Factors associated with intramammary infection in dairy cows caused by coagulase-negative staphylococci,Staphylococcus aureus,Streptococcus uberis,Streptococcus dysgalactiae,Corynebacterium bovis,or Escherichia coli

The aim of this study was to determine risk factors for bovine intramammary infection (IMI) associated with the most common bacterial species in Finland. Large databases of the Finnish milk-recording system and results of microbiological analyses of mastitic milk samples from Valio Ltd. (Helsinki, Finland) were analyzed. The study group comprised 29,969 cows with IMI from 4,173 dairy herds. A cow with a quarter milk sample in which DNA of target species was detected in the PathoProof Mastitis PCR Assay (Thermo Fisher Scientific, Waltham, MA) was determined to have IMI. Only cows with IMI caused by the 6 most common pathogens or groups of pathogens, coagulase-negative staphylococci (CNS), Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, Corynebacterium bovis, and Escherichia coli, were included. The control group comprised 160,176 IMI-free cows from the same herds as the study group. A multilevel logistic regression model was used to study herd- and cow-specific risk factors for incidence of IMI. Pathogen-specific results confirmed those of earlier studies, specifically that increasing parity increases prevalence of IMI regardless of causative pathogen. Holsteins were more susceptible to IMI than Nordic Reds except when the causative pathogen was CNS. Occurrence of IMI caused by C. bovis was not related to milk yield, in contrast to IMI caused by all other pathogens investigated. Organic milk production was associated with IMI only when the causative pathogen of IMI was Staph. aureus; Staph. aureus IMI was more likely to occur in conventional than in organic production. Cows in older freestall barns with parlor milking had an increased probability of contracting an IMI compared with cows in tiestall barns or in new freestall barns with automatic milking. This was the case for all IMI, except those caused by CNS, the prevalence of which was not associated with the milking system, and IMI caused by Staph. aureus, which was most common in cows housed in tiestall barns. A better breeding index for milk somatic cell count was associated with decreased occurrence of IMI, indicating that breeding for improved udder health has been successful in reducing the incidence of IMI caused by the most common pathogens in Finland. In the Finnish dairy sector, the importance of other measures to control IMI will increase as the Holstein breed progressively takes the place of the Nordic Red breed. Attention should be paid to hygiene and cleanliness, especially in old freestall barns. Based on our results, the increasing prevalence of automatic milking is not a reason for special concern.     

Mastitis diagnostics: quantitative PCR for Staphylococcus aureus genotype B in bulk tank milk

A novel real-time quantitative PCR assay for detecting the pathogenic and contagious Staphylococcus aureus genotype B (GTB) in bulk tank milk was developed and evaluated. The detection of this pathogen in bulk tank milk would greatly facilitate its control, as it is responsible for great economic loss in Swiss dairy herds. The assay is based on the simultaneous detection of 3 GTB-typical target sequences, including 2 enterotoxin genes and a polymorphism within the leucotoxin E gene. A variety of mastitis-associated bacteria was used to validate the assays, resulting in an analytical specificity of 100% and high repeatability. The analytical sensitivity in milk was 40 cfu/mL. An exponential association between simulated cow prevalence and quantitative PCR result was observed. An initial field study revealed 1 GTB-positive herd among the 33 studied herds. This novel assay for bulk tank milk analysis is suitable for routine purposes and is expected to be an effective tool for minimizing Staph. aureus GTB in Swiss dairy herds.     

Simultaneous detection of mastitis pathogens, Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae by multiplex real-time polymerase chain reaction

The objective of this study was to develop a multiplex real-time polymerase chain reaction (PCR) method for simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus uberis directly from milk. A genetic marker specific for Staph. aureus was used for primers and dual-labeled probe design. The target for Strep. agalactiae primers and dual-labeled probe was selected from the cfb gene encoding the Christie-Atkins-Munch-Petersen factor. The plasminogen activator gene was the target for primers and dual-labeled probe design for Strep. uberis. Quarter milk samples (n = 192) were analyzed by the multiplex real-time PCR assay and conventional microbiological methods. An additional 57 quarter milk samples were analyzed in a separate real-time PCR assay for Strep. agalactiae only. Using an overnight enrichment step, the real-time PCR technique correctly identified 96.4% of all quarter milk samples; 91.7% of Staph. aureus, 98.2% of Strep. agalactiae, and 100% of Strep. uberis. Results of conventional microbiological methods were used to determine the sensitivity and specificity of the multiplex real-time PCR procedure. The sensitivity of the procedure to correctly identify Staph. aureus, Strep. agalactiae, and Strep. uberis directly from milk was 95.5%, and the specificity was 99.6%. Results of this study indicate that the multiplex real-time PCR procedure has the potential to be a valuable diagnostic technique for simultaneous identification of Staph. aureus, Strep. agalactiae, and Strep. uberis directly from quarter milk samples.     

A PCR-based method for the detection of Streptococcus agalactiae in milk

Bovine mastitis caused by Streptococcus agalactiae is mainly subclinical and therefore can be diagnosed only in the laboratory. We developed a polymerase chain reaction (PCR)-based method for specific and sensitive detection of S. agalactiae in raw milk. The specificity of the PCR reaction is based on unique S. agalactiae DNA sequences within the 16S subunit of the rRNA genes. Two pairs of sequences were used as positive controls; general streptococci primers, which anneal to conserved areas within the 16S rRNA subunit gene, and primers, which anneal to sequences within bovine mitochondrial DNA. The method of detection includes selective enrichment of S. agalactiae in the milk sample, followed by DNA extraction using a rapid and simple procedure developed for this purpose, and specific PCR reaction with appropriate controls. The method enables the detection of one bacterium in 1 ml of raw milk. The method developed can be easily incorporated as part of routine screening of bulk milk collection tanks for early detection of infected cows in a herd.     

Application of a dot blot hybridization assay for genotyping Streptococcus uberis from Brazilian dairy herds

Streptococcus uberis is a major cause of environmental mastitis in many regions, and it is associated with clinical and subclinical infections. Although the main source of infection is the environment, reports of strains with a contagious profile have been described. Dot blot hybridization analysis allows the rapid identification of S. uberis population structures within and between herds, and it helps to identify strain diversity as well as possible clonal lineages that directly affect the control of bovine mastitis caused by this pathogen. The aim of this study was to evaluate the diversity of S. uberis isolates obtained from clinical (n = 22) and subclinical (n = 22) cases of mastitis in dairy herds (n = 13) in Brazil over a period of 12 mo. We submitted 44 S. uberis isolates to dot blot hybridization followed by automatic data analysis. We identified 8 different hybridization patterns using genetic markers associated with virulence factors and taxonomy, indicating diversity of S. uberis within the population and suggesting environmental transmission. However, the evidence of identical dot blot patterns in different mammary quarters from the same animal also suggested local contagious transmission. Of the virulence genes evaluated, we found a high prevalence of the genes sua, pauA, and gapC, highlighting the importance of these virulence factors for the adhesion, invasion, and multiplication of S. uberis in subclinical and clinical intramammary infections.     

速冻食品中沙门氏菌和金黄色葡萄球菌多重PCR检测方法的建立与应用

为实现速冻食品中沙门氏菌(Salmonella spp.)和金黄色葡萄球菌(Staphylococcus aureus)的多重聚合酶链式反应(PCR)检测,首先优化多重PCR扩增的反应条件,比较基因组DNA提取方法,结果表明：退火温度采用60℃、各引物浓度200nmol/L及扩增循环35次,本多重PCR检测技术可以有效地将沙门氏菌和金黄色葡萄球菌同时检出,检测特异性为100%。3种DNA提取方法中试剂盒法纯度最高,检出限分别是31、26DNA copies/reaction。经过在人工污染致病菌的速冻水饺中应用试验后,该多重PCR方法经过4h的增菌培养即可从速冻水饺中同时检测出起始菌落数低至100CFU/g的沙门氏菌和金黄色葡萄球菌。     

Bayesian estimation of sensitivity and specificity of a rapid mastitis test kit,bacterial culture,and PCR for detection of Staphylococcus aureus,Streptococcus species,and coliforms in bovine milk samples

Our objectives were to evaluate the diagnostic accuracy of a rapid and novel immunochromatography-based mastitis kit that includes 3 independent tests to detect coliforms (Escherichia coli or Klebsiella pneumoniae), Streptococcus spp., and Staphylococcus aureus. The kit was developed to facilitate diagnostic-based mastitis treatment. Validation of the kit was based on 154 aseptically collected mastitis samples from 2 clinical herds (clinical population) and 120 milk samples from 3 nonclinical herds (nonclinical population) without clinical cases at the time of enrollment. One herd sampled at different times was common to both populations. A 3-test in 2-population Bayesian latent class model with uniform priors for all test parameters except specificity of culture, which was modeled informatively, was used to estimate sensitivity (Se) and specificity (Sp) of the test kit, culture, and PCR at the cow level. The mastitis test kit's 96.9% Sp for Streptococcus spp. had a low false positive percentage (3.1%), which, together with the kit's rapid turnaround time for results, makes it a suitable initial screening test that producers can use to identify clinical cows to treat based on Streptococcus spp. mastitis in kit-positive results. Due to the 60.4% kit Se, producers should follow up on Streptococcus spp. kit-negative cows using a confirmatory test such as PCR (Sp of 98.4%) or culture (Sp of 99.6%). In contrast, aerobic culture had Se of 76.5% and Sp of 99.6% for Streptococcus spp. Similarly, the Sp of the kit (98.2%) and culture (99.8%) for Staph. aureus were particularly high, and even though the kit's Se (61.0%) was lower than culture (88.4%; posterior probability of difference 98%), the kit could be beneficial before use of a confirmatory test for kit-negative samples due to its ease and rapid turnaround time. Mostly, quantitative real-time (q)PCR outperformed the kit's Se (37.7%) and Sp (92.9%) for coliforms, as well as the kit's Se (60.4%) for Streptococcus spp. However, qPCR may require more technical skills and turnaround time for final results. Use of the on-farm mastitis test kit evaluated in the present study could enhance sustainable antimicrobial drug use by rapidly identifying Streptococcus mastitis for targeted treatment. Furthermore, the kit may be used in a Staph. aureus outbreak where cows can be rapidly screened to identify cases for segregation or culling during an outbreak and kit-negative cows further confirmed by milk culture or qPCR. However, the cost-effectiveness of such an approach has not been investigated.     

Quantitative PCR coupled with sodium dodecyl sulfate and propidium monoazide for detection of viable Staphylococcus aureus in milk

Conventional quantitative PCR (qPCR) are unable to differentiate DNA of viable Staphylococcus aureus cells from dead ones. The aim of this study was to use sodium dodecyl sulfate (SDS) and propidium monoazide (PMA) coupled with lysostaphin to detect viable Staph. aureus. The cell suspensions were treated with SDS and PMA before DNA extraction. The SDS is an anionic surfactant, which can increase the permeability of dead cells to PMA without compromising the viability of live cells. The lysostaphin was applied to improve the effectiveness of DNA extraction. The reliability and specificity of this method were further determined by the detection of Staph. aureus in spiked milk. The results showed that there were significant differences between the SDS-PMA-qPCR and qPCR when a final concentration of 200 μg/mL of lysostaphin was added in DNA extraction. The viable Staph. aureus could be effectively detected when SDS and PMA concentrations were 100 µg/mL and 40 μM, respectively. Compared with conventional qPCR, the SDS-PMA-qPCR assay coupled with lysostaphin was more specific and sensitive. Therefore, this method could accurately detect the number of viable Staph. aureus cells.     

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.     

多重PCR技术在食源性病原细菌检测中的应用

食品中污染的病原细菌是引起食源性疾病的主要因素之一,快速检测食品中病原细菌是及时有效预防病原细菌传播及食物中毒的重要前提。多重PCR作为一种可同时检测多种病原细菌的方法应用日益广泛。本文介绍了多重PCR技术在食源性病原细菌检测中的应用现状及其多重PCR反应条件的优化,同时提出了存在的问题并对应用前景作了展望。     

多重PCR同时检测食品中4 种细菌与常见霉菌

建立一种同时检测食品中金黄色葡萄球菌、单核细胞增生李斯特氏菌、沙门氏菌、大肠杆菌O157:H7以及霉菌的多重聚合酶链式反应（polymerase chain reaction,PCR）检测方法。根据金黄色葡萄球菌的耐热核酸酶基因（nuc）、沙门氏菌的侵袭正调节蛋白基因（hilA）、大肠杆菌O157:H7鞭毛基因（flic）、单核细胞增生李斯特氏菌的毒力调控蛋白基因（prfA）及霉菌18S?rRNA基因V5区分别设计5?对特异性引物,并对PCR扩增反应体系和扩增条件进行优化,确定获得特异性良好的PCR扩增产物。而后在37?℃对人工污染致病菌的香肠、面包和豆腐进行增菌培养,5?种致病微生物在20?h内的检出限均可达到100?CFU/25?g。本实验建立的多重PCR检测方法适用于食品中4?种细菌与常见霉菌的同时检测,相较于传统检测方法,具有快速、简便、特异性高的优点。     

食品中3种致病菌多重PCR检测方法的建立及初步应用

建立用多重聚合酶链式反应(Multiplex polymerase chain reaction,mPCR)同时检测食品中沙门氏菌、金黄色葡萄球菌和单核细胞增生性李斯特菌的方法。以沙门氏菌的gyrB基因、单核细胞增生性李斯特菌的gyrB基因、金黄色葡萄球菌的coa基因作为目的基因,分别设计3对引物,通过优化反应体系,建立3种致病菌的多重PCR检测体系。采用单重PCR检测时,灵敏度可达0.423ng/mL(沙门氏菌)、2.5ng/mL(金黄色葡萄球菌)和0.36ng/mL(单核细胞增生性李斯特菌);而采用三重PCR检测时,灵敏度较单重PCR有所下降,分别为2.43ng/mL(沙门氏菌)、2.5ng/mL(金黄色葡萄球菌)、3.6ng/mL(单核细胞增生性李斯特菌)。初步建立能同时、快速、灵敏地检测食品中沙门氏菌、金黄色葡萄球菌和单核细胞增生性李斯特菌的三重PCR方法。     

The Use of Multiplex PCR to Determine the Prevalence of Enterotoxigenic Staphylococcus aureus Isolated from Raw Milk,Feta Cheese,and Hand Swabs

Staphylococcus aureus (S. aureus) can cause mastitis in cattle and, therefore, can be present in milk. This study was undertaken to determine the prevalence of coagulase positive S. aureus and its enterotoxin genes sea, seb, and sec in isolates recovered from raw milk, feta cheese, and human hand swabs of milk and cheese handlers in Beni‐Suef province, Egypt. A total of 100 samples of raw milk and 50 samples of pasteurized‐milk feta cheese were collected. In addition, 50 hand swabs from milk handlers and 25 hand swabs from cheese handlers were examined for the presence of coagulase positive S. aureus. The isolates were characterized by multiplex PCR for detection of sea, seb, and sec genes, and for resistance to 5 classes of commonly used antibiotics. Twelve (12/100), 12 (6/50), and 17% (13/75) of milk, cheese, and hand swab samples, respectively, were positive for coagulase positive S. aureus. One isolate was obtained from each positive sample (31 isolates), and none contained genes for SEA or SEC production. Twenty‐five percent, 33%, and 31%, respectively, of the isolates contained the genes for SEB, resulting in 3%, 4%, and 5% of samples being positive for toxin producing coagulase positive S. aureus, respectively. At least one isolate was resistant to each of the antibiotics tested. Despite the low potential for SEB production shown, preventative measures, such as maintenance of the cold‐chain and good hygienic practices should be implemented to further reduce the potential risk to public health from SEB, and to reduce the spread of antimicrobial resistance.     

Development of a new real-time quantitative PCR assay for the detection of Staphylococcus aureus genotype B in cow milk,targeting the new gene adlb

The specific and reliable diagnosis of mastitis pathogens is essential for successful sanitation programs. The aim of the present study was to develop and evaluate a new real-time quantitative PCR (qPCR) assay for the very sensitive and specific detection of Staphylococcus aureus genotype B in cow milk samples. This mastitis pathogen is contagious and particularly prevalent in Switzerland and other central European countries. The new test is based on a rapid preparation of bacteria, followed by DNA isolation and qPCR for a unique target gene coding for the adhesion-like bovine protein (adlb). The inclusivity of the new target gene was 97% and the exclusivity 98%, meaning that other genotypes and bacterial species could be excluded with high reliability. The limit of detection of the new assay was 235 staphylococcal cell equivalents/mL of culture. The new test shows high intra- and interassay repeatability. Results are available within 2 d after sampling, allowing farmers and veterinarians to apply sanitation measures immediately. Based on the results of a preliminary field study, the diagnostic sensitivity and specificity of the new qPCR assay are 99 and 100%, respectively. The new analytical procedure is straightforward and can be applied for routine diagnostics.     

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.     

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.     

石河子地区乳源性金黄色葡萄球菌毒力及耐药性的研究

本文研究了石河子地区乳源性金黄色葡萄球菌的污染情况、毒力特性及耐药性。对24份零售鲜奶样品进行金黄色葡萄球菌定量检测,PCR方法检测nuc、16S r RNA和7种毒力基因,利用Kirby-Bauer法对分离得到的金黄色葡萄球菌进行了12种抗生素的药物敏感试验。共分离55株疑似乳源性金黄色葡萄球菌,nuc和16S r RNA基因PCR扩增检测结果中有20株菌为阳性。毒力基因检测结果中有5株(5/20;25%)携带毒力基因,其中3株携带Coa(3/5;60%),1株携带Sei(1/5;20%),1株携带Sea+Sec+Coa(1/5;20%)。发现20株金黄色葡萄球菌中有19株对所测试的一种或多种抗生素具有抗性,且19株均对亚胺培南和氯霉素敏感。本文为指导抗生素在人类临床和动物饲养中的合理应用提供重要的参考性数据,为乳品中有害金黄色葡萄球菌的风险评估提供理论依据。     

Two-step large-volume magnetic separation combined with PCR assay for sensitive detection of Listeria monocytogenes in pasteurized milk

Immunomagnetic separation (IMS) is an effective tool for the preconcentration and purification of food-borne pathogens from complex food samples because of its high capture efficiency (CE). In conventional IMS, antibodies are usually conjugated on the surface of magnetic beads (MB); the random orientation and conformation changes of antibodies on the MB surface can decrease their bioactivity. Moreover, the Brownian motion of immobilized antibodies is weakened, thereby rendering their binding efficiency with bacteria lower than that of free antibodies. Thus, abundant antibodies are commonly required to ensure high CE for IMS, particularly for large volumes. In this study, a 2-step large-volume magnetic separation (10 mL) was proposed to preconcentrate Listeria monocytogenes from pasteurized milk. First, the biotinylated anti-L. monocytogenes monoclonal antibodies (mAb) were bound with L. monocytogenes in 10 mL of diluted milk through an antigen-antibody interaction, and then streptavidin-labeled MB were used to capture biotin-mAb coated with L. monocytogenes by biotin and streptavidin interaction. Under optimal conditions, the CE of 2-step magnetic separation was >90% with L. monocytogenes concentrations ranging from 8 × 10⁰ to 8 × 10⁴ cfu/mL, whereas the amount of biotin-mAb was 14 fold lower than that of the conventional IMS method. Coupled with a PCR assay, the detection limit of the proposed method was 8 × 10⁰ cfu/mL in pure culture and 8 × 10¹ cfu/mL in pasteurized milk without any pre-enrichment process. Moreover, the overall detection time, including sample preparation, large-volume magnetic separation, and PCR, took less than 7 h. In summary, the developed 2-step large-volume IMS combined with PCR was highly sensitive and low cost and, thus, has considerable potential for the rapid screening of food-borne pathogenic bacteria.     

Development of a method for the quantification of haddock (Melanogrammus aeglefinus) in commercial products using real-time PCR

A method is presented for the quantification of haddock in complex food matrices based on real-time PCR. The proportion of haddock muscle tissue to the numbers of a single copy gene has been shown to remain relatively constant, throughout the year and across a number of fishing grounds, using conventional muscle nitrogen content as a comparator. This indicates that quantification of fish, based upon nitrogen content or the numbers of a single copy gene, would return data with a similar degree of accuracy. Thus, a haddock-specific, real-time PCR primer and probe set was designed and optimised, and when used in conjunction with a haddock calibration curve was shown to be able to quantify model samples to within 7% of the true percentage. This is the first report of a real-time assay for the quantification of white fish, which would allow enforcement of the legislation in a complex food matrix containing haddock with other fish species.