Efficacy of two iodine teat dips during experimental challenge with Staphylococcus aureus and Streptococcus agalactiae

An experimental challenge trial was performed against Staphylococcus aureus and Streptococcus agalactiae following the procedures recommended by the National Mastitis Council. The efficacy of two teat dips, product 1 (Bovadine with I-Tech II) and product 2 (Bovadine with I-Tech, used as a positive control), was determined. Both teat dips contain 1% iodine and 10% glycerin. Product 1 established an 89.7% reduction in infections against Staph. aureus and 73.1% reduction in infections against Strep. agalactiae. Product 2 demonstrated an 86.2% reduction in infections against Staph. aureus and 78.4% reduction in infections against Strep. agalactiae. Teat skin and teat ends were evaluated before and after the trial. No significant change in teat condition was observed for either product.     

Efficacy of a 0.1% iodine teat dip against Staphylococcus aureus and Streptococcus agalactiae during experimental challenge

An experimental challenge trial was performed according to the guidelines recommended by the National Mastitis Council (NMC). A 0.1% iodine teat dip (Quartermate with I-Tech) was examined. This product gave an 87.9% reduction of new intramammary infections with Staphylococcus aureus and a 66.5% reduction for Streptococcus agalactiae compared with a negative control. Teat end and teat skin characteristics remained excellent throughout the trial.     

A longitudinal field study to evaluate the diagnostic properties of a quantitative real-time polymerase chain reaction-based assay to detect Staphylococcus aureus in milk

Bacteriological culture as a diagnostic tool for chronic infections with Staphylococcus aureus intramammary infection is not completely satisfactory. The cyclical shedding pattern of Staph. aureus with intervals of low excretion is considered to be the main reason. We recently developed a novel assay for Staph. aureus in milk, based on real-time quantitative PCR (QPCR). In a longitudinal study of chronically infected cows we evaluated the diagnostic properties of this test under field conditions. Diagnostic sensitivity of the novel test proved to be very high with a value of 99.4%; diagnostic specificity was 97.1%. In addition, the shedding patterns of Staph. aureus for the sampling period were analyzed. Using log₁₀-transformed QPCR data and plotting them across sampling time revealed a sinusoidal shedding pattern in 6 of 11 naturally infected quarters. Shedding patterns obtained by QPCR and by bacteriological culture were synchronous. In conclusion, the novel test has a very high diagnostic sensitivity and specificity so that quarters chronically infected with Staph. aureus are reliably detected, independent from their actual shedding quantity.     

Efficacy of an iodophore teat disinfectant against Staphylococcus aureus and Streptococcus agalactiae in experimental challenge

A 1.0% iodophore teat disinfectant (Full-Bac) was evaluated in comparison with a positive control (Bovadine), a commercially available 1.0% iodophore teat disinfectant. The study was conducted under conditions of experimental challenge, following the guidelines recommended by the National Mastitis Council. The test product and a positive control were compared in 41 cows, with 82 teats receiving each product at milking, during a 10-wk study period. There were no differences between the test product and the positive control in new intramammary infections due to Staphylococcus aureus, which averaged 13.4% in each of the 2 treatment groups. Additionally, no statistical difference was seen between the test product and positive control in new intramammary infections by Streptococcus agalactiae, which averaged 8.5 and 6.1% for the Full-Bac and Bovadine groups, respectively. Teat skin and teat end condition scores were statistically evaluated at wk 1, 5, and 9 of the study, and no significant differences were observed between the treatment groups. The test teat disinfectant provided similar germicidal activity to that of the positive control teat disinfectant with no adverse effects on teat skin or teat end condition during the warm-season study period.     

Evaluation of three different molecular markers for the detection of Staphylococcus aureus by polymerase chain reaction

The aim of this study was to target three genes of Staphylococcus aureus-fmhA (coding for a factor of unknown function), catalase and femA (coding for a factor essential for methicillin resistance) to establish and validate a PCR assay for the detection of this pathogen. Two pairs of primers were designed for fmhA and one pair each for catalase and femA genes. The PCR assays were standardized and found to give specific amplicons under similar reaction parameters. Target specificity of the primers was confirmed by DNA sequencing of the amplicons. While the initial inclusivity and exclusivity test reactions were in agreement in case of three of the primer pairs, one pair based on fmhA gene produced a non-specific product with a template DNA used in exclusivity test reactions. Forty-five strains of S. aureus were subjected to these PCR assays for their evaluation. Three among the four pairs of primers, one against each gene detected all the 45 strains precisely whereas one of the PCR assays using primers targeting the fmhA gene did not generate the specific amplicon with several of the strains. Seven unidentified strains of Gram-positive cocci subjected to these PCR assays produced negative results for each culture. Six of the strains were identified as Staphylococcus haemolyticus and one strain as Staphylococcus arlettae by 16S ribosomal gene analyses. All the three assay systems showed a detection limit of 100 cells per 20mul reaction assay. For validation of these assay systems, 80 coded samples of 11% skimmed milk spiked with different pathogens were received from NICED (National Institute of Cholera and Enteric Diseases), Kolkata and subjected to these PCR assays. All the three assays could detect S. aureus correctly in two of the samples. Amongst 150 raw milk samples, 36 (24%) were found positive for S. aureus. We conclude that fmhA, catalase and femA genes are conserved in S. aureus and, therefore, could be used as specific targets for its detection and identification by PCR. The protocols developed herein could be used for rapid and specific detection of this pathogen in food, clinical and environmental samples, especially milk.     

多重PCR技术在食源性致病菌检测中应用的研究进展

食源性致病菌的多重PCR检测技术是指能够同时扩增得到同种或多种致病菌的不同基因片段的技术。因该技术特异、灵敏且分析效率高,现已被广泛应用于食源性致病菌的检测工作中。本文介绍了多重PCR在食源性致病菌检测中应用的研究近况,并对影响因素及存在的问题进行了阐述。      

Conventional identification of Streptococcus uberis isolated from bovine mastitis in Argentinean dairy herds

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 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.     

Development of a highly sensitive and specific assay to detect Staphylococcus aureus in bovine mastitic milk

Diagnosis of udder infections with Staphylococcus aureus by bacteriological milk testing of quarter milk samples is often not satisfactory. To get reliable results, repeated sampling is necessary, which is normally too expensive. Therefore, we developed a test that allows the highly specific detection of Staph. aureus in bovine milk samples at very low concentrations. It is based on a fast procedure to prepare bacteria from milk, followed by DNA extraction and quantitative PCR. The whole analysis is done within 5 h. For clinical milk samples, the analytical sensitivity of the assay was 50.7 times and 507 times higher than conventional bacteriology with 100 and 10 μL, respectively. The diagnostic specificity was 100%. The test is further characterized by a low intra- and interassay variability as well as by a good recovery of Staph. aureus from raw milk. Furthermore, a high correlation (R = 0.925) between the agar plate counts and the quantitative PCR methodology over the whole range of measurement was found. In addition, our test revealed considerably more positive results than bacteriology. Due to its favorable properties, the assay might become an important diagnostic tool in the context of bovine mastitis caused by Staph. aureus.     

Extended ceftiofur therapy for treatment of experimentally-induced Streptococcus uberis mastitis in lactating dairy cattle

Streptococcus uberis is an important cause of mastitis in dairy cows throughout the world, particularly during the dry period, the period around calving, and during early lactation. Strategies for controlling Strep. uberis mastitis are poorly defined and are currently inadequate. Objectives of the present study were to evaluate efficacy of ceftiofur, a new broad-spectrum cephalosporin antibiotic, for treatment of experimentally induced Strep. uberis intramammary infections (IMI) in lactating dairy cows during early lactation and to determine whether extended therapy regimens enhanced efficacy of ceftiofur. Efficacy of extended ceftiofur intramammary therapy regimens was investigated in 37 mammary quarters of 23 dairy cows that developed clinical mastitis following experimental infection with Strep. uberis during early lactation. Cows that developed clinical mastitis during the challenge period were allocated randomly to 3 groups representing 3 different ceftiofur treatment regimens: 2-d (n = 7 mammary quarters), 5-d (n = 16 mammary quarters), and 8-d (n = 14 mammary quarters) treatment regimens. For all groups, 125 mg of ceftiofur hydrochloride was administered via intramammary infusion. A bacteriological cure was defined as an experimentally infected quarter that was treated and was bacteriologically negative for the presence of Strep. uberis at 7, 14, 21, and 28 d posttreatment. Percentage of Strep. uberis IMI eliminated was 43, 88, and 100% for the 2-, 5-, and 8-d ceftiofur treatment regimens, respectively. Both the 5- and 8-d ceftiofur extended therapy treatment regimens had significantly higher bacterial cure rates than the standard 2-d ceftiofur treatment regimen. The bacterial cure rate of the 8-d ceftiofur extended therapy group was marginally better (P = 0.052) than the 5-d ceftiofur extended therapy group. Results of this study indicate that ceftiofur therapy was effective for eliminating Strep. uberis experimental IMI, and 5- and 8-d extended ceftiofur therapy regimens were more effective than the standard 2-d treatment.     

微滴式数字聚合酶链式反应定量检测食品中金黄色葡萄球菌方法的研究

目的 建立微滴数字聚合酶链式反应(ddPCR)快速定量检测食品中金黄色葡萄球菌的方法.方法 以金黄色葡萄球菌nu基因为靶序列,筛选出同时适用于实时荧光定量PCR(qPCR)和微滴数字PCR(ddPCR)的引物探针,建立食品中金黄色葡萄球菌ddPCR快速定量检测方法,并对该方法进行特异性、灵敏度、准确性和重复性实验.结果...     

Efficacy of two hydrogen peroxide teat disinfectants against Staphylococcus aureus and Streptococcus agalactiae

An experimental teat dip containing 0.5% hydrogen peroxide as the active ingredient was compared with a teat disinfectant also containing 0.5% hydrogen peroxide that is commercially available throughout North America. The study was conducted under conditions of experimental challenge with a positive control following the guidelines recommended by the National Mastitis Council. The efficacy of the test product and the positive control product were compared in 45 cows, with 89 total teats receiving each product after milking during a 10-wk study period. There was no significant difference between the experimental hydrogen peroxide product and the positive control in new intramammary infections caused by Staphylococcus aureus (27.0 and 18.0% in the treatment groups, respectively). Additionally, there was no significant difference between the experimental product (6.7%) and the positive control groups (4.5%) in new intramammary infections caused by Streptococcus agalactiae. Traditional analysis of teat skin condition changes supported improved teat skin condition with the test disinfectant. After accounting for the correlation of teats within cow, significant differences were also observed between the treatment groups for teat skin condition. The experimental hydrogen peroxide-based teat disinfectant provided efficacy similar to that of the positive control teat disinfectant, with significant improvement in teat skin condition and no adverse effects on teat end condition.     

Genotypic and phenotypic detection of macrolide and lincosamide resistance in Streptococcus uberis

Streptococcus uberis isolates (n = 55) were obtained from milk samples of cases of mild clinical mastitis in 55 dairy cows from 35 herds serviced by one veterinary practice in Mayenne, France. Isolates were tested for macrolide and lincosamide resistance by using phenotypic and genotypic methods. Erythromycin resistance was detected in 12 of the 55 (22%) isolates based on agar disc diffusion testing and MIC measurements, and was encoded by ermB. This gene also conferred phenotypic resistance to pirlimycin based on MIC measurements, but the D-test was needed for detection of the resistance phenotype in the agar disc diffusion test. Isolates with ermB were also highly resistant to the macrolide antibiotic spiramycin. Seventeen of the 55 isolates (31%) were classified as resistant to spiramcyin only and as having intermediate susceptibility to spiramycin based on agar disc diffusion testing and MIC measurements, respectively. The genetic mechanism behind this phenotype and its clinical relevance are unknown. The efflux pump gene mefA was not detected in any of the 55 isolates in this study. Pirlimycin resistance without macrolide resistance was encoded by the lincosamide resistance gene linB in 4 isolates. Based on current guidelines, some linB-positive isolates would be classified as susceptible by using phenotypic tests, and alternative values for the interpretation of the agar disc diffusion test are suggested. We conclude that the agar disc diffusion test is a useful indicator for macrolide and lincosamide resistance of Strep. uberis in veterinary practice, provided that the D-test is used for detection of pirlimycin resistance.     

Simultaneous detection of cow and buffalo species in milk from China, India, and Pakistan using multiplex real-time PCR

Asian countries are major producers of cow and buffalo milk. For quality and authenticity purposes, a multiplex real-time PCR assay was developed to specifically and simultaneously detect DNA from these 2 bovine species. Targeting the cytochrome b gene of mitochondrial DNA, common PCR primers amplified a 105-bp fragment, and 2 fluorescent probes specific to either cow or buffalo were designed for their identification. Specificity was successfully tested on 6 other species, including sheep and goat, and sensitivity reached 1% of cow DNA in buffalo DNA and vice versa. As an evaluation, the method was tested using 119 freeze-dried Asian milk samples from regional industrial milk facilities. Although these samples did not cover the entire Asian zone, the multiplex assay indicated that approximately 20% of the samples (mainly from India) showed high levels of cross-contamination of cow milk by buffalo milk, and vice versa. Fast, sensitive, and straightforward, this method is fit-for-purpose for the authenticity control of Asian milk.     

多重聚合酶链式反应技术检测罗非鱼5种常见食源性致病菌

目的 建立一种可同时检测无乳链球菌(Streptococcus agalactiae)、嗜水气单胞菌(Aeromonas hydrophila)、霍乱弧菌(Vibrio cholerae)、大肠杆菌(Escherichia coli)和沙门菌(Salmonella) 5种罗非鱼常见食源性致病菌的多重聚合酶链式反应(polymerase chain reaction,PCR)方法。方法 根据5种致病菌特异性基因片段设计并合成引物,优化多重PCR体系条件,并对多重PCR体系的特异性、灵敏度以及人工模拟样品进行检测。结果 建立的多重PCR方法可同时扩增5种目的菌株的特异性条带,且不与非靶标细菌发生交叉反应。敏感性实验结果显示,该方法对无乳链球菌、嗜水气单胞菌、沙门菌、霍乱弧菌、大肠杆菌纯培养物基因组DNA的检出限均为0.4 ng/μL。人工模拟样品检测结果显示,该方法可以快速且准确地检测上述5种食源性致病菌,且检出限可达到2×10¹ CFU/g。结论 本研究建立了一种可同时检测无乳链球菌、嗜水气单胞菌、霍乱弧菌、大肠杆菌和沙门菌5种罗非鱼常见食源性致病菌的多重PCR检测...     

多重实时荧光定量PCR快速检测婴幼儿奶粉中沙门氏菌、克罗诺杆菌和金黄色葡萄球菌

目的建立多重实时荧光定量PCR(multiplex quantitative real-time PCR,multiplex qPCR)快速检测奶粉中金黄色葡萄球菌、沙门氏菌和克罗诺杆菌3种常见致病菌的方法。方法筛选目标菌株的特异性引物与探针,优化反应体系,建立稳定的多重q PCR反应体系。通过阳性菌株加标的方式验证体系的特异性,并确定人工污染奶粉的检出限。结果各对引物探针对目标菌株均能扩增,多重实时荧光PCR未发现交叉反应,对17株非目标菌进行检测均未检出,人工污染奶粉中克罗诺杆菌和沙门氏菌的检出限均为10~3 CFU/mL,金黄色葡萄球菌的检出限为10~4 CFU/mL。结论本研究方法可实现婴幼儿奶粉样品中3种致病菌qPCR高效率检测。     

A novel real-time polymerase chain reaction-based method for the detection and quantification of lactose-fermenting Enterobacteriaceae in the dairy and other food industries

The presence of lactose-fermenting Enterobacteriaceae and coliforms is routinely assessed to determine the hygienic quality of water and foods, particularly dairy products. This paper reports the use of lacZ-specific primers in an SYBR green I-based real-time PCR method for the easy and rapid detection of coliforms in dairy products. A large number of bacterial species were assayed to establish the specificity of the method. The sensitivity of the method was assessed using artificially contaminated cheeses. The limit of detection was 1 coliform cell in cheese samples enriched for 8 h in a culture medium. The entire procedure, including sample processing, enrichment, DNA extraction, and real-time PCR amplification, can be completed within 10 to 12 h, making it a single-day assay.     

Multiplex polymerase chain reaction as a mastitis screening test for Staphylococcus aureus,Streptococcus agalactiae,Streptococcus dysgalactiae and Streptococcus uberis in bulk milk samples

Effective diagnostic tools for screening herds for mastitis pathogens are important in development and monitoring of mastitis control programmes. A multiplex polymerase chain reaction (PCR) assay for simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis was used in preliminary studies to assess its applicability as an alternative method for monitoring mastitis caused by these organisms at the herd level. PCR was used to detect the presence of these organisms in bulk milk samples. Correlations with bulk milk somatic cell counts (BMCC), total bacteria counts and thermoduric bacteria counts were evaluated. A total of 176 bulk milk samples were collected from 42 herds on five consecutive occasions at approx. 10-d intervals. Str. uberis was the most common organism in these bulk milk samples. There was no relationship between presence of either Staph. aureus, Str. dysgalactiae or Str. uberis and BMCC, total bacteria counts or thermoduric bacteria counts. However, presence of Str. agalactiae was associated with high BMCC and total bacteria counts. The results of this study show that regular analysis of bulk milk using this multiplex PCR assay may be a useful tool for monitoring herd status with respect to Str. agalactiae, but is of less value for monitoring occurrence of Staph. aureus, Str. dysgalactiae and Str. uberis. Further investigations are needed to clarify the relationship between positive PCR results and the prevalence of infected cows in the herd.     

Technical note: Antimicrobial susceptibility of Portuguese isolates of Staphylococcus aureus and Staphylococcus epidermidis in subclinical bovine mastitis

To evaluate the antimicrobial resistance traits of staphylococci responsible for subclinical bovine mastitis in Portugal, the minimum inhibitory concentrations (MIC) of 7 antimicrobial agents, frequently administered for mastitis treatment, were determined for 30 Staphylococcus aureus and 31 Staphylococcus epidermidis field isolates. β-Lactamase production was detected through the use of nitrocefin-impregnated discs. The MIC that inhibited 90% of the isolates tested (MIC₉₀) of penicillin, oxacillin, cefazolin, gentamicin, sulfamethoxazole/trimethoprim, oxytetracycline, and enrofloxacin were, respectively, 4, 0.5, 1, 1, 0.25, 0.25, and 0.06 μg/mL for Staph. aureus and ≥64, 8, 1, 32, ≥64, ≥64, and 0.06 μg/mL for Staph. epidermidis. All Staph. aureus isolates showed susceptibility to oxacillin, cefazolin, gentamicin, sulphamethoxazole/trimethoprim, and enrofloxacin. β-Lactamase production was detected in 20 of these isolates (66.7%), all of which were resistant to penicillin. Of the 31 Staph. epidermidis tested, 24 (77.4%) were β-lactamase positive. All isolates were susceptible to both cefazolin and enrofloxacin. Nine Staph. epidermidis isolates were resistant to oxacillin, with MIC values ranging from 4 to 8 μg/mL. The MIC values of 5 antimicrobial agents tested were higher than those reported in other countries. Enrofloxacin was the only exception, showing lower MIC values compared with other reports. Overall, the antimicrobial agents tested in our study, with the exception of penicillin, were active against the 61 isolates studied.     

Antibiogram and coagulase diversity in staphylococcal enterotoxin-producing Staphylococcus aureus from bovine mastitis

We investigated antibiogram and coagulase gene diversity in staphylococcal enterotoxin (StE)-producing Staphylococcus aureus isolated from raw milk samples of cows infected with mastitis from 140 dairy farms in Korea between 1997 and 2004. Of the 696 Staph. aureus isolates collected in this study, 164 isolates (23.6%) produced one or more staphylococcal enterotoxins (A to D), and 19 isolates (2.7%) were methicillin-resistant. The percentage of StE-producing Staph. aureus (SES) isolates resistant to methicillin, kanamycin, neomycin, amikacin, and tetracycline was greater than that of non-SES. Ten coagulase genotype patterns were observed, including 4 main types comprising I (25.4%), II (13.9%), VII (13.2%), and VIII (17.8%). More than 4 Staph. aureus types were isolated from each of 82 dairy farms in different geographic locations, and only 1 coagulase genotype pattern was observed in 39 of the herds (47.6%). There was no significant correlation between coagulase genotypes harbored by Staph. aureus and their specific StE type. The percentage of isolates producing major StE types (A, B, AC, and ABCD) and being resistant to cephalothin and methicillin was greater among the Staph. aureus isolates with the 4 predominant coagulase genotypes (I, II, VII, and VIII) than among the isolates harboring the 6 rare coagulase types (III, IV, V, VI, IX, and X). Based on coagulase gene polymorphisms, our data indicate that a broad distribution of identical or closely related enterotoxin-producing Staph. aureus strains seem to contribute to bovine mastitis in the Republic of Korea.     

Vancomycin-modified poly-l-lysine magnetic separation combined with multiplex polymerase chain reaction assay for efficient detection of Bacillus cereus in milk

In this study, a new vancomycin (Van)-modified poly-l-lysine (PLL) magnetic bead (MB) technique was developed for isolation of gram-positive bacteria. The method combines magnetic separation with a multiplex PCR (mPCR) assay and gel electrophoresis for easy and rapid detection of Bacillus cereus. Vancomycin was used as a molecular ligand between the MB and the d-alanyl-d-alanine moieties on the cell wall surface of B. cereus. The PLL served as a flexible molecular tether between the MB and Van that reduced steric hindrance maintaining the biological activity of Van. The MB-PLL-Van capture nanoprobes exhibited excellent capture and isolation efficiency for B. cereus in spiked milk matrix samples without interference from the complex food matrix. The subsequent mPCR assay showed high specificity for the 4 target genes in B. cereus, the entFM, cesB, cer, and 16S rRNA genes, that were used to achieve efficient genotyping and detection. Under optimum conditions, the limit of detection reached 10³ cfu/mL, with a dynamic range of detection at 10³ to 10⁷ cfu/mL in pure culture. Application of the MB-PLL-Van mediated mPCR assay for B. cereus in milk matrix samples achieved results similar to those of the pure culture. In addition, with a 6-h pre-enrichment of B. cereus that was spiked in milk matrix samples, the limit of detection reached 10¹ cfu/mL. The MB-PLL-Van mediated mPCR assay developed in this study could be used as a universal technology platform for the efficient enrichment and genotyping of gram-positive bacteria.