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.     

Use of polymerase chain reaction for the differentiation of Group A bovine rotavirus G6, G8, and G10 genotypes in the North Island of New Zealand

AIM: To determine the presence of Group A rotavirus G6, G8, and G10 genotypes in calves in the North Island of New Zealand. METHODS: Faecal samples from 730 calves (<6 weeks old) with diarrhoea were collected during 2006 and 2007 from seven regions in the North Island of New Zealand. The samples were screened for the presence of Group A rotavirus antigen, using a commercial ELISA. Forty-one samples from different farms were randomly selected out of the 385 ELISA-positive samples and tested using PCR for the presence of G6, G8, and G10 genotypes of rotavirus. RESULTS: The PCR analysis of 41 antigen-positive field samples revealed that 37 (90%) contained genotype G6, three (7%) genotype G10, one sample (2%) had both G6 and G10 genotypes, and none contained genotype G8. CONCLUSIONS: Rotavirus genotype G6 was the predominant genotype found in this preliminary study and was present in all seven regions studied. Genotype G10 was also found in some regions of the North Island, whereas genotype G8 was not found in any sample. CLINICAL RELEVANCE: This is the first report on rotavirus G genotypes present in calves in the North Island of New Zealand.     

A serotype-specific polymerase chain reaction for identification of Pasteurella multocida serotype 1

A serotype-specific polymerase chain reaction (PCR) assay was developed for detection and identification of Pasteurella multocida serotype 1, the causative agent of avian cholera in wild waterfowl. Arbitrarily primed PCR was used to detect DNA fragments that distinguish serotype 1 from the other 15 serotypes of P. multocida (with the exception of serotype 14). Oligonucleotide primers were constructed from these sequences, and a PCR assay was optimized and evaluated. PCR reactions consistently resulted in amplification products with reference strains 1 and 14 and all other serotype 1 strains tested, with cell numbers as low as 2.3 cells/ml. No amplification products were produced with other P. multocida serotypes or any other bacterial species tested. To compare the sensitivity and further test the specificity of this PCR assay with traditional culturing and serotyping techniques, tissue samples from 84 Pekin ducks inoculated with field strains of P. multocida and 54 wild lesser snow geese collected during an avian cholera outbreak were provided by other investigators working on avian cholera. PCR was as sensitive (58/64) as routine isolation (52/64) in detecting and identifying P. multocida serotype 1 from the livers of inoculated Pekins that became sick or died from avian cholera. No product was amplified from tissues of 20 other Pekin ducks that received serotypes other than type 1 (serotype 3, 12 x 3, or 10) or 12 control birds. Of the 54 snow geese necropsied and tested for P. multocida, our PCR detected and identified the bacteria from 44 compared with 45 by direct isolation. The serotype-specific PCR we developed was much faster and less labor intensive than traditional culturing and serotyping procedures and could result in diagnosis of serotype 1 pasteurellosis within 24 hr of specimen submission.     

Re: Use of polymerase chain reaction for the differentiation of Group A bovine rotavirus G6, G8, and G10 genotypes in the North Island of New Zealand

Extract

In the paper by L Howe, H Sugiarto and RA Squires published in the New Zealand Veterinary Journal 56, 218–221, 2008, entitled, “Use of polymerase chain reaction for the differentiation of Group A bovine rotavirus G6, G8, and G10 genotypes in the North Island of New Zealand”, the authors concluded that “Currently, the rotavirus vaccine available in New Zealand contains only the G6 strain, and may not provide optimal protection for animals infected with G10 rotavirus. … This information may be of assistance in the development and optimisation of vaccines, and enhancement of effective control strategies.”     

Multiplex polymerase chain reaction used for bovine embryo sex determination

Widely used bovine sexing primers were compared in terms of suitability in determining the sex of bovine embryos. Under optimized multiplex PCR conditions, the ConBV/ConEY couple primers did not show accurate results when combined together in multiplex PCR, but worked well when the couple primers were used separately. The S4BF/S4BR primers showed accurate results; however, some unexpected bands were detected. When the BY/BSP couple primers were used to determine one-cell, two-cell, four-cell and eight-cell stage embryos of known sexed SCNT-derived embryos, the results showed 100% accuracy. The BY/BSP couple primers were also able to identify the sex of one-cell and two-cell IVF-derived embryos.     

Genotypic characterization by polymerase chain reaction of Staphylococcus aureus isolates associated with bovine mastitis

Staphylococcus aureus is recognized worldwide as a pathogen causing many serious diseases in humans and animals, and is the most common aetiological agent of clinical and subclinical bovine mastitis. The importance of evaluating the combination of S. aureus virulence factors has been emphasized both in human and veterinary medicine, and knowledge about the genetic variability within different S. aureus populations would help in the design of efficient treatments. The aim of the present study was to determine the genetic profiles of S. aureus strains isolated from milk of cows suffering from clinical and subclinical mastitis in Belgium. The presence of about forty virulence-associated genes was investigated by specific polymerase chain reaction (PCR) amplification. A high number of genotypic subtypes were observed, demonstrating further the large variation in the presence of virulence genes in S. aureus isolates and the considerable diversity of strains populations that are able to cause mastitis in cows. In accordance with other studies, we showed that some genes are associated with mastitis-causing S. aureus isolates, whereas others are absent or rarely present. We also further highlighted the presence of conserved gene combinations, namely the enterotoxigenic egc-cluster and the bovine pathogenicity island SaPIbov. Importantly, the presence of isolates carrying genes coding for toxins involved in important human infections makes the milk of cows with mastitis a potential reservoir for these toxins, and therefore a potential danger in human health, which strengthens the importance to consider raw milk consumption and its processing very carefully.     

Analysis of methanogens in the bovine rumen by polymerase chain reaction single-strand conformation polymorphism

The polymerase chain reaction single‐strand conformation polymorphism (PCR‐SSCP) method reported by Schwieger and Tebbe (1998) was used to analyze the diversity of methanogens inhabiting the rumen. Partial 16S rRNA gene fragments were amplified from DNA extracted from rumen contents by PCR with archaea‐specific primers, Ar1000F and Ar1500R, or methanogen‐specific primers, M301F and M915R, with one primer phosphorylated at the 5′ end. The amplified DNA fragments were analyzed by SSCP gel electrophoresis after the phosphorylated strands of the PCR products were digested with λ exonuclease. When we analyzed samples collected from the six Holstein cows used in a previous study, in which cows were given feed with or without α‐cyclodextrin‐horseradish oil complex (CD‐HR), nine and six bands were identified in the profiles generated by PCR products amplified with archaea‐specific and methanogen‐specific primers, respectively. While dendrogram analysis based on SSCP gel profiles found that the methanogens from each rumen showed a particular composition of methanogens, the profiles of the methanogens isolated from two of three cows fed with CD‐HR fell into the same branch in the dendrogram constructed from the profiles. Therefore, this study demonstrates the potential of the PCR‐SSCP method in the methanogenic community analysis of the rumen and in investigating changes in the methanogenic community due to the addition of CD‐HR to the rumen.     

Development of a quantitative-competitive polymerase chain reaction assay for serotype 1 Marek's disease virus

We have developed a quantitative-competitive (QC) polymerase chain reaction (PCR) for the detection of Marek's disease virus (MDV) DNA. The assay utilizes a competitor DNA that differs from the viral DNA of interest by having a small insertion. The competitor DNA acts as an internal standard for the estimation of viral DNA in an unknown sample. The amount of viral DNA in a sample is quantitated by coamplification in the presence of a known amount of competitor DNA. The same PCR primers that amplify the viral DNA also amplify the competitor DNA. When the amount of competitor is equal to the amount of viral DNA in a sample, there is equal amplification of the competitor and the virus. Thus, we are able to quantitate the viral DNA in an unknown sample. To establish the utility of this assay, in vivo correlations between virulence and virus replication were studied. Our data demonstrated that a more virulent strain of MDV (648A) replicated better in thymus during cytolytic infection than did a less virulent strain (GA). However, no differences in virus titer were observed when these two viruses were propagated in tissue culture. Our data are consistent with the generally held idea that "hot" strains of MDV replicate earlier and better in birds. Thus, QC-PCR is extremely specific and sensitive to measure MDV DNA over a wide range and can be applied to in vivo studies of viral pathogenesis.     

PCR技术检测饲料中沙门氏菌的应用研究

用聚合酶链反应(PCR)技术检测饲料中沙门氏菌,对已知被沙门氏菌污染的动物饲料培养物均能检出阳性,说明本方法对检测饲料中沙门氏菌具有特异性高、灵敏、快速等特点,适用于快速、准确地检测饲料中沙门氏菌的需要。     

Genetic and serological characterization of novel serotype G8 bovine group A rotavirus strains isolated in Japan

G8 bovine group A rotaviruses isolated in Japan were genetically and serologically characterized. The VP7 gene nucleotide and amino acid sequences revealed high identity with each other. All Japanese G8 strains were classified into the same lineage in the phylogenetic analysis based on VP7 gene sequences. Antisera to four Japanese G8 strains neutralized other G8 strains, but their neutralizing titers were between 8-fold lower and 2-fold higher than homologous strains. These results suggest that the VP7s of Japanese G8 strains have similar genetic and serologic characteristics. Observed differences in the neutralizing abilities of antisera for each strain appear to depend on differences in the P serotypes/genotypes.     

Detection of avian adenovirus by polymerase chain reaction

An avian adenovirus-specific polymerase chain reaction was developed. The origin of primers was from the DNA sequence data of the chicken embryo lethal orphan avian adenovirus virus genome. An avian adenovirus-specific 421-bp DNA product was amplified by these primers from group I of adenovirus containing 12 serotypes and serotypes of adenovirus from group II and group III. The adenovirus-specific DNA product was also amplified from the 19 field isolates of avian adenoviruses but not from the mammalian adenovirus and other avian pathogenic viruses and bacteria. As little as 1 fg of avian adenovirus DNA was detected by gel electrophoresis and Southern blot analysis.     

Detection of pigeon circovirus by polymerase chain reaction

Pigeon circovirus was identified by polymerase chain reaction (PCR) in young pigeons belonging to 12 different lofts. Viral DNA was extracted from formalin-fed, paraffin-imbedded tissues containing primarily bursa and occasionally liver and spleen with a commercial kit. PCR primers were selected from a published sequence for columbid circovirus and evaluated in a PCR assay. The histopathologic examination of various tissues revealed basophilic globular intracytoplasmic inclusions in the mononuclear cells of the bursa of Fabricius and occasionally in the spleen characteristic for a circovirus. Transmission electron microscopy of a few bursas of Fabricius revealed virus particles measuring 18-21 nm. All the samples were negative by PCR for psittacine beak and feather disease (PBFD) virus and chicken infectious anemia virus. The primers for both pigeon circovirus and PBFD virus did not react in PCR with the chicken anemia virus DNA. Most of the circovirus-infected pigeons had concurrent infections of Escherichia coli, Salmonella, Pasteurella, Aspergillus, candidiasis, nematodiasis, or capillariasis.     

The frequent detection of a treponeme in bovine digital dermatitis by immunocytochemistry and polymerase chain reaction

A study was carried out to determine whether spirochaetes are frequently associated with digital dermatitis in United Kingdom (UK) dairy cattle. Histopathological examination of lesions using a silver stain showed a large number of unidentified spirochaete-like organisms present in digital dermatitis hoof skin tissue in all examined biopsies. Immunocytochemical staining demonstrated that spirochaetes in skin lesions were identified by polyclonal antisera to Borrelia burgdorferi, Treponema denticola and Treponema vincentii (again all biopsies were positively stained), whereas monoclonal antibodies to B. burgdorferi and any Treponema pallidum did not stain any organisms in all biopsies. A PCR of 16S rRNA, previously shown to be specific for a new treponeme, was employed and produced positive results from 82.4% of digital dermatitis tissues. It is concluded that this spirochaete (or related spirochaetes), which is similar to human oral treponemes, is frequently associated with, and may be responsible for, pathological changes in digital dermatitis.     

Identification of duck plague virus by polymerase chain reaction

A polymerase chain reaction (PCR) assay was developed for detecting duck plague virus. A 765-bp EcoRI fragment cloned from the genome of the duck plague vaccine (DP-VAC) virus was sequenced for PCR primer development. The fragment sequence was found by GenBank alignment searches to be similar to the 3' ends of an undefined open reading frame and the gene for DNA polymerase protein in other herpesviruses. Three of four primers sets were found to be specific for the DP-VAC virus and 100% (7/7) of field isolates but did not amplify DNA from inclusion body disease of cranes virus. The specificity of one primer set was tested with genome templates from other avian herpesviruses, including those from a golden eagle, bald eagle, great horned owl, snowy owl, peregrine falcon, prairie falcon, pigeon, psittacine, and chicken (infectious laryngotracheitis), but amplicons were not produced. Hence, this PCR test is highly specific for duck plague virus DNA. Two primer sets were able to detect 1 fg of DNA from the duck plague vaccine strain, equivalent to five genome copies. In addition, the ratio of tissue culture infectious doses to genome copies of duck plague vaccine virus from infected duck embryo cells was determined to be 1:100, making the PCR assay 20 times more sensitive than tissue culture for detecting duck plague virus. The speed, sensitivity, and specificity of this PCR provide a greatly improved diagnostic and research tool for studying the epizootiology of duck plague.