design oligonucleotides coding for 2 regions of amino acid homology. The oligonucleotides were a mixed population including all possible codons for the conserved amino acids. These degenerate oligonucleotides were used in the polymerase chain reaction to detect viral RNA in cells infected in vitro, or in circulating blood leukocytes from infected animals. Virus was detected in over 60 samples from diverse isolates. The detection of BVDV by the polymerase chain reaction is a rapid, sensitive, and specific technique, which represents an improvement over existing technology.     

Detection of Mycoplasma mycoides subspecies mycoides in tissues from an outbreak of contagious bovine pleuropneumonia by culture, immunohistochemistry and polymerase chain reaction

Postmortem observations of 37 cattle from an outbreak of contagious bovine pleuropneumonia (CBPP) in north Italy in 1993 were made at the abattoir, where samples of lung and tracheobronchial lymph node tissues were taken for culture and identification of Mycoplasma mycoides subspecies mycoides (MmmSC), immunohistochemistry with the peroxidase anti-peroxidase (PAP) system, and molecular detection by the polymerase chain reaction (PCR) amplification of specific DNA from MmmSC. Nasal swabs were also taken for testing by PCR Lung pathology typical of CBPP was observed in 38 per cent of the animals, and MmmSC was isolated from 19 per cent DNA of MmmSC was detected by PCR in 64 per cent of lung samples and 35 per cent of the nasal swabs. Staining of lung tissue and lymph node tissue by PAP was positive in 27 per cent and 30 per cent of cases, respectively, and was a useful back-up test. These results suggest that PCR amplification from lung tissue may be used as a rapid and accurate confirmatory test for cases with pathology resembling CBPP.