Major Mastitis Research Articles

In vitro efficacy of teat antiseptics against Staphylococcus aureus strains isolated from bovine mastitis

The process of teat disinfection is a widely accepted component of successful mastitis control programs by reducing the number of bacteria on the teat skin and healing teat lesions. For contagious pathogens such as Staphylococcus aureus, post-milking teat disinfection remains a simple, effective and economical practice for prevention of new intramammary infections (IMIs) of lactating dairy cows. Despite the universal acceptance of teat dipping as a method of mastitis control, variations in the susceptibility and resistance profile of mastitis pathogens among antiseptics have been described. Thus, here we sought to explore the in vitro efficacy of the followings antiseptics against S. aureus isolated from IMIs: chlorhexidine (2.0%), chlorine (2.5%), quarternary ammonium (4.0%), lactic acid (2.0%) and iodine (0.6%). We used 50 S. aureus strains isolated from bovine IMIs from 50 dairy herds located at Minas Gerais, São Paulo, Paraná and Rio Grande do Sul States (Brazil). The antiseptics were evaluated at four different specific intervals (15, 30, 60 and 300 s). We found a higher activity for quarternary ammonium and chlorhexidine against S. aureus at all time-points, followed by iodine and then chlorine. Lactic acid treatment produced the worst results for all time-points and strains. Due to variations in the sensitivity and resistance profile of antiseptics against S. aureus isolated from IMIs, the effectiveness of the antiseptics against the major mastitis pathogens should be periodically evaluated in dairy farms in an attempt to reduce the rate of new IMIs in the herd.

PCR and PCR-RFLP genotyping of Staphylococcus aureus coagulase gene: convenience compared to pulse-field gel electrophoresis

Staphylococcus aureus belongs to the group of major contagious mastitis pathogens. We aimed to evaluate the usefulness and the convenience of the coagulase (coa) gene polymorphism to type S. aureus from bovine mastitis milk samples. A total of 22 S. aureus strains were collected from bovine cases of mastitis in four veterinary practices in the Rhone-Alpes region of France and were assessed using coagulase gene’s polymerase chain reaction (PCR) followed by AluI PCR-restriction fragment length polymorphism (PCR-RFLP). Coa gene PCR produced six profiles. The AluI PCR-RFLP displayed nine profiles, giving ten combined patterns. Two genotypes accounted for 50 % of the isolates. Although the results showed that cows from the studied regions were infected by S. aureus strains harboring more than one coagulase genotype, only one or two types predominated. Identification based on PCR-RFLP of the coa gene gave the same results than pulse-field gel electrophoresis (PFGE). Identification based on PCR-RFLP of the coa gene has been considered a more simple and accurate typing method for epidemiological investigations of bovine mastitis.

Prevalence of Bovine Mastitis Pathogens in Bulk Tank Milk in China.

The objectives of this study were to estimate the herd prevalence of major mastitis pathogens in bulk tank milk (BTM) in China dairy herds, to determine the relationship between the presence of mastitis pathogens and bulk tank milk somatic cell counts (BTSCC), and to investigate the impact of different dairy cattle farming modes and region on bacterial species. BTM samples collected from 894 dairy herds in China were examined for the presence of mastitis pathogens. The Flinders Technology Associates (FTA) cards were used for BTM sample collection, storage, and transportation and bacterial DNA amplification by real-time PCR. Among contagious pathogens, Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus dysgalactiae were detected in 50.1, 92.2, and 72.3% of the 894 BTM samples, respectively. Among environmental pathogens, E. coli, Streptococcus uberis, Enterococcus spp., Klebsiella spp., Serratia marcescens, Corynebacterium bovis, and Arcanobacterium pyogenes were detected in 28.6, 8.9, 35.7, 20.0, 1.3, 17.0, and 67.2% of the BTM samples, respectively. Staphylococcal β-lactamase gene was detected in 61.7% of the BTM samples. The presence of Staphylococcus aureus and Arcanobacterium pyogenes were significantly associated with high BTSCC, respectively. Significant differences were found in presence of Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus dysgalactiae in BTM sampled from the small household farms, dairy-farming communities, and large-scaled dairy farms. There were significant differences in the presence of Streptococcus agalactiae, Streptococcus dysgalactiae, Arcanobacterium pyogenes, staphylococcal β-lactamase gene, Staphylococcus spp., Klebsiella spp., Enterococcus spp., and Streptococcus uberis in BTM among Inner Mongolia, Heilongjiang, and Hebei province. In conclusion, contagious mammary pathogens are predominated among pathogens in BTM samples in China.

Short communication: N-Acetylcysteine-mediated modulation of antibiotic susceptibility of bovine mastitis pathogens

The aim of this study was to investigate the effects of N-acetylcysteine (NAC) on antibiotic susceptibility of bovine mastitis pathogens including Staphylococcus aureus, Streptococcus dysgalactiae, Escherichia coli, and Streptococcus agalactiae. Minimum inhibitory concentrations (MIC) were tested by the agar-based E-test method. The presence of 10mM NAC reduced the MIC of penicillin and ampicillin but enhanced the MIC of erythromycin and ciprofloxacin for all of the strains. In addition, NAC-mediated modulation of MIC of kanamycin, tetracycline, and vancomycin was diverse, depending on the target bacterial pathogen and antibiotic being used. The results suggest that NAC is an important modulator of antibiotic activity against the major bovine mastitis pathogens.

Interaction between bovine-associated coagulase-negative staphylococci species and strains and bovine mammary epithelial cells reflects differences in ecology and epidemiological behavior

Bacteria adherence seems to be an essential first stage for the internalization of bacteria into the cytoplasm of the host cell, which is considered an important virulence strategy enabling bacteria to occupy a microenvironment separated from host defense mechanisms. Thus, this study aimed to explore the difference in the capacity of 4 bovine-associated staphylococci species or strains to adhere to and internalize into bovine mammary epithelial cells (MEC). Three different isolates of coagulase-negative staphylococci (CNS) were used: one strain of Staphylococcus fleurettii isolated from sawdust and considered an environmental opportunistic bacterium, and 2 dissimilar Staphylococcus chromogenes isolates, one cultured from a heifer’s teat apex (Staph. chromogenes TA) and the other originating from a chronic intramammary infection (Staph. chromogenes IM). Also, one well-characterized strain of Staphylococcus aureus (Newbould 305) was used for comparison with a major mastitis pathogen. The CNS species and strains adhered to and internalized into MEC slower than did Staph. aureus. Still, we observed high variation in adhesion and internalization capacity among the different CNS, with Staph. chromogenes IM showing a greater ability to adhere to and internalize into MEC than the 2 CNS strains isolated from extramammary habitats. In conclusion, the 3 well-characterized bovine-associated CNS species and strains originating from distinct habitats showed clear differences in their capacity to adhere to and internalize into MEC. The observed differences might be related to their diversity in ecology and epidemiological behavior.

Lactoferrin and Immunoglobulin G Concentration in Bovine Milk from Cows with Subclinical Mastitis during the Late Lactation Period

Background: Lactoferrin and immunoglobulin G in milk have an important role in udder resistance to infection in the involution period. Both proteins express antimicrobial activity- lactoferrin by the binding and sequestration of iron ion; and immunoglobulin G by complement activation, bacterial opsonization and agglutination. Many factors affect lactoferrin and immunoglobulin G concentrations in bovine milk, such as the stage of lactation, milk production, and intramammary infections. The aim of this study was to determine concentrations of lactoferrin and immunoglobulin G in milk from healthy cows and subclinical mastitic cows during the late lactation period, and to evaluate the relationship between them.Materials, Methods & Results: A total of 150 quarter milk samples from 41 cows (Holstein-Friesian breed) in late lactation period were reviewed in this study. Milk samples were collected during morning milking, using aseptic techniques in sterile test tubes. From each sample, 0.1 mL of milk was plated on Columbia blood agar base with 5% defibrinated ovine blood, MacConkey agar and Sabouraud dextrose agar and incubated for 24 h - 48 h (bacteria) and 5 days (yeasts, mould) at 37oC. Milk samples for detection lactoferrin and immunoglobulin G concentration were skimmed at 1,400 g for 45 min and stored at -20°C until analysis. Lactoferrin concentration in bovine milk was determined using the Bovine Lactoferrin ELISA Quantitation Set. Milk samples were diluted at a ratio of 1:10,000. Plates were read at 450 nm absorbence values. Immunoglobulin G concentration was determined by the immunodiffusion method using radial immunodiffusion (RID) plates. Milk samples were diluted in a ratio of 1:30. Reading of results was done after incubation for 48 h by measuring the diameter of the precipitation ring. The highest mean lactoferrin concentration was observed in udder quarters infected with contagious pathogens (Streptococcus agalactiae and Staphylococcus aureus), while the highest mean immunoglobulin G concentration was detected in milk samples where minor mastitis pathogens (coagulase-negative staphylococci and Corynebacterium spp.) were isolated. Milk samples where Staphylococcus aureus was isolated had the lowest immunoglobulin G concentration, and the lowest lactoferrin concentration was observed in samples infected with enviromental pathogens (Streptococcus dysgalactiae).Discussion: This study showed that lactoferrin and immunoglobulin G concentrations are higher in milk samples from subclinical mastitic cows than in milk from normal lactating cows. Lactoferrin concentrations in milk samples from udder quarters infected with major mastitis pathogens were significantly higher than in milk infected with minor mastitis pathogens. The lowest concentration of immunoglobulin G was detected in milk samples where Staphylococcus aureus was isolated, while the highest immunoglobulin G concentration was observed in milk samples from quarters infected with minor mastitis pathogens. Lactoferrin and immunoglobulin G concentrations were significantly and positively correlated in all milk samples. This means that cows with high lactoferrin concentrations have high immunoglobulin G concentrations. In quarter milk samples infected with Staphylococcus aureus, lactoferrin and immunoglobulin G concentrations were negatively correlated. The cause of these findings could be the suppression of local immune response of mammary gland.

Cytokines Expression Associated with E.coli Infection In bovine mammary glands

Abstract -In order to characterize the expression of genes associated with immune response mechanisms to mastitis, we quantified the relative expression of the IL-4, IL-6, IL-8 and IL-10genes in milk cells of healthy cows and cows with clinical mastitis. Gene expression was analyzed by real-time PCR. E. coli is among the major mastitis pathogens responsible for clinical mastitis in dairy cows, but the infection are normally cleared by the immune system with in a few days. Mastitis tolerance/susceptibility is difficult to measure directly and hence milk somatic cell count (SCC) or milk somatic cell score (SCS) is used as an indicator trait for mastitis as both traits are highly positively correlated.

Lactococcus lactis V7 inhibits the cell invasion of bovine mammary epithelial cells by Escherichia coli and Staphylococcus aureus.

Bovine mastitis, an inflammatory disease of the mammary gland often associated to bacterial infection, is the first cause of antibiotic use in dairy cattle. Because of the risk of antibioresistance emergence, alternative non-antibiotic strategies are needed to prevent or to cure bovine mastitis and reduce the antibiotic use in veterinary medicine. In this work, we investigated Lactococcus lactis V7, a strain isolated from the mammary gland, as a probiotic option against bovine mastitis. Using bovine mammary epithelial cell (bMEC) culture, and two representative strains for Escherichia coli and for Staphylococcus aureus, two major mastitis pathogens, we investigated L. lactis V7 ability to inhibit cell invasion (i.e. adhesion and internalization) of these pathogens into bMEC. L. lactis V7 ability to modulate the production of CXCL8, a key chemokine IL-8 responsible for neutrophil influx, in bMEC upon challenge with E. coli was investigated by an ELISA dosage of CXCL8 in bMEC culture supernatants. We showed that L. lactis V7 inhibited the internalisation of both E. coli and S. aureus strains into bMEC, whereas it inhibited the adhesion of only one out of the two S. aureus strains and of none of the E. coli strains tested. Investigation of the bMEC immune response showed that L. lactis V7 alone induced a slight increase in CXCL8 production in bMEC and that it increased the inflammatory response in bMEC challenged with the E. coli strains. Altogether these features of L. lactis V7 make it a potential promising candidate for a probiotic prevention strategy against bovine mastitis.

An investigation of the dynamics of intramammary infections acquired during the dry period on European dairy farms

The dry period is acknowledged as playing a key role in mastitis epidemiology and yet surprisingly few studies have explored dry period infection dynamics in detail. The aim of this study was to investigate the dynamics of intramammary infection across a cohort of dairy herds in Europe. Five hundred and twenty-two cows were recruited from 12 farms in 6 European countries. All cows received antibiotic dry cow therapy but teat sealants were not used. All quarters of all cows were sampled for bacteriology at drying off and in the week immediately postcalving. Two ipsilateral quarters were also sampled for bacteriology in each cow 2 and 6wk after drying off. Cows were body condition scored and teats assessed for cleanliness at all sampling time points and for the presence of a keratin plug during the dry period. Other cow-level parameters such as historic somatic cell counts and milk yields before drying off were collated from farm records. Univariable and multivariable analyses were undertaken to investigate the etiology, prevalence, and dynamics of infection during the dry period and associated influential factors. In summary, environmental mastitis pathogens predominated. Although gram-positive major pathogens were typically well controlled and did not increase in prevalence across the dry period, gram-negative pathogens generally increased in prevalence. There was an increase in the number of quarters that yielded no growth across the dry period, although this was driven by minor rather than major mastitis pathogen control. Other than the presence of a gram-positive or gram-negative pathogen 6wk after drying off, the measured parameters were not influential when considering their effect on the presence of pathogens postcalving. Analysis also suggested that the early and mid dry period may be more important with respect to the timing of acquisition of infection than previously thought. We observed substantial variation in the etiology and prevalence of different pathogens on different farms with, in all cases, at least one of the 12 herds experiencing the opposite of the others with respect to increases and decreases in pathogen prevalence. Overall, this study confirms the importance of the dry period in mastitis epidemiology but highlights the importance of assessing and understanding infection dynamics on individual units. The lack of influence of the cow and quarter factors measured in this study suggests that herd and management factors may be more influential.

Detection of major mastitis pathogens by multiplex polymerase chain reaction assay in buffalo milk

In the present study multiplex PCR assay was standardized for simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae, S. dysgalactiae and Escherichia coli associated with mastitis. The target sequence 16S to 23S rRNA inter spacer regions was used. Primers used were chosen to have approximately same Tm value, common annealing temperature and easily differentiable specific amplified products. The performance of the assay was examined on 92 milk samples collected from subclinically and clinically infected buffaloes and the diagnostic sensitivity and specificity of multiplex PCR was compared with culture examination. Out of total milk samples, 16 were diagnosed for mixed infections of Staphylococcus aureus + S. dysgalactiae (43.75%), S. aureus + S. agalactiae(12.5%), S. aureus + E. coli (25%), S. dysgalactiae + E. coli (12.5%) and S. aureus + S. dysgalactiae + E. coli (6.25%). Multiplex PCR assay was more promising option than culture methods. Milk culture method is cumbersome and more time consuming and it may yield no bacteria due to the presence of very low number of pathogens or due to residual therapeutic antibiotics concentration in milk. The assay has an added advantage over simplex PCR that it can simultaneous detect and type different species of bacteria. Multiplex PCR assay is rapid, sensitive and specific assay which can be used as a routine diagnostic tool to detect major mastitis pathogens.

Seasonal Prevalence and Antibiogram Profile of Bacterial Isolates from Bovine Mastitis

The current study examines the prevalence and seasonal occurrence of major bacterial pathogens and effect of different antibiotics on bacteria isolated from 935 bovine mastitic milk samples in northern region of India for characterization and culture sensitivity against different antibiotics. A major proportion of mastitis samples- 412 (44%) were recorded during rainy season, while, samples in equal shares, 276 (28%) and 247 (27%) were documented in winter and summer seasons, respectively. Out of total 935 mastitic milk samples, 889 (95%) samples showed microbial growth, while, 46 (5%) samples were found negative for any bacterial growth. Among total isolated bacteria, Gram positive- 471 (53%) shared a major proportion, followed by Gram negative 341 (38%), while a small part of 53 (6%) and 24 (2%) samples yielded mixed unidentified cultures and Candida species, respectively. In antibiogram study, gentamicin (91.21%), ciprofloxacin (89.60%), enrofloxacin (88.28%) and tetracycline (71.30%) were found to be highly effective antibiotics, while, penicillin (86%), colistin (83.30%), cloxacillin (78.62%), amoxycillin (70.71%) and ampicillin (62.51%) showed least effect against both Gram positive and negative bacteria. The present study showed that there was close association between season, bacterial pathogens and occurrence of of bovine mastitis. Overall, Staphylococcus spp., Streptococcus spp. and E. coli contributed as major mastitis dweller bacteria.

Effect of Prostaglandin F2α on Growth of Mycoplasma bovis Associated with Bovine Mastitis

Mycoplasma bovis (M. bovis) is a major mastitis pathogen that has been reported to be refractory to antibiotic treatment. Certain fatty acids have been shown to inhibit the growth of mastitis pathogens such as Staphylococcus aureus (S. aureus). In vitro experiments were conducted to determine the effects of prostaglandin F2α (PGF2α) on growth of M. bovis. Five strains of M. bovis of bovine origin were selected for the study. Two strains were reference strains (ATCC 25025 and 25523) and the other strains were isolated from diseased cattle. Isolates were cultured and suspended in saline to achieve an optical density of 0.2 at 520 nm, a suspension of approximately 1 × 107 to 2 × 108 CFU/ml. Subsequently, M. bovis suspensions were incubated in culture media containing PGF2α (dinoprost tromethamine) at final concentrations of 0 [control), 2, 4, and 8 mg/ml, for 8 h at 37°C. A sample from each treatment group was obtained and cultured on agar plates for 10 d and bacterial growth assessed as CFU/ml. The entire experiment was repeated four times using duplicate tubes per PGF2α concentrations for each strain. Data were analyzed by ANOVA and the model included the effect of treatment, strain, and their interaction. Treatment affected (P<0.01) M. bovis growth, and mean CFU/ml decreased with concentrations of PGF2α at 4 and 8 mg/ml but not 2 mg/ml (43.6, 42.1, 24.3, 7.8 [±1.1] for 0, 2, 4, 8 mg/ml, respectively). However, an effect of treatment by strain interaction on mean CFU/ml was detected (P<0.05), indicating that the effect of PGF2α on bacterial growth was not consistent across strains. Overall, the 2 mg/ml PGF2α decreased CFU/ml in only one strain compared with control, whereas 4 and 8 mg/ml PGF2α decreased CFU/ml in all strains compared with control. These in vitro results provide evidence, for the first time, that the fatty acid PGF2α, in the form of dinoprost tromethamine, has inhibitory effects on growth of M. bovis, and this bacteriostatic effect appears to be strain and dose dependent.

Accuracy of the composite somatic cell count to detect intra-mammary infection in dairy cows using latent class analysis

The somatic cell count (SCC) is considered an important indicator of intra-mammary infection (IMI). The purpose of this study was to determine the accuracy of both SCC and culture to detect IMI and their conditional dependence by means of latent class methods. This study involved 175 dairy cows from 2 herds with different udder infection prevalences. Quarter and composite milk samples were collected for SCC and bacteriological culture. Latent-class models using Bayesian methods were used to estimate test sensitivity (Se) and specificity (Sp) and population prevalence. The models ran involved only major mastitis pathogens and composite SCC (CSCC). Five thresholds between 100,000 and 300,000cells/mL were evaluated and the receiver operating characteristics (ROC) curve analysis was performed. Fifty-five percent of the cows had CSCC ≥200,000cells/mL and 95.4% of the cows had at least one infected quarter either with minor or major pathogens. Considering a threshold of 150,000cells/mL, the estimated Se and Sp for the CSCC were, 0.80 (95% CrI 0.71–0.88) and 0.57 (95% CrI 0.44–0.71), respectively. The estimated culture Se and Sp were 0.83 (95% CrI 0.73–0.93) and 0.89 (95% CrI 0.74–0.98), respectively. There was no evidence of dependence between CSCC and culture. The area under curve for CSCC was 0.72. To the best of our knowledge, this is the first report of the CSCC accuracy to detect IMI for major pathogens considering the effect of culture misclassification. The estimates provided here could help to examine the performance of sampling schemes based on CSCC to manage udder health.

Prevalence of mastitis pathogens in milk from clinically healthy cows

A total of 669 individual cow milk samples originating from asymptomatic cows from 16 dairy farms were examined for the presence of microorganisms with the potential to cause mastitis. Coagulase-negative staphylococci clearly predominated (53.5% positive samples) followed by streptococci and enterococci (both occurring in 16.1% samples). Among streptococci, so-called mastitis streptococci (S. uberis, S. dysgalactiae and S. agalactiae) prevailed (11.7% positive samples). Enterobacteriaceae were found in 10.0% samples, most of which (6.6% samples) were positive for Escherichia coli. Yeasts (mainly Candida spp.) were found in 8.2% samples. One of the major mastitis pathogens, Staphylococcus aureus subsp. aureus, was isolated from 9.0% of samples. S. aureus isolates were further characterised in terms of their capability to form biofilm, antimicrobial susceptibility and clonality (PFGE). All S. aureus isolates were capable of biofilm formation and were generally susceptible to the majority of tested antibiotics. The exception was ampicillin, resistance to which was observed in 27.7% isolates. Therefore, the relatively frequent occurrence of S. aureus could be attributed to persistent intramammary infections due to biofilm formation rather than low efficacy of particular antibiotics. PFGE analysis revealed clonal spread of certain S. aureus isolates within and between farms indicating that certain lineages of S. aureus mastitis strains are particularly successful.

Relationship between previous history ofStreptococcus uberisinfection and response to a challenge model

Streptococcus uberis is the most common cause of clinical mastitis at calving in pasture-based dairy cows. Results of experimental inoculations were compared with cows' previous history of infection to help define a model for susceptibility to Str. uberis mastitis. Cows used had either no apparent history of intramammary infection (IMI) by Str. uberis or other major mastitis pathogens throughout their productive lifetime ('apparently uninfected'; AUI), or had a confirmed history of Str. uberis IMI ('historically infected'; HI). Cows were exposed to Str. uberis in sequential steps: dipping of the teat end (DIP; n=53 cows); a teat canal inoculation (TCI; n=33 cows); and, finally, intramammary inoculation challenge (IC; n=7 cows). Only cows that remained free of infection at each step progressed to the next phase. Infection rates were similar between AUI or HI cows following the DIP (9 and 17% respectively), or the TCI (75 and 68% respectively). Physical and biochemical traits of cows were examined. Analysis of traits prior to inoculations implied that HI cows produced more milk fat, while AUI cows tended to have longer teat canals. Analysis of traits for cows that became infected following DIP, implied that there was a positive association with milk fat production and negative association with somatic cell count (SCC), while there was a positive association with the duration of p.m. milking, and negative association with SCC in those cows that became infected following TCI. Only AUI cows became infected following the IC inoculation. Similarity in response to experimental inoculation between the two groups suggests that the current dip or teat canal inoculation (using a 3-mm depth of inoculation) models are not good predictors of natural resistance to Str. uberis. However, a population of cows was identified that remained uninfected after DIP, TCI and IC, and may comprise a resistant phenotype.

Escherichia coli- and Staphylococcus aureus-induced mastitis differentially modulate transcriptional responses in neighbouring uninfected bovine mammary gland quarters

BackgroundThe most important disease of dairy cattle is mastitis, caused by the infection of the mammary gland by various micro-organisms. Although the transcriptional response of bovine mammary gland cells to in vitro infection has been studied, the interplay and consequences of these responses in the in vivo environment of the mammary gland are less clear. Previously mammary gland quarters were considered to be unaffected by events occurring in neighbouring quarters. More recently infection of individual quarters with mastitis causing pathogens, especially Escherichia coli, has been shown to influence the physiology of neighbouring uninfected quarters. Therefore, the transcriptional responses of uninfected mammary gland quarters adjacent to quarters infected with two major mastitis causing pathogens, E. coli and Staphylococcus aureus, were compared.ResultsThe bacteriologically sterile, within-animal control quarters exhibited a transcriptional response to the infection of neighbouring quarters. The greatest response was associated with E. coli infection, while a weaker, yet significant, response occurred during S. aureus infection. The transcriptional responses of these uninfected quarters included the enhanced expression of many genes previously associated with mammary gland infections. Comparison of the transcriptional response of uninfected quarters to S. aureus and E. coli infection identified 187 differentially expressed genes, which were particularly associated with cellular responses, e.g. response to stress. The most affected network identified by Ingenuity Pathway analysis has the immunosuppressor transforming growth factor beta 1 (TGFB1) at its hub and largely consists of genes more highly expressed in control quarters from S. aureus infected cows.ConclusionsUninfected mammary gland quarters reacted to the infection of neighbouring quarters and the responses were dependent on pathogen type. Therefore, bovine udder quarters exhibit interdependence and should not be considered as separate functional entities. This suggests that mastitis pathogens not only interact directly with host mammary cells, but also influence discrete sites some distance away, which will affect their response to the subsequent spread of the infection. Understanding the underlying mechanisms may provide further clues for ways to control mammary gland infections. These results also have implications for the design of experimental studies investigating immune regulatory mechanisms in the bovine mammary gland.

Incidence of Subclinical Mastitis and Prevalence of Major Mastitis Pathogens in Organized Farms and Unorganized Sectors

Subclinical mastitis (SCM) represents a major proportion of the burden of mastitis. Determining somatic cell count (SCC) and electrical conductivity (EC) of milk are useful approaches to detect SCM. In order to correlate grades of SCM with the load of five major mastitis pathogens, 246 milk samples from a handful of organized and unorganized sectors were screened. SCC (>5×10(5)/mL) and EC (>6.5 mS/cm) identified 110 (45%) and 153 (62%) samples, respectively, to be from SCM cases. Randomly selected SCM-negative samples as well as 186 samples positive by either SCC or EC were then evaluated for isolation of five major mastitis-associated bacteria. Of the 323 isolates obtained, 95 each were S. aureus and coagulase-negative staphylococci (CoNS), 48 were E. coli and 85 were streptococci. There was no association between the distribution of organisms and (a) the different groups of SCC, or (b) organised farms and unorganised sectors. By contrast, there was a significant difference in the distribution of CoNS, and not other species, between organized farms and unorganized sectors. In summary, bacteria were isolated irrespective of the density of somatic cells or the type of farm setting, and the frequency of isolation of CoNS was higher with organized farms. These results suggest the requirement for fine tuning SCC and EC limits and the higher probability for CoNS to be associated with SCM in organized diary sectors, and have implications for the identification, management and control of mastitis in India.

Examining the effect of intramammary infections with minor mastitis pathogens on the acquisition of new intramammary infections with major mastitis pathogens—A systematic review and meta-analysis

Major mastitis pathogens such as Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, Streptococcus dysgalactiae, and the coliforms are usually considered more virulent and damaging to the udder than minor mastitis pathogens such as Corynebacterium bovis and coagulase-negative staphylococci (CNS). The current literature contains several studies detailing analyses with conflicting results as to whether intramammary infection (IMI) with the minor pathogens decreases, increases, or has no effect on the risk of a quarter acquiring a new intramammary infection (NIMI) with a major pathogen. To investigate the available scientific evidence regarding the effect of IMI with minor pathogens on the acquisition of NIMI with major pathogens, a systematic review and meta-analysis were conducted. The total extant English- and French-language literature in electronic databases was searched and all publications cited by relevant papers were investigated. Results from 68 studies were extracted from 38 relevant papers. Random-effects models were used to investigate the effects of CNS and C. bovis on acquisition of new IMI with any of the major pathogens, as well as individually for the minor pathogens and Staph. aureus. Significant heterogeneity among studies exists, some of which could be accounted for by using meta-regression. Overall, observational studies showed no effect, whereas challenge studies showed strong and significant protective effects, specifically when major pathogens were introduced into the mammary gland via methods bypassing the teat end. Underlying risk can account for several unmeasured factors, and studies with higher underlying risk found more protective effects of minor pathogens. Larger doses of challenge organisms reduced the protective effect of minor pathogens, and studies with more stringent diagnostic criteria for pathogen IMI identified less protection. Smaller studies (those utilizing fewer than 40 cows) also showed a greater protective effect than larger studies.

Pathogen-group specific association betweenCXCR1polymorphisms and subclinical mastitis in dairy heifers

The chemokine (C-X-C motif) receptor 1 (CXCR1) gene encodes the homonymous receptor for interleukin 8 (IL8) on polymorphonuclear neutrophilic leucocytes (PMNL). Binding causes migration from blood to milk, activation and prolonged survival of PMNL, a crucial process in the innate immune defence of the bovine mammary gland against invading mastitis-causing pathogens. The main objective of this study was to screen the entire coding region of the CXCR1 gene for polymorphisms and to analyse their association with udder health of dairy heifers. One-hundred-and-forty Belgian Holstein heifers originating from 20 commercial dairy farms were genotyped by DNA sequencing. Detailed phenotypic data on udder health was available including quarter bacteriological culture results and somatic cell count (SCC) in early lactation and composite milk SCC during first lactation. In total, 16 polymorphisms (including 8 missense mutations) were detected. Polymorphism c.980A>G was associated with pathogen-group specific IMI: heifers with genotype AG were less likely to have an IMI due to major mastitis pathogens compared with heifers with genotype GG but did not have less IMI by coagulase-negative staphylococci, so-called minor pathogens. CXCR1 genotype was neither associated with quarter SCC in early lactation nor with composite SCC during lactation. Although mastitis susceptibility is influenced by many factors, some genetic polymorphisms potentially have major effects on udder health of heifers, as was shown here. These results trigger us to further study the relationship between CXCR1 polymorphisms and mastitis susceptibility in both observational and experimental trials.

Evaluation of minor pathogen intramammary infection, susceptibility parameters, and somatic cell counts on the development of new intramammary infections with major mastitis pathogens

Major mastitis pathogens such as Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, and coliforms are usually considered more virulent and damaging to the udder than minor mastitis pathogens such as Corynebacterium spp. and coagulase-negative staphylococci (CNS). The current literature comprises several studies (n=38) detailing analyses with conflicting results as to whether intramammary infections (IMI) with the minor pathogens decrease, increase, or have no effect on the risk of a quarter acquiring a new IMI (NIMI) with a major pathogen. The Canadian Bovine Mastitis Research Network has a large mastitis database derived from a 2-yr data collection on a national cohort of dairy farms, and data from this initiative were used to further investigate the effect of IMI with minor pathogens on the acquisition of new major pathogen infections (defined as a culture-positive quarter sample in a quarter that had been free of that major pathogen in previous samples in the sampling period). Longitudinal milk samplings of clinically normal udders taken over several 6-wk periods as well as samples from cows pre-dry-off and postcalving were used to this end (n=80,397 quarter milk samples). The effects of CNS and Corynebacterium spp. on the major mastitis pathogens Staph. aureus, Strep. uberis, Strep. dysgalactiae, and coliform bacteria (Escherichia coli and Klebsiella spp.) were investigated using risk ratio analyses and multilevel logistic regression models. Quarter-, cow- and herd-level susceptibility parameters were also evaluated and were able to account for the increased susceptibility that exists within herds, cows and quarters, removing it from estimates for the effects of the minor pathogens. Increased quarter-level susceptibility was associated with increased risk of major pathogen NIMI for all pathogens except the coliforms. Increased somatic cell count was consistently associated with elevated risk of new major pathogen infections, but this was assumed to be a result of low sensitivity of bacteriology to diagnose major pathogen NIMI expediently and accurately. The presence of CNS in the sample 2 samplings before the occurrence of a NIMI increased the odds of experiencing a Staph. aureus NIMI 2.0 times, making the presence of CNS a risk factor for acquiring a Staph. aureus NIMI. Even with this extensive data set, power was insufficient to make a definitive statement about the effect of minor pathogen IMI on the acquisition of major pathogen NIMI. Definitively answering questions of this nature are likely to require an extremely large data set dedicated particularly to minor pathogen presence and NIMI with major pathogens.