Evaluation of PCR test for detecting major pathogens of bubaline mastitis directly from mastitic milk samples of buffaloes

Present study is aimed to evaluate the PCR test for detecting the major pathogens of bubaline mastitis (BM) directly from the mastitic milk samples of the buffaloes. Cell lysates of the enriched mastitic milk samples were directly used as template DNA in PCR and the reactivity of genus and/or species specific primers against the selected pathogens of BM was tested in individual simplex PCR assays. Out of the 60 mastitic milk samples tested 30 were positive for E. coli, 21 were positive for S. aureus, 4 were positive for S. agalactiae, 9 were positive for S. dysgalactiae and 7 were positive for S. uberis. Certain samples were positive for more than one pathogen thus indicating the mixed infection of the udder. Although certain mastitic milk samples reacted with Staphylococcus genus specific primers, they didn’t react with S. aureus specific primers in PCR, which indicates the implication of other species of Staphylococcus in BM. The processing of mastitic milk samples was also simplified by eliminating the use of expensive reagents. The detection limits of PCR with the cell lysates are sensitive enough for PCR to be used as diagnostic tool for identifying the pathogens of BM.     

Bacteriological diagnosis with Petrifilm of mastitis pathogens in milk samples from each quarter and bulk milk samples

Three experiments were conducted to evaluate four different Petrifllm products (3M, Neuss) for detection of mastitis pathogens in quarter and bulk milk samples, comparing them to the results of standard microbiological techniques. The aim of experiment 1 was to determine the sensitivity of 3M Rapid Coliform Count Plate in identifying clinical mastitis cases caused by coliform bacteria. Within 12 h of incubation, three times more coliform bacteria could be identified with Petrifilm than with the standard technique. For a valid result, milk samples must be free of contamination. Experiment 2 focused on whether Petrifilm was able to monitor S. aureus on bulk milk level in herds being infected with this pathogen. In relation to the gold standard (combination of both procedures (standard and Petrifilm, prevalence 52%), sensitivity for the standard procedure amounted to 15.4% and to 94% for Petrifilm. In Experiment 3 the combination of several Petrifilm (RUEGG, 2004) was compared with the standard diagnostic technique (gold standard). Sensitivity of the Petrifilm method approached the assumed gold standard to 43% and specificity to 29%. The positive predictive value of 28% showed that both procedures are not directly comparable with each other. Due to the definition of a gold standard, the weaknesses of the classical technique can be interpreted as a disadvantage of the Petrifilm procedure. The strength of the available Petrifilm as mastitis diagnostic tools is the identification of S. aureus and coliform microorganisms, moreover E. coli.     

Detection of virulence-associated genes in Staphylococcus aureus isolated from bovine clinical mastitis milk samples in Guangxi

Staphylococcus aureus is recognized worldwide as a pathogen causing many serious diseases in humans and animals and is one of the most common etiological agents of clinical and subclinical bovine mastitis. The purpose of this study was to determine the presence of genes encoding clfA, fnbA, fnbB, cap5, cap8, hla, hlb, nuc, sea, and tst of S. aureus strains (n?=?39) isolated from bovine clinical mastitis in Guangxi by polymerase chain reaction amplification. The results of the present study indicated that all isolates were found to contain one or more virulence-associated genes. The most frequently encountered genes were fnbA (97?%) and nuc (90?%), followed by hla (85?%) and hlb (82?%), respectively. None of the investigated S. aureus strains harbored fnbB and sea genes. The data in the present study showed a relatively wide distribution of the genes fnbA and nuc among the investigated isolates, indicating that they play an important role on bovine mastitis pathogenesis. The study provides a valuable insight into the virulence-associated genes of this important pathogen.     

Culture results from milk samples submitted to veterinary diagnostic laboratories from August 2003 to December 2006 in New Zealand

Abstract

AIMS: To determine the pattern of isolation of major mastitis-causing organisms isolated from milk samples submitted to five veterinary diagnostic laboratories in New Zealand.

METHODS: The culture results of 25,288 milk samples that were collected from dairy cows throughout New Zealand from August 2003 to December 2006 and submitted to a group of veterinary diagnostic laboratories were assembled, reviewed and summarised. Logistic regression was used to analyse the effect of year, region (i.e. North vs South Island), and season on the probability of isolating the two most common organisms.

RESULTS: The most commonly isolated mastitis causing organisms from all samples were: Streptococcus uberis (23.6%), Staphylococcus aureus (23.5%), coagulase-negative staphylococci (CNS; 7.2%), Strep. dysgalactiae (6.2%), Bacillus spp. (4.0%), and coliforms (3.7%). The percentage of samples with isolates of Strep. uberis or Staph. aureus was affected by island, year and season (p<0.001). For most of the year, except in late winter and early spring when Strep. uberis was much more common, the percentage of isolates of Strep. uberis and Staph. aureus were not apparently different despite the former being an environmental pathogen and the other a contagious one.

CONCLUSION: The pattern of isolation of major mastitis-causing organisms, as determined from culture of milk samples submitted to diagnostic laboratories in New Zealand, has changed significantly over the last 40 years, with a substantial increase in the percentage of isolates that are Strep. uberis and a decrease in isolates of Strep. agalactiae. There is a clear seasonal pattern to the isolation of both Strep. uberis and Staph. aureus, particularly the former.

CLINICAL RELEVANCE: Knowledge of the aetiological agents causing bovine mastitis on a farm is of value in determining the choice of treatment. This dataset shows that, although there is seasonal pattern to the isolation of mastitis-causing organisms in New Zealand, both Strep. uberis and Staph. aureus are isolated throughout the year, so bacteriology is of value in determining aetiology even in late winter/early spring.     

Reliability of three bulk-tank antimicrobial residue detection assays used to test individual milk samples from cows with mild clinical mastitis.

OBJECTIVE: To determine the likelihood of false-positive results when testing milk samples from individual cows by use of 3 commercially available assays (Penzyme MilkTest and the SNAP beta-lactam and Delvo-SP assays) labeled for use with commingled milk. SAMPLE POPULATION: Milk samples from 111 cows with mild clinical mastitis. PROCEDURE: Cows were randomly assigned to the control (no antimicrobials) or intramammary treatment group. Posttreatment milk samples were collected at the first milking after the labeled withholding period or an equivalent time for controls, randomly ordered, and tested twice by use of each assay and once by use of high-performance liquid chromatography. Sensitivity, specificity, and positive and negative predictive values were determined for each assay. Concordance of results for the same sample was assessed for each assay by calculating kappa. RESULTS: Sensitivities of the Delvo-SP and SNAP lactam assays were > 90%, whereas the sensitivity of the Penzyme Milk Test was 60%. Positive predictive values (range, 39.29 to 73.68%) were poor for all 3 assays. Concordance of test results was excellent for the SNAP beta-lactam and Delvo-SP assays (kappa = 0.846 and 0.813, respectively) but was less for the Penzyme MilkTest (kappa = 0.545). CONCLUSIONS AND CLINICAL RELEVANCE: Because of the low positive predictive values, these 3 assays may not be useful for detecting violative antimicrobial residues in individual milk samples from cows treated for mild clinical mastitis. However, repeatability of each assay was considered good to excellent.     

Effect of preculture freezing and incubation on bacteriological isolation from subclinical mastitis samples

In this study the sensitivity of three methods of isolation of udder pathogens from milk samples from subclinical mastitis cases was compared. For analysis 1827 quarter milk samples were selected. Milk was cultured using a standard culture technique (0.01 ml of fresh milk streaked on a sheep blood agar plate and on Edward's medium). In addition, an inoculum of 0.01 ml of the original milk sample was incubated for 24h at 37 degrees C in broth, followed by culture using the standard culture technique. In the third method, the whole milk sample was frozen for 24h, and then incubated for 24h at 37 degrees C, followed by culture using standard culture technique. The isolation percentage of Staphylococcus aureus was 4.7% for standard culture technique, 14.2% for incubation in broth, and 21.5% for the combination of freezing plus incubation. Isolation percentage of Streptococcus dysgalactiae and Streptococcus agalactiae was highest using the standard culture technique, while isolation rate of Streptococcus uberis was not different among the three methods used. With increasing somatic cell count, the likelihood of S. aureus, S. dysgalactiae and S. uberis isolation increased.Based on the relative sensitivity, defined as the isolation rate using a single technique compared to the isolation rate of the three techniques together, a combination of standard culture technique and freezing plus incubation was most attractive for achieving a high isolation rate of S. agalactiae and S. dysgalactiae. Relative sensitivity of S. uberis isolation was highest using the standard culture technique and incubation in broth, while S. aureus was most often isolated using a combination of incubation in broth and freezing plus incubation. A combination of the three methods increased the isolation rate for S. dysgalactiae, S. uberis and S. aureus. The standard culture technique, together with the combination of freezing plus incubation, can be recommended for isolating major udder pathogens. If S. aureus is the pathogen of main interest, using incubation in broth together with the combination of freezing plus incubation performed best.     

Comparison of the epidemiological behavior of mastitis pathogens by applying time-series analysis in results of milk samples submitted for microbiological examination

The objective of this study is to examine and compare the trends of mastitis pathogens in quarter milk samples (n?=?240,232) submitted for microbiological examination at the Milk Analysis Laboratory (L.I.G.A.L.) at Galicia, Spain from June 2005 to September 2011. Autoregressive Integrated Moving Average (ARIMA) models and multivariate statistical techniques such as Cluster Analysis were used in order to detect seasonal trends and similarities between the series trends and to classify mastitis pathogens into relatively homogeneous groups. The decrease of bulk milk somatic cell counts achieved by the mastitis control program, developed in recent years in this region, is the result of the decrease in IMI caused by a limited number of mastitis pathogens. The obtained results reflect a greater complexity in the behavior of mastitis pathogens, unlike the traditional classification into contagious or environmental. Staphylococcus aureus showed a trend similar to Streptococcus dysgalactiae, a mastitis pathogen can behave in both a contagious and an environmental manner. Among the traditionally considered environmental mastitis pathogens, Strep. uberis showed a different behavior to Escherichia coli and Klebsiella pneumoniae. Coagulase-negative staphylococci (CNS) species and Streptococcus other than Strep. agalactiae showed differences in the trend model. Time-series analysis and multivariate statistical techniques, such as Cluster Analysis, could be powerful tools to assess the isolation trend of mastitis pathogens because of their ability to cope with stochastic dependence of consecutive data. Furthermore, they could be used to identify the epidemiological behavior of mastitis pathogens using the results of milk samples submitted for routine microbiological examination, by classifying them into relatively homogeneous groups.     

Occurrence and characterization of methicillin-resistant staphylococci from bovine mastitis milk samples in Finland

Background

Methicillin-resistant staphylococci (MRS) are increasingly being isolated in bovine mastitis. The aim of our study was to evaluate the occurrence of MRS in Finnish mastitis milk samples and characterize the MRS isolates using molecular methods.

Results

Methicillin-resistant S. aureus (MRSA) was a rare finding in bovine mastitis in Finland. Only two out of 135 (1.5%) S. aureus isolates were positive for mec genes. One of these carried mecA and was of spa type t172, SCCmec type IV and ST375, and the other harboured mecC, being spa type t3256, and ST130. MRSA ST375 is common among human MRSA isolates in Finland, but this is the first report in the country of bovine mecC MRSA. In coagulase-negative staphylococci (CoNS) originating from bovine mastitis, methicillin resistance was more common. In the two CoNS collections studied, 5.2% (17/324) and 1.8% (2/110) of the isolates were mecA positive. Eighteen of these were methicillin-resistant S. epidermidis (MRSE), which were divided into 6 separate PFGE clusters. One pulsotype was detected in different parts of the country, indicating clonal spread. Most MRSE (13/18) were of SCCmec type IV, one was of type V and four were non-typeable. Comparison with a human staphylococcal database indicated that bovine MRSE strains were not closely related to human MRSE isolates.

Conclusions

The occurrence of MRS, especially MRSA, in bovine mastitis in Finland was low. Most methicillin-resistant bovine CoNS are MRSE, and we found evidence of a bovine MRSE strain that may spread clonally. This is the first report of a Finnish bovine isolate of MRSA_mecC ST130. The study provides a baseline for further MRS monitoring.     

Genotypical differentiation of Prototheca isolates of milk samples from mastitis affected cattle in Germany

To elucidate the spectra of Prototheca species and P. zopfii genotypes in milk samples from mastitis affected cattle, 200 Prototheca-isolates of 57 German dairy cattle barns were analyzed by species- and genotype- specific PCR and RFLP analysis. The investigations showed that 177 (88.5%) of all isolates represented P. zopfii genotype 2, 21 (10.5%) isolates represented P. blaschkeae and two (1.0%) P. zopfii genotype 1. Therefore mainly P. zopfii genotype 2 but also P. blaschkeae act in the epidemiology of mastitis in cattle, whereas the role of P. blaschkeae in development of bovine protothecal mastitis should be further explored in epidemiological studies. In this study P. blaschkeae was demonstrated for the first time in association with bovine mastitis in Germany.     

Application of an indirect ELISA to milk samples to identify cows with Mycoplasma bovis mastitis

An indirect ELISA was used to detect antibodies to Mycoplasma bovis in milk samples collected from a herd with M bovis mastitis. Antibodies were detected in samples from nine cows which had developed clinical M bovis mastitis. Milk from only three consistently antigen-negative cows tested positive for M bovis antibodies. These results indicate the potential value of the indirect ELISA for the detection of cows which have recently developed M bovis mastitis during the early stages of an outbreak.     

Studies of bovine Mycoplasma mastitis. 2. Testing of various culture media and culture methods for the isolation of Mycoplasma from milk samples]

The recipes Medium-I broth, Medium-B broth, and Weissenseemycoplasma broth as well as Medium-I agar, Medium-B agar, and MRL agar were tested for their applicability to culturing mycoplasma from milk samples, using the direct and indirect techniques. No dependable information on the occurrence of mycoplasma was obtainable from tested material unless several nutritive media and techniques were combined. Medium I, for which almost no imported substances were needed, was in no way inferior to common international nutritive substrates. Its use in conjunction with the indirect culturing technique is recommended for routine diagnosis.     

The correlation between California mastitis test results and the presence of mastitis pathogens in composite milk samples

Extract

In this, the Ira Cunningham Memorial Lecture, I will not detail all the Professor's achievements. He did so much so well, that I would be hard put to do justice to his record in the time available. What I will do is reflect on his professionalism and on the way he took that professionalism out to work in society.     

Resistance situation and enterotoxin production capacity of Staphylococcus aureus strains from bovine mastitis milk samples]

In total, 63 S. aureus strains from mastitis milk samples of different animals in 57 farms were isolated. In 14 (22%) of the S. aureus strains resistancies against one or several of the examined antibiotics could be observed whereby six resistance patterns were found. 14.3% of the strains were penicillin resistant. 34 (54%) of the 63 S. aureus produced enterotoxins (SE). Three strains formed SEA, 21 SEC, three SED and seven strains 2 SE, SEAC, SEAD or SEBD.     

Observational study on antimicrobial resistance in Escherichia coli and Salmonella isolates from Ontario calf samples submitted to a diagnostic laboratory from 2007 to 2020

The objectives of this study were to i) describe Escherichia coli and Salmonella isolates; ii) investigate the temporal trends in antimicrobial resistance (AMR) profiles; and iii) evaluate the impact of season and age on these AMR profiles from diagnostic and post-mortem samples in Ontario calves ≤ 2-months-old submitted from 2007 to 2020 to the Animal Health Laboratory in Guelph, Ontario, Canada. Antimicrobial susceptibility testing results were measured by the Kirby-Bauer disk diffusion method. A total of 1291 isolates with AMR profiles were obtained from calves, with E. coli (n = 434) and Salmonella (n = 378) being the most common bacteria characterized for AMR. For E. coli, 79% of isolates tested showed a positive result in F5/K99, whereas for Salmonella isolates, S. Typhimurium (33%) and S. Dublin (22%) were the 2 most common serotypes identified. Multivariable logistic regression models were built to evaluate AMR profiles for E. coli (n = 414) and Salmonella (n = 357) to each antimicrobial tested. Most E. coli isolates (91%) and Salmonella isolates (97%) were resistant to at least one of the antimicrobials tested. In general, E. coli and Salmonella had higher odds of resistance in calves aged ≥ 2 wk compared to 1-week-old calves, and little difference was seen in the level of resistance over the years observed or between seasons in most of the antimicrobials tested. Prospective research should investigate potential risk factors for the development of AMR in calves examples being antimicrobial use and farm management practices.     

Antimicrobial resistance of Staphylococcus aureus and coagulase negative staphylococci isolated from mastitis milk samples from sheep and goats

Staphylococcus aureus and coagulase negative staphylococci (CNS) were isolated from ovine and caprine mastitis milk samples originating from more than 40 Swiss farms. CNS dominated as causal microorganisms of mastitis in small ruminants. By restriction fragment length polymorphism (RFLP) analysis of the groEL gene and sequencing of 16S rDNA, various CNS species were identified, albeit certain of them predominated. For susceptibility testing of mastitis pathogens to selected antibiotics, minimal inhibitory concentrations were determined. Of the 67 S. aureus and 208 CNS strains, 31.3 % and 8.2 % were resistant to penicillin, 29.9 % and 1.0 % to ampicillin, 1.5 % and 10.6 % to erythromycin, and 3.0 % and 7.7 % to tetracycline, respectively. Moreover, 10 CNS strains (4.8 %) were resistant to oxacillin and one CNS strain to sulfamethoxazole/trimethoprim. The results obtained describe for the first time the resistance situation of mastitis pathogens from sheep and goats in Switzerland. However, accompanying and preventing measures are also of importance in mastitis control of small ruminants.