Agreement of conventional microbiological and molecular identification of streptococci isolated from bovine milk (original) (raw)
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Journal of Dairy Science, 2006
The objective of this study was to evaluate a conventional scheme for identifying Streptococcus uberis strains isolated from bovine mastitis. Seventy-five gram-positive, catalase-negative cocci were collected from cows with mastitis from 19 dairy herds located in the east-central region of Argentina. Five American Type Culture Collection strains and bovine isolates were identified by the API 20 Strep system and by restriction fragment length polymorphism analysis of 16S rDNA. A conventional scheme based on 11 biochemical tests was selected for identification of Strep. uberis strains: the Christie-Atkins-Munch-Petersen reaction; hydrolysis of Arg, esculin, and sodium hippurate; growth in inulin, mannitol, raffinose, salicin, and sorbitol; and growth at 45°C and in 6.5% NaCl. Reference strains and 25 bovine isolates were classified accurately to the species level by the conventional scheme in a blind assay. Each reference strain and each bovine isolate were identified as belonging to the same species following these 3 methods. The remaining 50 isolates identified as Strep. uberis by the API 20 Strep system and 16S rDNA RFLP were assayed by the conventional scheme. This scheme correctly identified 47 (94%) of 50 isolates as Strep. uberis by comparing their biochemical profile with that of the reference strain. Three (6%) of the 50 isolates were classified as Strep. uberis by the API 20 Strep system and by 16S rDNA RFLP and were identified as Enterococcus faecalis by the conventional scheme. Thirty percent of the Strep. uberis strains showed biochemical profiles identical to the Strep. uberis American Type Culture Collection 27958 strain. Seventy percent of the Strep. uberis strains demonstrated variability compared with the reference strain, resulting in 19 different biochemical profiles. The conventional scheme proposed in this study resulted in a relatively low number of misidentifications and could 3886 biochemically identify not only typical, but also atypical Strep. uberis strains. This conventional scheme can be considered an adequate method for identifying Strep. uberis strains isolated from bovine mastitis because of its affordable cost in developing countries, and it may contribute to determining the frequency of isolation of Strep. uberis strains in Argentinean dairy herds.
Identification of Streptococcus canis Isolated from Milk of Dairy Cows with Subclinical Mastitis
Journal of Clinical Microbiology, 2005
Streptococcus canis was isolated from 31 milk samples from 11 cows in a dairy herd (with 49 lactating cows) affected by subclinical mastitis in north Rhine-Westphalia, Germany. Thirty-one isolates from the infected udder quarters were further characterized for their phenotypic and molecular properties. Most isolates (83.9%) produced α-galactosidase, and all were negative for β- d -glucuronidase. Amplification of the 16S rRNA gene by the PCR method and digestion with the restriction enzymes RsaI, MspI, and AvaII yielded species-specific patterns. Additional identification by species-specific amplification of the 16S rRNA gene, the 16S-23S rRNA gene intergenic spacer region, the CAMP factor-encoding gene cfg , and the internal fragments of the sodA gene was consistent with S. canis . Macrorestriction analysis of the chromosomal DNA by pulsed-field gel electrophoresis showed that the S. canis isolates originated from a single clone or were very closely related.
Laboratory Differentiation between Streptococcus Species Isolated from Different Sources
Alexandria Journal of Veterinary Sciences, 2014
Streptococcusa Galactia streptococcus pyogenes Milk, This study was planned to throw light on the most important Streptococcus bacteria that isolated from the milk and pus and the most important laboratory tests that used in differentiation between the streptococcal bacterial species. This study was carried-out on a total number of 100 random samples of milk which were collected from different areas at Behera Governorate.. Also, 100 pus samples from closed abscesses from different parts of cattle body where collected and examined bacteriologically for streptococcus introduction. Our results concluded that, the streptococcus agalactiae and streptococcus pyogenes are the most important bacterial isolates that causes severe losses to milk industry, also the streptococcus pyogenes of zoonotic importance as it transmitted to human. The catalase test, oxidatse test and hemolytic test are the main tests used for diagnosis and differentiation between streptococcus agalactiae and streptococcus pyogenes. The bacitracin sensitivity test is main test for differentiation between streptococcus agalactiae and streptococcus pyogenes The results also indicated that the level of streptococcus agalactiae in milk higher than that of streptococcus pyogenes and reached to 7 % and 3 % for streptococcus agalctiae and pyogenes, while, in pus its levels reached to 0 and 40 % for streptococcus agalactiae and pyogenes in pus. Also the results cleared that the PCR method for detection of mastitis considered as the best method
Frontiers in Veterinary Science, 2023
Introduction: Streptococci are the major etiology in mastitis in dairy cattle, a cause of huge economic losses in the dairy industries. This study was aimed to determine the diversity of Streptococcus spp. isolated from clinical mastitis of cattle reared in Bangladesh. Methods: A total of 843 lactating cattle reared in four prominent dairy farms and one dairy community were purposively included in this study where 80 cattle were positive to clinical mastitis (CM) based on gross changes in the udder (redness, swelling, and sensitive udder) and/or milk (flakes and/or clots). Milk samples were collected from all the eighty cattle with clinical mastitis (CCM) and twenty five apparently healthy cattle (AHC). Samples were enriched in Luria Bertani broth (LB) and one hundred microliter of the enrichment culture was spread onto selective media for the isolation of Staphylococcus spp., Streptococcus spp., Enterococcus spp., Escherichia coli and Corynebacterium spp., the major pathogen associated with mastitis. Isolates recovered from culture were further confirmed by species specific PCR. Results and Discussion: Out of 105 samples examined 56.2% (59/105), 17.14% (18/105), 9.52% (10/105) and 22.9% (24/105) samples were positive for Staphylococcus, Streptococcus, Enterococcus faecalis and E. coli, respectively. This study was then directed to the determination of diversity of Streptococcus spp. through the sequencing of 16S rRNA. A total of eighteen of the samples from CCM (22.5%) but none from the AHC were positive for Streptococcus spp. by cultural and molecular examination. Sequencing and phylogenetic analysis of 16S rRNA identified 55.6, 33.3, 5.6 and 5.6% of the Streptococcus isolates as Streptococcus uberis, Streptococcus agalactiae, Streptococcus hyovaginalis and Streptococcus urinalis, respectively. Considering the high prevalence and worldwide increasing trend of S. uberis in mastitis, in-depth molecular characterization of S. uberis was performed through whole genome sequencing. Five of the S. uberis strain isolated in this study were subjected to WGS and on analysis two novel ST types of S. uberis were identified, indicating the presence of at least two different genotypes of S. uberis in the study areas. On virulence profiling, all the isolates harbored at least 35 virulence and putative virulence genes probably associated with intramammary infection (IMI) indicating all the S. uberis isolated in this study are potential mastitis pathogen. Overall findings
Molecular Detection of Streptococcus Species Isolated from Cows with Mastitis
Streptococcal mastitis is considered as one of the most common infectious diseases in the dairy cattle, which threatens the dairy industry all over the world. The aim of this study was to determine the prevalence of Streptococcus species in mastitic cows with molecular investigation to detect the presence of some virulence genes of the recovered isolates by PCR. A total of 150 milk samples were collected from dairy cattle with clinical and subclinical mastitis from different areas in El-Gharbia governorate, Egypt. Streptococcus species were isolated with an incidence of 38%. S. agalactiae, S. dysgalactiae, S. uberis, S. pyogenes, S. pneumoniae and S. faecalis were isolated from the milk samples of the examined cows with the percentage of 14.7%, 6%, 9.3%, 4.7%, 1.3% and 2%, respectively. Molecular investigation of virulence associated genes revealed that sip, cfb and bca genes of S. agalactiae were found with the percentage of 77.7%, 88.8% and 33.3%, respectively. The mig gene of S. dysgalactiae was found with an incidence of 77.8%. Of the examined S. uberis isolates, 55.5%, 22.2% and 33.3% were carrying the cfu, oppF and has A genes, respectively. The present study revealed the prevalence of Streptococci and distribution of virulence associated genes among the isolates. The high frequency of virulence genes in the isolates suggests an important role of these virulence genes in the pathogenesis of Streptococci in cattle mastitis.
Journal of Veterinary Science, 2003
In the present study 130 S. uberis strains and one S. parauberis strain isolated from bovine milk samples of 58 different farms of various locations in Hesse, Germany, as well as two reference strains of each species were comparatively investigated for cultural, biochemical, serological and molecular properties. All S. uberis strains produced the enzyme β-D-glucuronidase, while the S. parauberis strains were negative. The S. uberis and S. parauberis 16S rRNA genes were amplified by polymerase chain reaction and subsequently digested with the restriction enzymes RsaI and AvaII yielding speciesspecific restriction patterns. Both species were additionally identified by amplifying species-specific parts of the genes encoding the 16S rRNA, the 23S rRNA and the 16S-23S rDNA intergenic spacer region, respectively. The CAMP factor gene cfu, a potential virulence factor of S. uberis, was amplified, corresponding to a phenotypically positive CAMP-reaction, using cfu-specific oligonucleotide primers. In addition the streptokinase/ plasminogen activator encoding genes skc/pauA, a second potential virulence factor, could be amplified for 126 of the 130 S. uberis but not for S. parauberis. A DNA fingerprinting of S. uberis strains, performed by macrorestriction analysis of their chromosomal DNA by pulsed-field gel electrophoresis, revealed that most of the isolates were not related to each other. However, identical DNA patterns were noted for some of the isolates within different quarters of an individual cow and also for different cows within the same farm. The generally unrelated DNA patterns indicated that S. uberis is a pathogen with multiple environmental habitats and that infections are caused by a great variety of strains.
Small Ruminant Research, 2019
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Mastitis is a common, serious and easily disseminated infection in dairy herds and constitutes a major cause for economic losses worldwide. Among incriminated pathogens, streptococci are considered as common relevant agents. Unfortunately, the routine diagnosis of streptococcal infections via phenotypic and serological methods is a difficult and time-consuming process. 16S rRNA gene sequencing is well established extensively used as a universal gold standard method for the microbial identification and phylogenetic classification of prokaryotic species, genera, and families. In this sense, a comparative identification of 24 isolates of mastitic origin previously recognized as Streptococcus sp. via automated biochemical VITEK-2 system, has been done against the gold standard, 16S rRNA gene sequencing. The identification of the most relevant Streptococcus spp. involved in bovine mastitis has not shown high accuracy and reliability by VITEK-2 system as obtained by 16S rRNA gene sequenci...