Major Mastitis Pathogens Research Articles

Exploring antimicrobial activity of Lactobacillus spp. (probiotics) isolated from raw cow’s milk against Staphylococcus aureus causing bovine mastitis

Bovine mastitis is a costly disease in the dairy farms globally. The control of such disease is generally based on the prevention by the strict hygienic measures during milking. Other approaches include vaccination and the application of antibiotics. Regardless to these procedures, mastitis is not entirely under the control, thus increasing the need for alternative tactics. This study was conducted to isolate and identify lactic acid bacteria (LAB) from fresh cow’s milk which possess antibacterial activity that could be used for mastitis control. 146 isolates were recognized as (LAB) from105 milk tanks samples after being cultured anaerobically on de Man, Rogosa and Sharpe (MRS) agar plates for 48 hours at 37 oC and identified by general bacteriological investigation. Afterwards, 24 isolates were identified to belong to genus Lactobacillus using polymerase chain reaction (PCR), and for species level recognition MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) was used resulted in : L. fermentum (5), L. brevis (3), L. plantarum (4), L. paracasei (2), L. rhamnosus (3), L. pentosus (2), L. casei (3), L. raffinolactis (1) and L. mesenteroids (1). The antimicrobial activity of these strains against one of the major mastitis pathogens, S. aureus,was detected by the agar well diffusion assay and the modified double layer method , where L. casei, L. fermentum andL. plantarum possess the most inhibiting effect besides they have no hemolytic nor gelatin liquefaction activity when their safety profiles were evaluated. The result of the antibiotic susceptibility test revealed that these isolates were resistant to vancomycin (VA), neomycin (N) and gentamycin (CN). On the other hands, they were highly sensitive to amoxicillin clavulanic acid (AMC), levamisole (LEV), tetracycline (TE) and penicillin (P).The study suggests that L. casei, L. fermentum and L. plantarum are perfect candidates to be used as probiotics to help in preventing and controlling bovine mastitis caused by Staphylococcus aureus as they were proven to be safe and have antimicrobial activity againstthe organism.

An Understanding of the Global Status of Major Bacterial Pathogens of Milk Concerning Bovine Mastitis: A Systematic Review and Meta-Analysis (Scientometrics).

In this study, the major mastitis pathogen prevalence in the cattle and buffalo of the world was estimated by a meta-analysis. Staphylococcus (S) species, Streptococcus (St) species, and Escherichia coli (Ec) prevalence studies reported during 1979–2019 were collected using online databases, and offline resources. A meta-analysis of these data was done with the meta package in R-Software. The Staphylococcus aureus was the major mastitis pathogen, mostly causing subclinical mastitis, Ec causing clinical mastitis and St causing subclinical and clinical mastitis. The pooled prevalence estimates of S, St, and Ec were 28%, 12%, and 11% in the world from 156, 129, and 92 studies, respectively. The S, St, and Ec prevalences were high in Latin America (51%), Oceania (25%), and Oceania (28%), respectively. Higher S, St, and Ec prevalences were observed by molecular methods, signifying high sensitivity and usefulness for future studies. Among bacterial species, S. aureus (25%) followed by coagulase-negative Staphylococcus species (20%), Escherichia coli (11%), St. agalactiae (9%), St. uberis (9%) were the important pathogens present in the milk of the world. We hypothesize that there is a urgent need to reduce mastitis pathogen prevalence by ensuring scientific farm management practices, proper feeding, therapeutic interventions to augment profits in dairying, and improving animal and human health.

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

The 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.

Risk Factors for the Occurrence of Methicillin-Resistant Staphylococcus aureus in Dairy Herds: An Update.

In dairy cows, Staphylococcus aureus is a major mastitis pathogen and methicillin-resistant S. aureus (MRSA) has been reported from dairy farms around the world. The risk of foodborne zoonotic infections with bovine MRSA strains seems to be low since MRSA prevalence is low in dairy herds and milk is commonly heat treated before consumption. However, bovine mastitis caused by MRSA is an important issue in veterinary medicine since treatment options with non-β-lactam antibiotics are limited. For the development of effective MRSA prevention strategies, it is necessary to know which factors increase the risk for MRSA transmission into and within dairy herds. Therefore, the aim of this review is to summarize the risk factors for the occurrence of MRSA in dairy herds and to identify the respective knowledge gaps. MRSA was more frequently detected in conventional dairy farms than in organic farms and in larger farms than in smaller farms. Dairy farms housing pigs along with cattle are more frequently affected by MRSA. Moreover, humans carrying MRSA can probably infect dairy cows. Consequently, pigs and humans may introduce new MRSA strains into dairy herds. MRSA transmission within dairy herds was associated with improper milking hygiene procedures. Furthermore, methicillin-resistant coagulase-negative staphylococci (MR-CoNS) were repeatedly isolated from dairy farms. This is an important issue since MR-CoNS may transfer resistance genes to S. aureus. The role of antimicrobial exposure as a risk factor for the occurrence of MRSA within dairy herds needs to be further investigated.

Monitoring and Characteristics of Major Mastitis Pathogens from Bulk Tank Milk in Korea.

Simple SummaryThe monitoring of milk quality and the presence of major mastitis pathogens is an important part of milk quality assurance program. Bulk tank milk has been used in many countries to identify multiple milk quality problems and mastitis pathogens that might exist in a dairy herd. This study aimed to compare the presence of mastitis pathogens and the antimicrobial resistance of the isolates from bulk tank milk by dairy companies. The results showed that the prevalence of mastitis pathogens and the antimicrobial resistance of the isolates were significantly different among factories, and support the development of strong monitoring and prevention programs in dairy operations.In many countries, bulk tank milk (BTM) has been used for examining milk and analyzed as an important part of milk quality assurance programs. The objectives of this study were to investigate milk quality and the presence of major mastitis pathogens in BTM, and to compare the characteristics of BTM by dairy factory or company. A total of 1588 batches of BTM samples were collected from 396 dairy farms of seven dairy factories owned by four companies in Korea. The means of individual bacterial counts (IBC) and somatic cell count (SCC) were 3.7 × 104 cells/mL and 1.1 × 105 cells/mL, respectively, and no significant differences among dairy factories were observed. The most common pathogen was Staphylococcus spp. (60.1%), followed by E. faecalis (53.8%), E. coli (37.6%) and Streptococcus spp. (22.5%). Enterococcus spp. showed the highest resistance to tetracyclines (51.1% to 73.9%) and macrolides (46.5%). S. aureus and coagulase-negative staphylococci (CNS) showed the highest resistance to penicillin (28.4% and 40.2%, respectively), and three (3.2%) S. aureus and seven (3.3%) CNS were also methicillin-resistant. These data show the diverse prevalence and characteristics of major mastitis pathogens among factories, and support the development of strong monitoring and prevention programs of mastitis pathogens by commercial dairy operations.

Antibacterial properties of lauric acid in combination with organic acids against major pathogens causing dairy mastitis

The objective of this study was to determine the antibacterial properties of lauric acid in combination with acetic acid and lactic acid against major dairy mastitis pathogens including Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, Escherichia coli and Klebsiella spp. The antibacterial effect of each acid and the acid mixtures was evaluated by their minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC) using broth microdilution method. The differences in, MIC and MBC values of lauric acid and acid mixtures for each pathogen were calculated by applying the one-way analysis of variance (ANOVA) and Turkey’s multiple-range tests were used for pairwise comparison. Results demonstrated that acetic acid had the highest inhibitory and bactericidal effect against all tested pathogens with the lowest MIC and MBC values of 0.125% and 0.25-1 %w/v, respectively. The mixture of lauric acid with acetic and lactic acid exhibited significant higher inhibitory and bactericidal effects by having the lower MIC and MBC values against all tested pathogens when compared with lauric acid alone (P < 0.05). In conclusion, acetic and lactic acid can enhance antibacterial properties of lauric acid against major mastitis pathogens.

Proteomic Characterization of Antibiotic Resistance, and Production of Antimicrobial and Virulence Factors in Streptococcus Species Associated with Bovine Mastitis. Could Enzybiotics Represent Novel Therapeutic Agents Against These Pathogens?

Streptococcus spp. are major mastitis pathogens present in dairy products, which produce a variety of virulence factors that are involved in streptococcal pathogenicity. These include neuraminidase, pyrogenic exotoxin, and M protein, and in addition they might produce bacteriocins and antibiotic-resistance proteins. Unjustifiable misuse of antimicrobials has led to an increase in antibiotic-resistant bacteria present in foodstuffs. Identification of the mastitis-causing bacterial strain, as well as determining its antibiotic resistance and sensitivity is crucial for effective therapy. The present work focused on the LC–ESI–MS/MS (liquid chromatography–electrospray ionization tandem mass spectrometry) analysis of tryptic digestion peptides from mastitis-causing Streptococcus spp. isolated from milk. A total of 2706 non-redundant peptides belonging to 2510 proteins was identified and analyzed. Among them, 168 peptides were determined, representing proteins that act as virulence factors, toxins, anti-toxins, provide resistance to antibiotics that are associated with the production of lantibiotic-related compounds, or play a role in the resistance to toxic substances. Protein comparisons with the NCBI database allowed the identification of 134 peptides as specific to Streptococcus spp., while two peptides (EATGNQNISPNLTISNAQLNLEDKNK and DLWC*NM*IIAAK) were found to be species-specific to Streptococcus dysgalactiae. This proteomic repository might be useful for further studies and research work, as well as for the development of new therapeutics for the mastitis-causing Streptococcus strains.

Effect of bismuth subnitrate on in vitro growth of major mastitis pathogens

The mode of action of bismuth subnitrate in teat sealant formulations as a preventative for intramammary infections during the dry period is unknown. Although previous studies proposed an action mechanism-creating a physical barrier in the teat canal to prevent bacterial invasion-it has not been proven experimentally. We hypothesized that bismuth subnitrate has an inhibitory effect on bacterial growth, in addition to its barrier effect. The objective of this study was to assess the effect of bismuth subnitrate on bacterial growth of major mastitis-causing agents. A strain of Streptococcus uberis (SR115), 2 strains of Staphylococcus aureus (SA3971/59 and SA1), and a strain of Escherichia coli (P17.14291) were tested in vitro for their ability to grow in the presence or absence of bismuth subnitrate. Disk diffusion testing, impedance measurement, and evaluation of bacterial growth in shaking conditions were the methods used to test this hypothesis. A reduction of growth in the presence of bismuth subnitrate occurred for all the strains tested. However, we observed strain and species variations in the extent of growth inhibition. These results suggest that an inhibitory effect on bacterial growth by bismuth subnitrate could partially explain the efficacy of bismuth-based formulations for preventing intramammary infections over the dry period. Further research is required to test the effect of teat sealant formulations on bacterial growth.

Short communication: Barrier characteristics of 3 external teat sealants to prevent bacterial penetration under in vitro conditions using rubber calf-feeding nipples

The main objective of this study was to evaluate the barrier characteristics of 3 external teat sealants for dry cows in preventing bacterial penetration by 3 common major mastitis pathogens (Escherichia coli, Staphylococcus aureus, and Streptococcus uberis) via a novel in vitro simulation model using rubber calf-feeding nipples. All feeding nipples were filled with a sterile cotton plug soaked in sterile broth heart infusion medium and were treated as follows: rubber teats 1 and 5 were sealed with Ubera Dry (Inovet, Arendonk, Belgium); rubber teats 2 and 6 were sealed with T-Hexx Dry (Huvepharma Livestock, St. Louis, MO); rubber teats 3 and 7 were sealed with Uddergold Dry (Ecolab Food and Beverage Division, St. Paul, MN); and rubber teats 4 and 8 remained unsealed and served as positive and negative controls, respectively. After the dips had dried, rubber teats 1 to 4 were immersed in a suspension of E. coli (experiment 1), Staph. aureus (experiment 2), or Strep. uberis (experiment 3; ≥1.5 × 108 cfu/mL) for 24 h, whereas rubber teats 5 to 8 were not exposed to the bacterial suspensions. All external teat sealants adhered well to the rubber teats. All cotton plugs collected from the teats that were not exposed to E. coli, Staph. aureus, or Strep. uberis (rubber teats 5 to 8) remained culture-negative, except for 1 (due to contamination). Of the rubber teats that were exposed to the major mastitis pathogens, all cotton plugs collected from the teats dipped with Ubera Dry and T-Hexx Dry remained culture-negative for the mastitis pathogen they were exposed to. The cotton plugs of the rubber teats that were sealed with Uddergold Dry and exposed to E. coli and Strep. uberis showed positive cultures for the respective bacteria, as demonstrated using strain-typing. The cotton plugs collected from the rubber teats that were not sealed with an external teat sealant and that served as positive controls became culture-positive for the mastitis pathogens they were exposed to. We conclude that Ubera Dry showed comparable and superior barrier performance against penetration of E. coli, Staph. aureus, and Strep. uberis compared with T-Hexx Dry and Uddergold Dry, respectively, under in vitro conditions using a novel in vitro simulation model. Although one should be aware that the method has not yet been validated to predict risk of intramammary infections, the proposed technique can be a meaningful starting point to evaluate and compare the barrier characteristics of external teat sealants in preventing bacterial penetration. A large-scale clinical trial is needed before any definite conclusions can be drawn as to the adherence, duration of adherence, barrier performance, and efficacy in protection against intramammary infections of the 3 external teat sealants under field conditions.

Genetic diversity and virulence characteristics of Staphylococcus aureus isolates from cases of bovine mastitis

Staphylococcus aureus is one of the major bacterial mastitis pathogens with significant effects on animal and human health. Some studies showed that S. aureus strains that infect different host species are genetically distinct, although most strains can infect a wide range of host species. However, there are no clearly defined clonal patterns of S. aureus strains that are known to infect a specific host. The objectives of this study were to evaluate the clonal diversity and virulence characteristics of S. aureus isolates from cases of bovine mastitis. Bacteriological tests were conducted on milk samples from cases of bovine mastitis from 11 dairy farms including some milk samples from unknown farms in Eastern Tennessee. Overall, a total of 111 S. aureus were isolated and identified, and further evaluated for their genetic diversity by pulsed-field gel electrophoresis (PFGE) and virulence characteristics by PCR. Genotypic virulence factors including staphylococcal enterotoxins, and toxic shock syndrome toxin 1 (tsst-1) were tested by PCR. In addition, the association among several known virulence factors of these isolates based on our current and previous studies in our lab were evaluated. Previously generated data that were included in the analysis of association among virulence factors were the presence of biofilm production associated genes in the ica operon such as icaA, icaD and icaAB, and phenotypic virulence characteristics such as hemolysis on blood agar, slime production and resistance or susceptibility to ten commonly used antimicrobials in dairy farms. The PFGE results showed the presence of 16 PFGE types (designated A – P) throughout farms, of which three pulsotypes, I, M and O were the most frequently isolated PFGE types from most farms. The PFGE type M was the most prevalent of all 16 PFGE types, with 64 isolates being present among nine farms. The PCR results of enterotoxin genes showed that out of the total 111 tested 84 (75.7%) were negative whereas 13 (11.7%), 2 (1.8%), 3 (2.7%), 1 (0.9%) and 8 (7.2%) were positive for seb, seb and sec, sec, see, and tsst-1, respectively. All 111 isolates were negative for sea and sej.Results of the evaluation of I, M and O strains adhesion to and invasion into mammary epithelial cells showed that the total count of each strain of bacteria adhered to and invaded into mammary epithelial cell line (MAC-T cells) was not significantly different (P > 0.05). This may be an indication that there is no significant difference in their ability to establish early host-pathogen interaction and colonization of the host.There were no statistically significant associations among PFGE types and other known virulence factors of these strains. However, PFGE types O and M tend to cluster with β-hemolysin, absence of enterotoxins and susceptibility to antimicrobials. In conclusion, there was not any association between pulsotype and genotypic and phenotypic virulence factors. S. aureus isolates from cases of bovine mastitis had diverse genotypes that possessed variable virulence factors.

Comparison of the population structure of Streptococcus uberis mastitis isolates from Austrian small-scale dairy farms and a Slovakian large-scale farm

Streptococcus uberis, a major mastitis pathogen associated with intramammary infections (IMI), can be found ubiquitously in the cow's environment. Although Strep. uberis is reported to be susceptible to most antimicrobials, in practice poor responses to treatment and recurrent mastitis are observed. This can be explained by reinfection or by persistence of strains. We hypothesized that among a heterogeneous group of Strep. uberis mastitis isolates, some predominant host-adapted clones might be recurrently isolated from IMI. Therefore, the aim of this pilot study was to determine the Strep. uberis genotype variety found among small-scale dairy herds (127 Austrian dairy farms) and compare this with a large-scale herd (a Slovakian dairy farm). We determined the occurrence and strain diversity of Strep. uberis (n = 309) isolates using molecular analysis. Streptococcus uberis isolates from aseptically collected quarter milk samples were genotypically characterized using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. The Strep. uberis strain set covered isolates from 4 Austrian federal areas [Lower Austria (n = 67), Upper Austria (n = 8), Salzburg (n = 51), and Styria (n = 1)] and the Bratislava Region of Slovakia (n = 1). The PFGE analysis resulted in 187 SmaI profiles with 151 unique profiles. Simpson's index of diversity was 0.988. Individual cows (n = 17) harbored up to 3 different PFGE types in the udder. Dairy cows shared distinct PFGE types within a farm. Seven PFGE types were widely distributed among Austrian dairy farms. In the Slovakian farm, 10 predominant PFGE types were recurrently isolated from the same quarters; these genotypes were assigned as persisters. We identified novel sequence types (ST) using multilocus sequence typing related to the global clonal complexes ST5 and ST143. We concluded that Strep. uberis IMI are caused by strains with a wide heterogeneity of PFGE types. This large number of unique subtypes indicates a high diversity of Strep. uberis in the environment. In the large herd, molecular epidemiological results revealed that specific strains might be involved in contagious transmission events and potentially lead to persistence.

Comparison of Staphylococcus aureus surface protein extraction methods and immunogenicity

Staphylococcus aureus is the major contagious bovine mastitis pathogen and has no effective vaccine. Strain variation and limited knowledge of common immunogenic antigen/s are among major constraints for developing effective vaccines. S. aureus cell surface proteins that are exposed to the host immune system constitute good vaccine candidates. The objective of this study was to compare two novel S. aureus surface protein extraction methods with biotinylation method and evaluate immune-reactivity of extracted proteins. Surface proteins were extracted from nine genetically distinct S. aureus strains from cases of bovine mastitis. After extraction, bacterial cell integrity was examined by Gram staining and electron microscopy to determine if extraction methods caused damage to cells that may release non-surface proteins. The extracted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and evaluated for immune-reactivity using western blot. Results showed that all three extraction methods provided multiple protein bands on SDS-PAGE. Western blot result showed several immunoreactive surface proteins, in which some proteins strongly (well-resolved, thick, dark, and intense band) reacted across the nine strains tested. The three methods are valid for the extraction of surface proteins and hexadecane, and cholic acid methods are more feasible than biotinylation since both are easier, cheaper, and have minor effects on the bacterial cell. Strongly immune-reactive surface proteins may serve as potential candidates for a vaccine to control S. aureus mastitis in dairy cows.

In vitro Antimicrobial Resistance of Causative Agents to Clinical Mastitis in Danish Dairy Cows.

This study was undertaken to investigate the antimicrobial resistance patterns of major causative agents to clinical mastitis in Danish dairy cows collected in 2016 to provide data on the current resistance patterns. Such data may subsequently serve as basis for a guideline for prudent use of antimicrobial agents in mastitis treatment. In addition, this study serves as a baseline for future comparison. The minimum inhibitory concentrations in Escherichia coli (n = 62), Klebsiella pneumoniae (n = 18), Staphylococcus aureus (n = 63), coagulase-negative Staphylococci (CNS) (n = 49), Streptococcus uberis (n = 61), Streptococcus dysgalactiae (n = 33), and Streptococcus agalactiae (n = 13) were determined to antimicrobial agents representing most classes relevant for treatment. The occurrence of resistance in the 299 bacterial isolates in total was evaluated using Clinical and Laboratory Standards Institute clinical breakpoints or in-house breakpoint values. For E. coli, low resistance levels were detected, 11.3% being resistant to ampicillin while resistance to other compounds was lower or zero. In contrast, K. pneumoniae revealed frequent ampicillin resistance (83.3%), but was susceptible to most other antimicrobial agents tested. Staphylococci were susceptible to the majority of antimicrobial agents tested, only 17.7% of the S. aureus isolates and 22.4% of the CNS being resistant to penicillin. Species distribution of the CNS isolates revealed that Staphylococcus simulans, Staphylococcus chromogenes, and Staphylococcus epidermidis were the most prevalent species. One S. aureus and one S. chromogenes isolate was found to be cefoxitin resistant and confirmed as methicillin resistant by polymerase chain reaction detection of the mecA gene, showing that methicillin resistance in staphylococci is present. All species of streptococci were susceptible to penicillin. No other critical resistance was found in any species, and resistance was in general low to all clinically relevant compounds. We emphasize the need for continuous surveillance of antibiotic resistance in major mastitis pathogens and the need for harmonization of methods and interpretations.

Characterization of Staphylococcus Species Isolated from Bovine Quarter Milk Samples.

Simple SummaryStaphylococci are the most prevalent bacteria isolated from bovine mammary secretions. They not only originate from cases of intramammary infections, but also from teat canal, skin and other environmental sources. They are usually divided into coagulase-negative staphylococci (CNS) and Staphylococcus (S.) aureus. In contrast to the contagious nature of most S. aureus infections, the epidemiology of CNS is less clear. Results of our observational study suggest that both, CNS and S. aureus, can be associated with clinical and subclinical mastitis but may also appear as colonizers and remain undetected in cows without inflammatory signs. As a result, the consequences differ, especially with the increased emphasis on reducing antibiotic use as a means of limiting antimicrobial resistance (AMR). A positive S. aureus test result requires antibiotic treatment of infected cows after evaluation of the probability of bacteriological cure, and, where necessary, implementation of management strategies to limit new infections. In contrast, treatment of CNS in cows without increase in somatic cell count should be avoided. Thus, these findings emphasize the value of regular bacteriological examination of clinical and subclinical cases and a thorough evaluation of any staphylococcal presentation before treatment.Staphylococcus (S.) aureus is considered as a major mastitis pathogen, with considerable epidemiological information on such infections while the epidemiology of coagulase-negative staphylococci (CNS) is more controversial. The aim of this study was to use matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology for identification of staphylococci isolated from bovine milk at species level and to characterize them in reference to presentation, somatic cell count (SCC), bacterial shedding (cfu) and antimicrobial resistance patterns. A total of 200 staphylococcal isolates (S. aureus n = 100; CNS n = 100) originating from aseptically collected quarter milk samples from different quarters of dairy cows were included in the study. They originated from cases of clinical (CM) and subclinical mastitis (SCM) or were isolated from milk with SCC ≤ 100,000 cells/mL in pure culture. We found staphylococci predominantly in cases of SCM (n = 120). In low-SCC cows, 12 S. aureus and 32 CNS isolates were detected. Eighteen percent of each were associated with CM. Eleven CNS species were identified, S. chromogenes (n = 26) and S. xylosus (n = 40) predominated. CNS, particularly those in low-SCC cows, showed higher MIC90 (minimal inhibitory concentration) values for penicillin, ampicillin, cefoperazone, pirlimycin and marbofloxacin. Based on the present results, a careful interpretation of laboratory results is recommended to avoid antimicrobial therapy of staphylococci without clinical relevance and to ensure prudent use of antimicrobials.

A PRELIMINARY SURVEY OF STAPHYLOCOCCUS AUREUS INFECTION IN GOATS WITH MASTITIS IN “NOUQRA” VALLEY OF ASWAN GOVERNORATE, SOUTH EGYPT

Prevalence of mastitis in goats located in Nouqra valley, Aswan Governorate, by indirect method (California Mastitis Test) in paralleling with culturing and molecular (PCR) procedures for detection of Staphylococcus aureus as a major mastitis pathogen. A total of 148 raw milk samples were subsequently collected from apparently healthy native breed goats, with different age and parity, and subjected to California Mastitis Test. By CMT, our results indicated that 117 (79.05 %) of the tested—samples were positive. Culturally using Baired Parker media, pure strains of Staphylococcus aureus was isolated from CMT—positive samples with a percentage of 1.35% and 77.7% of the samples showed fair growth, which classified as other non-aureus staphylococci. Coagulase test revealed 7 (4.7%) samples were positive and 141 (95.3%) were negative. These isolates were molecularly tested using 16s Rrna (Staphylococcus genus specific), nuc gene (S.aureus species specific) and mecA gene (methicillin resistance gene) by Multiplex PCR Technique. Their results indicated that 87.5% were positive for 16s Rrna, 25% were positive for nuc gene, 75% were positive for mec A gene and 12.5% were negative. The in-vitro antibiotic sensitivity testing revealed that the resistant percentages to penicillin were surprise (100% resistance). Amoxicillin, cefaclore, colistin, oxolinic acid, neomycin, erythromycin, and lincomycin were also examined with various resistant results. Approx. 85 % (85.71%) of the tested strains were Ciprofloxacin—sensitive

Comparison of Staphylococcus aureus Surface Protein ExtractionMethods

Staphylococcus aureus is the major contagious bovine mastitis pathogen and has no effective vaccine. Strainvariation and limited knowledge of common immunogenic antigen/s are among major constraints for developingeffective vaccines. S. aureus cell surface proteins that are exposed to the host immune system constitute goodvaccine candidates. The objective of this study was to compare S. aureus surface protein extraction methods andevaluate immune-reactivity of extracted proteins. Surface proteins were extracted from nine genetically distinct S.aureus strains from cases of bovine mastitis. After extraction, bacterial cell integrity was examined by Gram stainingand electron microscopy to determine if extraction methods caused damage to cells that may release non-surfaceproteins. The extracted proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) and evaluated for immune-reactivity using western blot. Results showed that all three extractionmethods provided multiple protein bands on SDS-PAGE. Western blot result showed several immuno-reactivesurface proteins, in which some proteins strongly (well-resolved, thick, dark and intense band) reacted across thenine strains tested. The three methods are valid for the extraction of surface proteins and hexadecane and cholicacid methods are more feasible than biotinylation since both are easier, cheaper and have minor effects on thebacterial cell. Strongly immune-reactive surface proteins may serve as potential candidates for a vaccine to controlS. aureus mastitis in dairy cows.

Case study: Evaluating quarter and composite milk sampling for detection of subclinical intramammary infections in dairy cattle

Our objective was to evaluate a 200,000 cells/mL somatic cell count (SCC) cut-point on both the quarter and composite level to determine its effectiveness at identifying subclinical mastitis infections in one commercial dairy herd in Central New York. Milk samples from 107 Holstein cows were used for analysis. All cows were eligible for enrollment provided they had 4 working udder quarters, were >14 and <365 d in milk, and had no clinical mastitis event or treatment with intramammary antibiotics ≤14 d prior to d of sampling. A total of 428 quarter and 107 composite samples from 34 primiparous and 73 multiparous animals were analyzed for total SCC and aerobic culture. Performance of SCC for identification of subclinically infected animals was evaluated against the gold standard aerobic culture. Sensitivities for a 200,000 cells/mL cut-point on both the quarter and composite basis were 28.6%, and specificities were 91.5% and 87.3% on the quarter and composite basis, respectively. Receiver operating characteristic (ROC) curves determined on a quarter basis found the cut-point that optimized the sensitivity and specificity of a positive culture was 32,000 cells/mL, with a sensitivity of 76.2%, a specificity of 62.4%, and an area under the curve (AUC) of 0.73. The ROC curve cut-point that optimized the sensitivity and specificity for the composite samples was 75,000 cells/mL, with a sensitivity of 57.1%, a specificity of 78.9%, and an AUC of 0.67. A large proportion of culture positive primiparous cows (38%) had low SCC (median of 101,000 cells/mL on the quarter and 80,000 cells/mL on the composite level), and therefore, when multiparous cows were examined separately, the cut-point that optimized sensitivity and specificity on the quarter basis increased to 645,000 cells/mL with a corresponding sensitivity of 34.8%, specificity of 97.5%, and AUC of 0.65. On the composite basis, the cut-point based on multiparous cows only was 152,000 cells/mL, with a corresponding sensitivity of 60.0%, and specificity of 82.0%, and an AUC of 0.65. Our data indicate that the 200,000 cells/mL cut-point was inefficient in identifying subclinically infected animals, regardless of whether quarter or composite sampling was used. The low prevalence of subclinical infections as well as the large proportion of minor pathogens, especially in primiparous animals, contributed to this inefficiency. This case study provides evidence that, with continued improvement upon mastitis control and reduction in major mastitis pathogens, blanket cut-points may no longer provide the same diagnostic usefulness as they once did.

Identificación molecular y frecuencia de patógenos aislados de mastitis bovina en establos de la Península de Baja California, México

La mastitis bovina es una enfermedad de alto impacto económico para la industria lechera, y algunos de los agentes etiológicos que la provocan también son de interés en el ámbito de salud pública. El objetivo de este estudio fue identificar las especies bacterianas aisladas de casos de mastitis bovina, provenientes de siete establos lecheros ubicados en la Península de Baja California. Se tomaron 316 muestras de leche de igual número de cuartos, pertenecientes a 186 vacas en producción que a la prueba de California tuvieron reacción positiva. Se obtuvieron 182 aislados bacterianos de 163 cuartos pertenecientes a 106 vacas y se identificaron por PCR y secuenciación, dando un total de 20 especies diferentes. Además, se obtuvieron las frecuencias relativas, siendo los agentes causales más frecuentes: Staphylococcus aureus (58.8 %), Streptococcus agalactiae (13.2 %), Staphylococcus chromogenes (8.8 %), Escherichia coli (2.2 %) y Streptococcus uberis (2.2 %). El 6.13 % (10/163) de los cuartos con aislamiento presentaron infección mixta, siendo la combinación más frecuente S. aureus con S. agalactiae 30 % (3/10). Estos resultados indican una alta frecuencia y diversidad de patógenos de carácter contagioso y ambiental que provocan mastitis en ganado lechero en la región de estudio, siendo algunos de importancia para la salud pública. Los resultados observados, muestran que las causas de la mastitis son diversas, por lo que es indispensable mejorar las medidas de control y prevención, pero también establecer el diagnóstico de rutina para lograr controlar la mastitis.

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

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 aureus showed 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.

Canadian National Dairy Study: Herd-level milk quality

The objective of this study was to estimate Canadian national milk quality parameters and estimate the bulk tank milk (BTM) prevalence of 4 mastitis pathogens, Staphylococcus aureus, Streptococcus agalactiae, Mycoplasma bovis, and Prototheca spp., on Canadian dairy farms. A questionnaire was sent to all Canadian dairy producers. Of the 1,062 producers who completed the questionnaire, 374 producers from across the country were visited and milking hygiene was assessed. Farm-level milk quality data for all Canadian dairy producers was collected from the provincial marketing boards and combined with the questionnaire and farm visit data. In addition, a BTM sample was collected either during the farm visit or by the marketing board in November of 2015 and was tested for 4 major mastitis pathogens using the PathoProof Mastitis Major 4 PCR Assay (Thermo Fisher Scientific Inc., Waltham, MA). Apparent herd-level prevalence was 46% for S. aureus, 6% for Prototheca spp., 0% for M. bovis, and 0% for Strep. agalactiae. Due to the low prevalence of M. bovis and Strep. agalactiae and a lack of significant factors associated with farms testing positive for Prototheca spp., an association analysis could only be carried out for Staph. aureus-positive farms. Factors associated with Staph. aureus-positive farms were not fore-stripping cows before milking (odds ratio = 1.87), milking with a pipeline system (odds ratio = 2.21), and stall bases made of a rubberized surface (mats and mattresses), whereas protective factors were using blanket dry cow therapy (odds ratio = 0.49) and applying a tag or visible mark on cows known to have chronic mastitis infections (odds ratio = 0.45). The Canadian national production-weighted geometric mean somatic cell count was determined to be 208,000 cells/mL. This is the first national dairy study conducted in Canada. Participating farms had higher milk yield; were more likely to have a loose housing system, parlor, or automated milking system; and had lower weighted mean BTM somatic cell count than the national level. Sampling larger farms with better milk quality means the apparent prevalence of the 4 mastitis pathogens likely underestimates the true levels.