Development of molecular methods for identification of Streptococcus bovis from human and ruminal origins

Whitehead, Terence R.

doi:10.1016/s0378-1097(99)00596-0

Cited by 19 publications

(28 citation statements)

References 12 publications

(22 reference statements)

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“…Thus, the possibilities for designing only S. bovisspecific primers were limited, and our primer pair reacted with streptococci of other ecological origin, e.g., with S. infantarius and S. equinus. The close similarity of 16S rDNA sequences within the Streptococcus bovis-Streptococcus equinus complex has also been established in other investigations (8,37), which suggests that less-conserved sequences, such as the intergenic region between 16S and 23S rDNAs, may be necessary for more accurate discrimination between them. In the present study, we assumed that the streptococci of other ecological origin are unlikely to be present in the rumen, but this requires further research.…”

Section: Discussionsupporting

confidence: 64%

“…These primers were rigorously verified with DNA of 19 rumen bacterial species and E. coli ( Table 2). The majority of PCR procedures were highly specific for the target species, except for S. bovis, which cross-reacted with S. equinus (Table 2), which is not surprising, since these strains are likely members of the same species (8,37). The S. ruminantium-M. multiacida primer set amplified the targeted sequences from these two species of bacteria as expected (Table 2).…”

Section: Resultsmentioning

confidence: 66%

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Diet-Dependent Shifts in the Bacterial Population of the Rumen Revealed with Real-Time PCR

Tajima¹,

Aminov

Nagamine³

et al. 2001

Appl Environ Microbiol

595

479

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A set of PCR primers was designed and validated for specific detection and quantification of Prevotella ruminicola, Prevotella albensis, Prevotella bryantii, Fibrobacter succinogenes, Selenomonas ruminantium-Mitsuokella multiacida, Streptococcus bovis, Ruminococcus flavefaciens, Ruminobacter amylophilus, Eubacterium ruminantium, Treponema bryantii, Succinivibrio dextrinosolvens, and Anaerovibrio lipolytica. By using these primers and the real-time PCR technique, the corresponding species in the rumens of cows for which the diet was switched from hay to grain were quantitatively monitored. The dynamics of two fibrolytic bacteria, F. succinogenes and R. flavefaciens, were in agreement with those of earlier, culture-based experiments. The quantity of F. succinogenes DNA, predominant in animals on the hay diet, fell 20-fold on the third day of the switch to a grain diet and further declined on day 28, with a 57-fold reduction in DNA. The R. flavefaciens DNA concentration on day 3 declined to approximately 10% of its initial value in animals on the hay diet and remained at this level on day 28. During the transition period (day 3), the quantities of two ruminal prevotella DNAs increased considerably: that of P. ruminicola increased 7-fold and that of P. bryantii increased 263-fold. On day 28, the quantity of P. ruminicola DNA decreased 3-fold, while P. bryantii DNA was still elevated 10-fold in comparison with the level found in animals on the initial hay diet. The DNA specific for another xylanolytic bacterium, E. ruminantium, dropped 14-fold during the diet switch and was maintained at this level on day 28. The concentration of a rumen spirochete, T. bryantii, decreased less profoundly and stabilized with a sevenfold decline by day 28. The variations in A. lipolytica DNA were not statistically significant. After an initial slight increase in S. dextrinosolvens DNA on day 3, this DNA was not detected at the end of the experiment. S. bovis DNA displayed a 67-fold increase during the transition period on day 3. However, on day 28, it actually declined in comparison with the level in animals on the hay ration. The amount of S. ruminantium-M. multiacida DNA also increased eightfold following the diet switch, but stabilized with only a twofold increase on day 28. The real-time PCR technique also uncovered differential amplification of rumen bacterial templates with the set of universal bacterial primers. This observation may explain why some predominant rumen bacteria have not been detected in PCR-generated 16S ribosomal DNA libraries.The complex symbiotic microbiota of the rumen is responsible for breakdown of plant fiber, an ability the ruminal host animals lack. This microbiota is highly responsive to changes in diet, age, antibiotic use, and the health of the host animal and varies according to geographical location, season, and feeding regimen (reviewed in references 4, 12, 21, and 31). In the early days of rumen microbiology, these changes were monitored by cultivation-based techniques, but limitations inherent in ...

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Section: Discussionsupporting

confidence: 64%

Section: Resultsmentioning

confidence: 66%

Diet-Dependent Shifts in the Bacterial Population of the Rumen Revealed with Real-Time PCR

Tajima¹,

Aminov

Nagamine³

et al. 2001

Appl Environ Microbiol

595

479

View full text Add to dashboard Cite

show abstract

“…Molecular methods are very efficient tools for the development of newly improved diagnosis tests. When methods such as ribotyping are laborious, new methods using PCR based on the 16S or the 23S rDNA region sequences have been successfully applied for the identification of many bacteria (5,7,12,19,23,24,26,31). The major advantages of PCR lay in the possibility of using only nanograms of nucleic acid samples, allowing the elimination of culture, rapidity, and easy analysis.…”

Section: Discussionmentioning

confidence: 99%

Development of a Rapid and Sensitive Test for Identification of Major Pathogens in Bovine Mastitis by PCR

et al. 2001

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Bovine mastitis is the most important source of loss for the dairy industry. A rapid and specific test for the detection of the main pathogens of bovine mastitis is not actually available. Molecular probes reacting in PCR with bacterial DNA from bovine milk, providing direct and rapid detection of Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus parauberis, and Streptococcus uberis, have been developed. Two sets of specific primers were designed for each of these microorganisms and appeared to discriminate close phylogenic bacterial species (e.g., S. agalactiae and S. dysgalactiae). In addition, two sets of universal primers were designed to react as positive controls with all major pathogens of bovine mastitis. The sensitivities of the test using S. aureus DNA extracted from milk with and without a pre-PCR enzymatic lysis step of bacterial cells were compared. The detection limit of the assay was 3.125 ؋ 10 2 CFU/ml of milk when S. aureus DNA was extracted with the pre-PCR enzymatic step compared to 5 ؋ 10 3 CFU/ml of milk in the absence of the pre-PCR enzymatic step. This latter threshold of sensitivity is still compatible with its use as an efficient tool of diagnosis in bovine mastitis, allowing the elimination of expensive reagents. The two PCR tests avoid cumbersome and lengthy cultivation steps, can be performed within hours, and are sensitive, specific, and reliable for the direct detection in milk of the six most prevalent bacteria causing bovine mastitis.Bovine mastitis (BM) is an inflammation of the mammary gland, usually due to a microbial infection (28), which causes North American dairy producers to lose billions of dollars every year. These losses are primarily due to lower milk yields, reduced milk quality, and higher production costs. BM often becomes chronic, and it is important to identify quickly the new clinical cases in order to control infection in the herd. The bacteria responsible for BM can be classified as environmental (Escherichia coli, Streptococcus dysgalactiae, Streptococcus parauberis, and Streptococcus uberis) or contagious (Staphylococcus aureus and Streptococcus agalactiae) depending of their primary reservoir (environment versus infected mammary gland quarter) (11,25).The suitability of a detection method for routine diagnosis depends on several factors, such as specificity, sensitivity, expense, amount of time, and applicability to large numbers of milk samples. The most common but unspecific method (2) to identify potential chronic infections is a somatic cell count: the California Mastitis Test in field conditions and the automated method in the diagnosis laboratory. Currently, the method of identification of the mammary gland pathogens is by in vitro culture, which provides the "gold standard"; however, this technique is labor-intensive and time-consuming. Two other problems can be encountered when these methods of identification are used: first, 2 to 3 days are required to grow, isolate, and identify the pathoge...

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“…identified as having the ability to decarboxylate one or more of the amino acids tested, all were found by sequence analysis to be of the species S. bovis (43). S. bovis has been implicated in the pathogenesis of acute laminitis in the horse, being one of the principal species found to overgrow in the ceca of horses given corn starch to experimentally induce this condition (1).…”

Section: Soc)mentioning

confidence: 99%

Identification of Equine Cecal Bacteria Producing Amines in an In Vitro Model of Carbohydrate Overload

Bailey¹,

Baillon²,

Rycroft

et al. 2003

Appl Environ Microbiol

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Acute laminitis has been associated with the overgrowth of gram-positive bacteria within the equine hindgut, causing the release of factor(s) leading to ischemia-reperfusion of the digits. The products of fermentation which trigger acute laminitis are, as yet, unknown; however, vasoactive amines are possible candidates. The objectives of this study were to use an in vitro model of carbohydrate overload to study the change in populations of cecal streptococci and lactobacilli and to establish whether certain species of these bacteria were capable of producing vasoactive amines from amino acids. Cecal contents from 10 horses were divided into aliquots and incubated anaerobically with either corn starch or inulin (fructan; both at 1 g/100 ml). Samples were taken at 6-h intervals over a 24-h period for enumeration of streptococci, lactobacilli, and gram-negative anaerobes by a dilution method onto standard selective growth media. The effects of the antibiotic virginiamycin (1 mg/100 ml) and calcium hydrogen phosphate (CaHPO 4 ; 0.3 g/100 ml) were also examined. Fermentation of excess carbohydrate was associated with increases in numbers of streptococci and lactobacilli (2-to 3.5-log unit increases; inhibited by virginiamycin) but numbers of gram-negative anaerobes were not significantly affected. A screening agar technique followed by 16S rRNA gene sequence analysis enabled the identification of 26 different bacterial strains capable of producing one or more vasoactive amines. These included members of the species Streptococcus bovis and five different Lactobacillus spp. These data suggest that certain bacteria, whose overgrowth is associated with carbohydrate fermentation, are capable of producing vasoactive amines which may play a role in the pathogenesis of acute laminitis.Fermentation of carbohydrate in the hindgut has been recognized as one of the primary events associated with acute laminitis in the horse (14). The ability of the equine small intestine to digest starch may be overwhelmed if starch is present in excess (Ͼ0.4% body weight [35]), leading to its metabolism by cecal and colonic bacteria and the production of lactic acid. Fructans, the oligosaccharides present in grass which act as the storage form of carbohydrate, do not appear to be digested by the small intestine (21) and so are also substrates for fermentative bacteria. The variation of fructan levels in grasses under different climatic conditions has been suggested to account for the seasonal incidence of acute laminitis (A. C. Longland, A. J. Cairns, and M. O. Humphreys, Proc. 16th Equine Nutr. Physiol. Soc., p. 258-259, 1999).Gram-positive bacteria have been implicated in the pathogenesis of laminitis in the carbohydrate overload model since they have been shown to overgrow in the cecum of horses given corn starch by stomach tube (16). This overgrowth, particularly of streptococci and lactobacilli, was associated with lactic acid accumulation and the death of gram-negative bacteria leading to endotoxin release, both of which were postulated t...

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Development of molecular methods for identification of Streptococcus bovis from human and ruminal origins

Cited by 19 publications

References 12 publications

Diet-Dependent Shifts in the Bacterial Population of the Rumen Revealed with Real-Time PCR

Diet-Dependent Shifts in the Bacterial Population of the Rumen Revealed with Real-Time PCR

Development of a Rapid and Sensitive Test for Identification of Major Pathogens in Bovine Mastitis by PCR

Identification of Equine Cecal Bacteria Producing Amines in an In Vitro Model of Carbohydrate Overload

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