scholarly journals A Novel Polymerase Chain Reaction Assay for the Detection of Seven Mycoplasma Species of Cattle Origin

2021 ◽  
Author(s):  
Jyhmirn Lai ◽  
Hengching Lin ◽  
Peichuan Hsu ◽  
Satoshi Gondaira ◽  
Hidetoshi Higuchi ◽  
...  
Keyword(s):  
Annealing Temperature ◽  
Bovine Mastitis ◽  
High Sensitivity ◽  
Mycoplasma Species ◽  
Clinical Settings ◽  
Coagulase Negative Staphylococci ◽  
Streptococcus Uberis ◽  
The Common ◽  
Polymerase Chain ◽  
Mastitis Pathogens

Abstract The aim of this study was to develop a pair of primers for detecting ruminant mycoplasma pathogens. We designed a set of primers based on the most similar sequences within 16sRNA regions of seven Mycoplasma spp. These primers have high sensitivity for detecting Mycoplasma dispar, M. arginine, M. canadense, M. bovis, M. alkalescens, M. californicum, and M. bovisgenitalium within the annealing temperature range of 46°C to 48°C. The minimum amount of DNA that can be detected using the protocol is 250 ng, which is equivalent to 2,000 cfu/mL. The primers can detect mycoplasma from DNA extracted directly from milk samples. The common bovine mastitis pathogens of Staphylococcus aureus coagulase-negative staphylococci, Escherichia coli, Streptococcus uberis, Klebiella pneumonia, Korucia rosea, and Acholeplasma spp. were not detected by the primers. We believe the high sensitivity and specificity of these primers make them useful for detecting infection with seven Mycoplasma species in ruminants, allowing the primers to be used in clinical settings.

scholarly journals A Novel Polymerase Chain Reaction Assay for the Detection of Seven Mycoplasma Species of Cattle Origin

2021 ◽  
Author(s):  
Jyhmirn Lai ◽  
Hengching Lin ◽  
Peichuan Hsu ◽  
Satoshi Gondaira ◽  
Hidetoshi Higuchi ◽  
...  
Keyword(s):  
Annealing Temperature ◽  
Bovine Mastitis ◽  
High Sensitivity ◽  
Mycoplasma Species ◽  
Clinical Settings ◽  
Coagulase Negative Staphylococci ◽  
Streptococcus Uberis ◽  
The Common ◽  
Polymerase Chain ◽  
Mastitis Pathogens

Abstract The aim of this study was to develop a pair of primers for detecting ruminant mycoplasma pathogens. We designed a set of primers based on the most similar sequences within 16sRNA regions of seven Mycoplasma spp. These primers have high sensitivity for detecting Mycoplasma dispar, M. arginine, M. canadense, M. bovis, M. alkalescens, M. californicum, and M. bovisgenitalium within the annealing temperature range of 46°C to 48°C. The minimum amount of DNA that can be detected using the protocol is 250 ng, which is equivalent to 2,000 cfu/mL. The primers can detect mycoplasma from DNA extracted directly from milk samples. The common bovine mastitis pathogens of Staphylococcus aureus coagulase-negative staphylococci, Escherichia coli, Streptococcus uberis, Klebiella pneumonia, Korucia rosea, and Acholeplasma spp. were not detected by the primers. We believe the high sensitivity and specificity of these primers make them useful for detecting infection with seven Mycoplasma species in ruminants, allowing the primers to be used in clinical settings.

scholarly journals Antimicrobial Resistance Profiles and Genes in Streptococcus uberis Associated With Bovine Mastitis in Thailand

2021 ◽  
Vol 8 ◽  
Author(s):  
Tingrui Zhang ◽  
Guoyi Niu ◽  
Sukolrat Boonyayatra ◽  
Duangporn Pichpol
Keyword(s):  
Antimicrobial Resistance ◽  
Inhibitory Concentration ◽  
Bovine Mastitis ◽  
Tetracycline Resistance ◽  
Detection Rates ◽  
Microdilution Method ◽  
Streptococcus Uberis ◽  
Stewardship Program ◽  
Polymerase Chain ◽  
Mastitis Pathogens

Streptococcus uberis is recognized as an environmental mastitis pathogen in dairy cattle. The varied success rate of antibiotic treatment for S. uberis intramammary infection may be associated with the antimicrobial resistance (AMR) of these bacteria. This observational study aimed to analyze 228 S. uberis strains associated with bovine mastitis in northern Thailand from 2010 to 2017. AMR and AMR genes were determined by the minimum inhibitory concentration (MIC) using a microdilution method and polymerase chain reaction, respectively. The majority of S. uberis strains were resistant to tetracycline (187/228, 82.02%), followed by ceftiofur (44/228, 19.30%), and erythromycin (19/228, 8.33%). The MIC50 and MIC90 of ceftiofur in 2017 were 2–4-fold higher than those in 2010 (P < 0.01). Resistance to tetracycline and ceftiofur significantly increased between 2010 and 2017 (P < 0.05). The most common gene detected in S. uberis was tetM (199/228, 87.28%), followed by ermB (151/228, 66.23 %) and blaZ (15/228, 6.58 %). The association between tetracycline resistance and tetM detection was statistically significant (P < 0.01). The detection rates of tetM significantly increased, while the detection rates of tetO and ermB significantly decreased during 2010–2017. AMR monitoring for bovine mastitis pathogens, especially S. uberis, is necessary to understand the trend of AMR among mastitis pathogens, which can help create an AMR stewardship program for dairy farms in Thailand.

scholarly journals Influence of nutrient availability on in vitro growth of major bovine mastitis pathogens

2021 ◽  
Vol 88 (1) ◽  
pp. 80-88
Author(s):  
Remo Stürmlin ◽  
Josef J. Gross ◽  
Olga Wellnitz ◽  
Lea A. Wagner ◽  
Camille Monney ◽  
...  
Keyword(s):  
Bacterial Growth ◽  
Nutrient Availability ◽  
Bovine Mastitis ◽  
Milk Composition ◽  
B Vitamins ◽  
E Coli ◽  
Streptococcus Uberis ◽  
Whole Milk ◽  
Mastitis Pathogens

AbstractThe aim of the present study was to investigate the effects of milk composition changes on the in vitro growth of bovine mastitis pathogens. Nutritional requirements of three major bovine mastitis pathogens Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Streptococcus uberis (S. uberis) were investigated in vitro. We used ultra-high temperature (UHT) treated milk with different contents of fat, protein, and carbohydrates to test the influence of the availability of various milk constituents on pathogen growth characteristics. Additionally, the bacterial growth was investigated under experimentally modified nutrient availability by dilution and subsequent supplementation with individual nutrients (carbohydrates, different nitrogen sources, minerals, and different types of B vitamins) either to milk or to a conventional medium (thioglycolate broth, TB). Varying contents of fat, protein or lactose did not affect bacterial growth with the exception of growth of S. uberis being promoted in protein-enriched milk. The addition of nutrients to diluted whole milk and TB partly revealed different effects, indicating that there are media-specific growth limiting factors after dilution. Supplementation of minerals to diluted milk did not affect growth rates of all studied bacteria. Bacterial growth in diluted whole milk was decreased by the addition of high concentrations of amino acids in S. aureus, and by urea and additional B vitamins in E. coli and S. aureus. The growth rate of S. uberis was increased by the addition of B vitamins to diluted whole milk. The present results demonstrate that growth-limiting nutrients differ among pathogen types. Because reduced bacterial growth was only shown in diluted milk or TB, it is unlikely that alterations in nutrient availability occurring as a consequence of physiological changes of milk composition in the cow's udder would directly affect the susceptibility or course of bovine mastitis.

scholarly journals Molecular identification of coagulase-negative staphylococci isolated from bovine mastitis and detection of β-lactam resistance

2015 ◽  
Vol 9 (09) ◽  
pp. 1022-1027 ◽  
Author(s):  
Mariela Elizabeth Srednik ◽  
Mario Andres Grieben ◽  
Adriana Bentancor ◽  
Elida Raquel Gentilini
Keyword(s):  
Fragment Length Polymorphism ◽  
Meca Gene ◽  
Bovine Mastitis ◽  
Economic Loss ◽  
Economic Losses ◽  
Dairy Herds ◽  
Coagulase Negative Staphylococci ◽  
Milk Samples ◽  
Gap Gene ◽  
Mastitis Pathogens

Introduction: Bovine mastitis is a frequent cause of economic loss in dairy herds. Coagulase-negative staphylococci (CoNS) are increasing in importance as causeof bovine intramammary infection (IMI) throughout the world in recent years. CoNShave been isolated from milk samples collected from cows with clinical andsubclinical mastitis in several countries. Identification of mastitis pathogensis important when selection appropriate antimicrobial therapy.Methodology: A total of 93 strains of Staphylococcusspp isolated from bovine clinical andsubclinical mastitis in Argentina during 2010-2013 were identified by Restriction Fragment Length Polymorphism (RFLP)-PCR using the gap gene. The isolates were tested by PCR for the presence of blaZ gene and mecA gene and were tested by disk diffusion for the susceptibilityto penicillin and cefoxitin. Results: The most common CoNS species was S.chromogenes 46.2% (43/93), followed by S. devriesei 11.8% (11/93) and S. haemolyticus 9.7% (9/93). The blaZ gene was detected in 19 (20.4%) but only 16 (17.2%) isolates were resistant to penicillin; the mecA was detected in6 (6.5%) isolates but only 4 (4.3) were resistant to cefoxitin. The 6 mecA-positive isolates showed oxaxillinMICs ≥ 0.5 μg/ml. Discussion: CoNSare important minor mastitis pathogens and can be the cause of substantial economic losses. Despite the low resistance to PEN in Argentina, the presenceof MR isolates found in this study emphasize the importance of identificationof CoNS when an IMI is present because of the potentially risk of lateraltransfer of resistance genes between staphylococcal species.

Effect of bovine lactoferrin on the internalization of coagulase-negative staphylococci into bovine mammary epithelial cells under in-vitro conditions

2009 ◽  
Vol 76 (2) ◽  
pp. 144-151 ◽  
Author(s):  
Paula Hyvönen ◽  
Sari Käyhkö ◽  
Suvi Taponen ◽  
Atte von Wright ◽  
Satu Pyörälä
Keyword(s):  
Cell Model ◽  
Bovine Mastitis ◽  
Mammary Epithelial ◽  
Bovine Lactoferrin ◽  
Intracellular Replication ◽  
Coagulase Negative Staphylococci ◽  
In Vitro Susceptibility ◽  
Intramammary Infection ◽  
Mastitis Pathogens

Coagulase-negative staphylococci (CNS) have emerged as bovine mastitis pathogens in many countries. CNS mastitis is generally mild but can persist in the udder for long periods. Pathogenesis of CNS intramammary infection is not well understood. In the present study, adhesion, invasion and intracellular replication of twenty-two CNS strains isolated from bovine mastitis and the effect of bovine lactoferrin (bLf) on the internalization were studied in vitro in a bovine mammary epithelial (BME) cell model. The CNS strains were ofStaphylococcus chromogenes, Staph. simulans, Staph. epidermidis, Staph. haemolyticusandStaph. cohnii urealyticus; two strains ofStaph. aureuswere used as controls. Seven of the CNS strains originated from persistent and five from transient mastitis infections. The in-vitro susceptibility of the strains to bLf was also investigated. All CNS species examined had an adhesive ability equal to that ofStaph. aureus, but internalization varied among staphylococcal strains. The antagonistic effect of bLf on the adhesion and invasion of CNS strains was weak, but bLf significantly decreased intracellular replication and replication rates of CNS. No correlation between the in-vitro susceptibility of the strain to bLf or internalization among clinical signs of mastitis was established. No difference between the persistent and transient CNS strains in adhesion, invasion or replication rate was recorded. This in-vitro BME cell model can be used to study the virulence potential of mastitis pathogens, although the severity and persistence of eventual infections shall be further investigated in vivo. The role of bLf in intramammary infection caused by CNS may be limited.

scholarly journals Molecular Detection and Sensitivity to Antibiotics and Bacteriocins of Pathogens Isolated from Bovine Mastitis in Family Dairy Herds of Central Mexico

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Ma. Fabiola León-Galván ◽  
José E. Barboza-Corona ◽  
A. Arianna Lechuga-Arana ◽  
Mauricio Valencia-Posadas ◽  
Daniel D. Aguayo ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Bovine Mastitis ◽  
Dairy Herd ◽  
Subclinical Mastitis ◽  
Herd Size ◽  
Clinical Mastitis ◽  
Dairy Herds ◽  
Coagulase Negative Staphylococci ◽  
California Mastitis Test ◽  
Streptococcus Uberis

Thirty-two farms (n=535cows) located in the state of Guanajuato, Mexico, were sampled. Pathogens from bovine subclinical mastitis (SCM) and clinical mastitis (CLM) were identified by 16S rDNA and the sensitivity to both antibiotics and bacteriocins ofBacillus thuringiensiswas tested. Forty-six milk samples were selected for their positive California Mastitis Test (CMT) (≥3) and any abnormality in the udder or milk. The frequency of SCM and CLM was 39.1% and 9.3%, respectively. Averages for test day milk yield (MY), lactation number (LN), herd size (HS), and number of days in milk (DM) were 20.6 kg, 2.8 lactations, 16.7 animals, and 164.1 days, respectively. MY was dependent on dairy herd (DH), LN, HS, and DMP<0.01, and correlations between udder quarters from the CMT were around 0.49P<0.01. Coagulase-negative staphylococci were mainly identified, as well asStaphylococcus aureus,Streptococcus uberis,Brevibacterium stationis,B. conglomeratum, andStaphylococcus agnetis. Bacterial isolates were resistant to penicillin, clindamycin, ampicillin, and cefotaxime. Bacteriocins synthesized byBacillus thuringiensisinhibited the growth of multiantibiotic resistance bacteria such asS. agnetis,S. equorum,Streptococcus uberis,Brevibacterium stationis, andBrachybacterium conglomeratum, but they were not active againstS. sciuri, a microorganism that showed an 84% resistance to antibiotics tested in this study.

Efficacy of a teat dip containing the bacteriocin lacticin 3147 to eliminate Gram-positive pathogens associated with bovine mastitis

2009 ◽  
Vol 77 (2) ◽  
pp. 231-238 ◽  
Author(s):  
Katja Klostermann ◽  
Fiona Crispie ◽  
Jim Flynn ◽  
William J Meaney ◽  
R Paul Ross ◽  
...  
Keyword(s):  
Bovine Mastitis ◽  
Gram Positive ◽  
Lactating Cows ◽  
Streptococcus Uberis ◽  
Colony Forming Units ◽  
Lacticin 3147 ◽  
Mastitis Pathogens ◽  
Teat Canal

On most dairy farms teat dips are applied to the teats of cows either before or after milking in order to prevent pathogens from gaining access to the mammary gland via the teat canal. In the present experiments, a natural teat dip was developed using a fermentate containing the live bacteriumLactococcus lactisDPC 3251. This bacterium produces lacticin 3147, a two-component lantibiotic which was previously shown to effectively kill Gram-positive mastitis pathogens. Lacticin 3147 activity in the fermentate was retained at 53% of its original level following storage for 3 weeks at 4°C. In the initial experiments in vitro, 105colony-forming units/ml (cfu/ml) of eitherStaphylococcus aureus,Streptococcus dysgalactiaeorStreptococcus uberiswere introduced into the lacticin-containing fermentate. NeitherStaph. aureusnorStr. dysgalactiaecould be detected after 30 min or 15 min, respectively, whileStr. uberiswas reduced approximately 100-fold after 15 min. Following these trials, preliminary experiments were performed in vivo on teats of lactating dairy cows. In these experiments, teats were coated with each of the challenge organisms and then dipped with the lacticin-containing fermented teat dip. Following a dip contact time of 10 min, staphylococci were reduced by 80% when compared with the undipped control teat. Streptococcal challenges were reduced by 97% forStr. dysgalactiaeand by 90% forStr. uberis. These trials showed that the teat dip is able to reduce mastitis pathogens on the teats of lactating cows.

scholarly journals Antibacterial Activities of Acetic Acid against Major and Minor Pathogens Isolated from Mastitis in Dairy Cows

Pathogens ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 961
Author(s):  
Noppason Pangprasit ◽  
Anyaphat Srithanasuwan ◽  
Witaya Suriyasathaporn ◽  
Surachai Pikulkaew ◽  
John K. Bernard ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Saturated Fatty Acids ◽  
Bovine Mastitis ◽  
Antibacterial Activities ◽  
Antimicrobial Activities ◽  
Culture Broth ◽  
Diffusion Method ◽  
Coagulase Negative Staphylococci ◽  
Microdilution Method ◽  
Mastitis Pathogens

The present study evaluated the antimicrobial activities of acetic acid against bovine mastitis pathogens compared to lactic acid and lauric and caprylic saturated fatty acids. Eleven mastitis pathogens were isolated from sub-clinical and clinical bovine mastitis cases for the study. An initial screening of their antibacterial activities by agar well diffusion method was performed. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of each acid were obtained using a microdilution method; each acid was diluted from stock solution and then were diluted with culture broth to reach concentrations ranging from 4 to 0.004% w/v. The results showed acetic acid had the highest zone of inhibition against all pathogens except Escherichia coli compared with lauric and caprylic acids. The MIC and MBC were lowest for acetic acid against both Gram-positive (except Staphylococcus chromogenes from the coagulase negative staphylococci (CNS) group) and Gram-negative pathogens, intermediate for lactic and caprylic acids and greatest for lauric acid. In conclusion, acetic acid had antimicrobial activities against most mastitis pathogens compared with other acids. Further studies are needed to optimize the formulation and concentration of acetic acid for teat-dipping agent in the future.

scholarly journals Prevalence of mastitis pathogens in South African pasture-based and total mixed ration-based dairies during 2008 and 2013

2018 ◽  
Vol 85 (1) ◽  
Author(s):  
David Blignaut ◽  
Peter Thompson ◽  
Inge-Marié Petzer
Keyword(s):  
South Africa ◽  
South African ◽  
Relative Number ◽  
Cow Milk ◽  
Coagulase Negative Staphylococci ◽  
Total Mixed Ration ◽  
Herd Prevalence ◽  
Streptococcus Uberis ◽  
Relative Prevalence ◽  
Mastitis Pathogens

Recent years have seen a change in the relative prevalence of environmental and contagious intramammary pathogens, as well as a change in the relative number of total mixed ration (TMR)-based and pasture (PAS)-based dairies in South Africa. The objectives of the study were to determine and compare the prevalence of mastitis pathogens in TMR and PAS dairies in South Africa during 2008 and 2013; furthermore, the within-herd prevalence of Streptococcus uberis in Str. uberis-positive herds was determined and compared. The prevalence of each pathogen, as well as the within-herd prevalence of Str. uberis, were compared between the two years and the two management systems using bacterial culture results from routinely collected composite cow milk samples submitted to the Onderstepoort Milk Laboratory, Faculty of Veterinary Science, University of Pretoria. Coagulase-negative staphylococci had the highest prevalence in both TMR and PAS dairies for both 2008 (29.60% [95.00% CI: 28.80% – 30.40%] and 26.90% [95.00% CI: 25.50% – 28.30%], respectively) and 2013 (20.20% [95.00% CI: 19.30% – 21.10%] and 22.70% [95.00% CI: 22.20% – 23.10%], respectively), which decreased significantly from 2008 to 2013 in both TMR and PAS dairies (p < 0.001). Streptococcus uberis showed an increase in prevalence in both TMR (p = 0.002) and PAS dairies (p = 0.001) from 2008 (2.36% [95.00% CI: 2.10% – 2.65%] and 2.63% [95.00% CI: 2.16% – 3.16%], respectively) to 2013 (3.10% [95.00% CI: 2.72% – 3.51%] and 3.64% [95.00% CI: 3.45% – 3.83%], respectively). Staphylococcus aureusshowed a significant decrease in both TMR (p = 0.011) and PAS (p < 0.001) dairies from 2008 (4.71% [95.00% CI: 4.34% – 5.10%] and 5.62% [95.00% CI: 4.94% – 6.36%], respectively) to 2013 (3.95% [95.00% CI: 3.52% – 4.40%] and 1.71% [95.00% CI: 1.58% – 1.84%], respectively). The median within-herd prevalence of Str. uberis for the combined dairy systems showed a significant increase from 2008 (1.72% [IQR: 0.88% – 5.00%]) to 2013 (3.10% [IQR: 1.72% – 4.70%]) (p < 0.001). Statistically significant differences were found in the prevalence of most of the major contagious and environmental mastitis pathogens between 2008 and 2013 and between TMR and PAS dairies. The within-herd prevalence of Str. uberis increased from 2008 to 2013, with the highest within-herd prevalence in PAS dairies in 2013.

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