Genes and pathways revealed by whole transcriptome analysis of milk derived bovine mammary epithelial cells after Escherichia coli challenge.

Mastitis, inflammation of the mammary gland, is the costliest disease in dairy cattle and a major animal welfare concern. Mastitis is usually caused by bacteria, of which staphylococci, streptococci and Escherichia coli are most frequently isolated from bovine mastitis. Bacteria activate the mammary immune system in variable ways, thereby influencing the severity of the disease. Escherichia coli is a common cause of mastitis in cattle causing both subclinical and clinical mastitis. Understanding of the molecular mechanisms that activate and regulate the host response would be central to effective prevention of mastitis and breeding of cows more resistant to mastitis. We used primary bovine mammary epithelial cell cultures extracted noninvasively from bovine milk samples to monitor the cellular responses to Escherichia coli challenge. Differences in gene expression between control and challenged cells were studied by total RNA-sequencing at two time points post-challenge. In total, 150 and 440 (Padj < 0.05) differentially expressed genes were identified at 3 h and 24 h post-challenge, respectively. The differentially expressed genes were mostly upregulated at 3 h (141/150) and 24 h (424/440) post-challenge. Our results are in line with known effects of E. coli infection, with a strong early inflammatory response mediated by pathogen receptor families. Among the most significantly enriched early KEGG pathways were the TNF signalling pathway, the cytokine-cytokine receptor interaction, and the NF-kappa B signalling pathway. At 24 h post-challenge, most significantly enriched were the Influenza A, the NOD-like receptor signalling, and the IL-17 signaling pathway.

A comparison of sedative effects of xylazine alone or combined with levomethadone or ketamine in calves prior to disbudding.

OBJECTIVE: To compare the sedative effects of intramuscular xylazine alone or combined with levomethadone or ketamine in calves before cautery disbudding. STUDY DESIGN: Randomized, blinded, clinical trial. ANIMALS: A total of 28 dairy calves, aged 21 ± 5 days and weighing 61.0 ± 9.3 kg (mean ± standard deviation). METHODS: Calves were randomly allocated to three groups: xylazine (0.1 mg kg-1) and levomethadone (0.05 mg kg-1; group XL), xylazine (0.1 mg kg-1) and ketamine (1 mg kg-1; group XK) and xylazine alone (0.2 mg kg-1; group X). Local anaesthesia (procaine hydrochloride) and meloxicam were administered subcutaneously 15 minutes after sedation and 15 minutes before disbudding. The calves' responses to the administration of local anaesthesia and disbudding were recorded. Sedation was assessed at baseline and at intervals up to 240 minutes postsedation. Times of recumbency, first head lift and first standing were recorded. Drug plasma concentrations were measured. RESULTS: Data were obtained from 27 animals. All protocols resulted in sedation sufficient to administer local anaesthesia and to perform disbudding. Sedation scores significantly correlated with drug plasma concentrations (p ≤ 0.002). Times to recumbency did not differ among protocols (2.8 ± 0.3, 3.1 ± 1.1 and 2.1 ± 0.8 minutes for groups XL, XK and X, respectively), whereas interval from drug(s) administration until first head lift was significantly shorter in group XK than X (47.3 ± 14.1, 34.4 ± 5.3 and 62.6 ± 31.9 minutes for groups XL, XK and X, respectively). The area under the time-sedation curve was significantly greater in group X than XK or XL (754 ± 215, 665 ± 118 and 1005 ± 258 minutes for groups XL, XK and X, respectively). CONCLUSIONS AND CLINICAL RELEVANCE: Levomethadone or ketamine with a low dose of xylazine produced short but sufficient sedation for local anaesthesia and disbudding with minimum resistance.

Bovine milk microbiome: a more complex issue than expected.

The aim of this study was to analyze bacterial profiles of bovine mastitic milk samples and samples from healthy quarters using Next Generation Sequencing of amplicons from 16S rRNA genes and to compare results with microbiological results by PCR assays of the same samples. A total of 49 samples were collected from one single dairy herd during the same day. The samples were divided in two sample sets, which were used in this study. The DNA extraction as well as the library preparation and sequencing of these two sets were performed separately, and results of the two datasets were then compared. The vast majority of genera detected appeared with low read numbers and/or in only a few samples. Results of PCR and microbiome analyses of samples infected with major pathogens Staphylococcus aureus or Streptococcus uberis were consistent as these genera also covered the majority of reads detected in the microbiome analysis. Analysis of alpha diversity revealed a much higher species richness in set 1 than in set 2. The dominating bacterial genera with the highest read numbers clearly differed between datasets, especially in PCR negative samples and samples positive for minor pathogens. In addition to this, linear discriminant analysis (LDA) was conducted between the two sets to identify significantly different genera/family level microbes. The genus Methylobacterium was much more common in set 2 compared to set 1, and genus Streptococcus more common in set 1. Our results indicate amplification of contaminating bacteria in excess in samples with no or minor amounts of pathogen DNA in dataset 2. There is a need for critical assessment of results of milk microbiome analyses.

Elimination of experimentally induced bovine intramammary infection assessed by multiplex real-time PCR and bacterial culture.

Diagnosis of bovine intramammary infection (IMI) has traditionally been based on bacterial culture, but currently IMI can also be detected with DNA based methods, such as multiplex real-time PCR. The aim of this study was to describe the elimination of bacteria in experimentally induced IMI on the quarter level, using conventional bacterial culture (BC) and multiplex real-time PCR. Two coagulase-negative staphylococcal species, Staphylococcus epidermidis and Staphylococcus simulans, were experimentally inoculated into 14 healthy quarters of 8 dairy cows during 4 consecutive study periods. Intramammary infections were followed with 20 milk samplings per each quarter. Milk somatic cell count was monitored to evaluate the inflammation process in the quarters. Four quarters cured spontaneously during the study period according to the culture. The PCR detected staphylococcal DNA from these quarters for several days after they were defined as cured in BC. Agreement between BC and PCR results varied from substantial to almost perfect agreement for the first 36 h postchallenge, decreasing to moderate levels toward the end of the sampling period. Based on this study, we recommend collecting possible follow-up samples to assess the bacteriological cure from IMI not until 2 to 3 wk after the onset of mastitis or after the quarter milk somatic cell count has normalized when PCR is used.

The effect of sampling technique on PCR-based bacteriological results of bovine milk samples.

The aim of the study was to evaluate the effect of sampling technique on the microbiological results of bovine milk samples using multiplex real-time PCR. Comparison was made between a technique where the milk sample was taken directly from the udder cistern of the udder quarter using a needle and vacuum tube and conventional sampling. The effect of different cycle threshold (Ct) cutoff limits on the results was also tested to estimate the amount of amplified DNA in the samples. A total of 113 quarters from 53 cows were tested pairwise using both techniques, and each sample was studied with real-time PCR. Sampling from the udder cistern reduced the number of species per sample compared with conventional sampling. In conventional samples, the number of positive Staphylococcus spp. results was over twice that of samples taken with the needle technique, indicating that most of the Staphylococcus spp. originated from the teat or environmental sources. The Ct values also showed that Staphylococcus spp. were present in most samples only in low numbers. Routine use of multiplex real-time PCR in mastitis diagnostics could benefit from critical evaluation of positive Staphylococcus spp. results with Ct values between 34.0 and 37.0. Our results emphasize the importance of a careful aseptic milk sampling technique and a microbiologically positive result for a milk sample should not be automatically interpreted as an intramammary infection or mastitis.

Comparative proteome profiling of bovine and human Staphylococcus epidermidis strains for screening specifically expressed virulence and adaptation proteins.

The present study reports a comparative proteome cataloging of a bovine mastitis and a human-associated Staphylococcus epidermidis strain with a specific focus on surfome (cell-wall bound and extracellular) proteins. Protein identification by 1DE coupled with LC-MS/MS analyses resulted in 1400 and 1287 proteins from the bovine (PM221) and human (ATCC12228) strains, respectively, covering over 50% of all predicted and more than 30% of all predicted surfome proteins in both strains. Comparison of the identification results suggests elevated levels of proteins involved in adherence, biofilm formation, signal transduction, house-keeping functions, and immune evasion in PM221, whereas ATCC12228 was more effective in expressing host defense evasion proteases, skin adaptation lipases, hemagglutination, and heavy-metal resistance proteins. Phenotypic analyses showed that only PM221 displays protein- and DNA-mediated adherent growth, and that PM221 was more efficient in cleaving tributyrin, a natural compound of milk fat under low CO2 conditions. These findings are in line with the identification data and suggest that distinct expression of lipases and adhesive surfome proteins could lead to the observed phenotypes. This study is the first extensive survey of S. epidermidis proteomes to date, providing several protein candidates to be examined for their roles in adaptation and virulence in vivo. All MS data have been deposited in the ProteomeXchange with identifier PXD000404 (http://proteomecentral.proteomexchange.org/dataset/PXD000404).

Genomics and Proteomics Provide New Insight into the Commensal and Pathogenic Lifestyles of Bovine- and Human-Associated Staphylococcus epidermidis Strains.

The present study reports comparative genomics and proteomics of Staphylococcus epidermidis (SE) strains isolated from bovine intramammary infection (PM221) and human hosts (ATCC12228 and RP62A). Genome-level profiling and protein expression analyses revealed that the bovine strain and the mildly infectious ATCC12228 strain are highly similar. Their genomes share high sequence identity and synteny, and both were predicted to encode the commensal-associated fdr marker gene. In contrast, PM221 was judged to differ from the sepsis-associated virulent human RP62A strain on the basis of distinct protein expression patterns and overall lack of genome synteny. The 2D DIGE and phenotypic analyses suggest that PM221 and ATCC12228 coordinate the TCA cycle activity and the formation of small colony variants in a way that could result in increased viability. Pilot experimental infection studies indicated that although ATCC12228 was able to infect a bovine host, the PM221 strain caused more severe clinical signs. Further investigation revealed strain- and condition-specific differences among surface bound proteins with likely roles in adhesion, biofilm formation, and immunomodulatory functions. Thus, our findings revealed a close link between the bovine and commensal-type human strains and suggest that humans could act as a reservoir of bovine mastitis-causing SE strains.

Bovine-associated CNS species resist phagocytosis differently.

BACKGROUND: Coagulase-negative staphylococci (CNS) cause usually subclinical or mild clinical bovine mastitis, which often remains persistent. Symptoms are usually mild, mostly only comprising slight changes in the appearance of milk and possibly slight swelling. However, clinical mastitis with severe signs has also been reported. The reasons for the differences in clinical expression are largely unknown. Macrophages play an important role in the innate immunity of the udder. This study examined phagocytosis and killing by mouse macrophage cells of three CNS species: Staphylococcus chromogenes (15 isolates), Staphylococcus agnetis (6 isolates) and Staphylococcus simulans (15 isolates). Staphylococcus aureus (7 isolates) was also included as a control. RESULTS: All the studied CNS species were phagocytosed by macrophages, but S. simulans resisted phagocytosis more effectively than the other CNS species. Only S. chromogenes was substantially killed by macrophages. Significant variations between isolates were seen in both phagocytosis and killing by macrophages and were more common in the killing assays. Significant differences between single CNS species and S. aureus were observed in both assays. CONCLUSION: This study demonstrated that differences in the phagocytosis and killing of mastitis-causing staphylococci by macrophages exist at both the species and isolate level.

Antimicrobial susceptibility of staphylococci from bovine milk samples in routine microbiological mastitis analysis in Finland.

The most frequent reason for antimicrobial use in dairy herds is mastitis and knowledge about mastitis-causing pathogens and their antimicrobial susceptibility should guide treatment decisions. The overall objective of this study was to assess antimicrobial resistance (AMR) of staphylococci in mastitic milk samples in Finland. MALDI-ToF MS identified a total of 504 Staphylococcus isolates (260 S. aureus and 244 non-aureus staphylococci, NAS) originating from bovine mastitic milk samples. Phenotypic susceptibility against cefoxitin, ceftiofur, enrofloxacin, gentamycin, oxacillin, penicillin, and tetracycline was evaluated by disk diffusion method and the presence of blaZ, mecA, and mecC genes investigated by PCR. Nitrocefin test assessed these isolates' beta-lactamase production. The most common NAS species were S. simulans, S. epidermidis, S. chromogenes, and S. haemolyticus. In total, 26.6% of the isolates (18.5% of S. aureus and 35.2% of all NAS) carried the blaZ gene. Penicillin resistance, based on disk diffusion, was lower: 18.8% of all the isolates (9.3% of S. aureus and 28.9% of all NAS) were resistant. Based on the nitrocefin test, 21.5% of the isolates produced beta-lactamase (11.6% of S. aureus and 32.0% of all NAS). Between the Staphylococcus species, the proportion of penicillin-resistant isolates varied, being lowest in S. simulans and highest in S. epidermidis. Resistance to antimicrobials other than penicillin was rare. Of the eight NAS isolates carrying the mecA gene, six were S. epidermidis. One S. aureus isolate carried the mecC gene. Agreement beyond chance, assessed by kappa coefficient, between phenotypic and genotypic resistance tests, was moderate to substantial. Some phenotypically penicillin-susceptible staphylococci carried the blaZ gene but isolates without blaZ or mec genes rarely exhibited resistance, suggesting that the more reliable treatment choice may depend upon genotypic AMR testing. Our results support earlier findings that penicillin resistance is the only significant form of antimicrobial resistance among mastitis-causing staphylococci in Finland.

Histopathological findings in a pilot study of dairy calves disbudded with hot cauterization or caustic paste.

We describe the histological tissue damage and compare the healing process in 16 dairy calves disbudded at a mean age of 6 days by cauterization or alkaline caustic paste application. Biopsies were taken 2 days (T2) and 2 weeks (T14) after disbudding from sedated calves treated with local anaesthetic and non-steroidal anti-inflammatory drugs. At T2, the cauterized horn buds generally had eosinophilic coagulative necrosis of the epidermis and superficial dermis, bordered basally by a neutrophilic demarcation zone. Lateral to the direct heat contact area, dermal blood vessels were thrombosed, with wall damage and perivascular neutrophils. In the caustic paste-treated horn buds, the epidermis and dermis had diffuse full-thickness liquefactive necrosis directly under the paste contact area. The necrosis spread laterally in the dermis beyond the area of paste contact and was bordered by a neutrophilic infiltrate. At T14, the cauterized horn buds had epidermal to superficial dermal ulceration and crusting, dermal neutrophilic infiltration and granulation tissue formation. In contrast, most of the caustic paste-treated horn buds consisted of a superficial dermal crust or predominantly necrotic tissue fragments. The remaining viable areas had histiocytic inflammation with peripheral neutrophils and early granulation tissue formation. Caustic paste disbudding caused poorly demarcated lesions that were more severe and extensive and took longer to heal than those due to cautery. Cauterization induced a more intense acute reaction adjacent to the primary lesion compared with caustic paste.

Staphylococcus agnetis sp. nov., a coagulase-variable species from bovine subclinical and mild clinical mastitis.

Thirteen Gram-positive-staining coagulase-variable staphylococci were isolated from subclinical and mild clinical mastitic bovine milk (n=12) and a teat apex (n=1). The results of sequence analysis of the 16S rRNA gene and two housekeeping genes, rpoB and tuf, and DNA fingerprinting with amplified fragment length polymorphism (AFLP) analysis showed that the isolates formed a separate branch within the genus Staphylococcus. The phylogenetically most closely related species were Staphylococcus hyicus and Staphylococcus chromogenes. DNA-DNA hybridization with S. hyicus DSM 20459(T) and S. chromogenes DSM 20674(T) confirmed that the isolates belonged to a separate species. The predominant fatty acids were i-C(15:0), ai-C(15:0), i-C(17:0) and C(20:0) and the peptidoglycan type was A3α L-Lys-Gly(5). Based on the results of genotypic and phenotypic analyses, it is proposed that the thirteen isolates represent a novel species, for which the name Staphylococcus agnetis sp. nov. is proposed. Strain 6-4(T) (=DSM 23656(T)=CCUG 59809(T)) is the type strain.

Somatic cell count in bovine quarter milk samples culture positive for various Staphylococcus species.

BACKGROUND: Non-aureus staphylococci (NAS) are the most prevalent group of bacteria isolated in bovine mastitis milk in Finland and many other countries. They usually cause subclinical or mild clinical mastitis. The increase in milk somatic cell count (SCC) during NAS intramammary infection varies from slight to marked, reflecting the severity of infection in the quarter. Limited evidence has indicated that NAS species may have different impact on milk SCC. We used a large data set originating from a prevalence study, including isolates from quarter milk samples and the SCCs of the respective quarters, to study the effect of different NAS species on quarter milk SCC. RESULTS: Staphylococcal species of a total of 1265 isolates, originally identified as NAS, were analysed with MALDI-TOF MS. The most prevalent NAS species were S. epidermidis, S. simulans, S. chromogenes and S. haemolyticus. Forty-two isolates appeared to be S. aureus. Geometric mean milk SCC of all quarter samples was 114,000 cells/mL and median 126,000 cells/mL. Staphylococcus species had a significant effect on the SCC of the quarter. The highest SCCs were caused by S. aureus, S. agnetis/S. hyicus (these two species cannot be distinguished with MALDI-TOF MS) and S. simulans. The mean SCCs of milk samples that were culture positive for these three species did not differ significantly from each other but were significantly higher than the mean SCCs of milk samples positive for any other species. The mean SCC of milk samples positive for S. chromogenes was significantly higher than those of milk samples positive for S. epidermidis or S. warneri. CONCLUSION: Our results confirm that different Staphylococcus species have different impacts on milk SCC, as shown in previous studies. S. aureus caused the highest SCC, as expected, but the SCCs caused by S. agnetis/S. hyicus and S. simulans did not differ significantly from that of S. aureus. Other Staphylococcus species may also cause high SCC but are often isolated also from quarters with SCC on the level of healthy quarters.

Innate immune response in experimentally induced bovine intramammary infection with Staphylococcus simulans and S. epidermidis.

Coagulase-negative staphylococci (CNS) are in several countries the most common bacteria isolated in subclinical mastitis. To investigate the innate immune response of cows to infections with two common mastitis-causing CNS species, Staphylococcus epidermidis and Staphylococcus simulans, experimental intramammary infection was induced in eight cows using a crossover design. The milk somatic cell count (SCC), N-acetyl-ß-D-glucosaminidase (NAGase) activity, milk amyloid A (MAA), serum amyloid A (SAA) and proinflammatory cytokines interleukin (IL)-1ß, IL-8, and tumor necrosis factor α (TNF-α) were determined at several time points before and after challenge. All cows became infected and showed mild to moderate clinical signs of mastitis. The spontaneous elimination rate of the 16 infections was 31.3%, with no difference between species. Infections triggered a local cytokine response in the experimental udder quarters, but cytokines were not detected in the uninfected control quarters or in systemic circulation. The innate local immune response for S. simulans was slightly stronger, with significantly higher concentrations of IL-1ß and IL-8. The IL-8 response could be divided into early, delayed, or combined types of response. The CNS species or persistency of infection was not associated with the type of IL-8 response. No significant differences were seen between spontaneously eliminated or persistent infections.

Genomic Analysis of Staphylococcus aureus Isolates Associated With Peracute Non-gangrenous or Gangrenous Mastitis and Comparison With Other Mastitis-Associated Staphylococcus aureus Isolates.

Staphylococcus aureus is a highly prevalent cause of mastitis in dairy herds worldwide, capable of causing outcomes that vary from subclinical to peracute gangrenous mastitis. We performed a comparative genomic analysis between 14 isolates of S. aureus, originating from peracute bovine mastitis with very severe signs (9 gangrenous, 5 non-gangrenous) and six isolates originating from subclinical or clinical mastitis with mild to moderate signs, to find differences that could be associated with the clinical outcome of mastitis. Of the 296 virulence factors studied, 219 were detected in all isolates. No difference in the presence of virulence genes was detected between the peracute and control groups. None of the virulence factors were significantly associated with only a single study group. Most of the variation in virulence gene profiles existed between the clonal complexes. Our isolates belonged to five clonal complexes (CC97, CC133, CC151, CC479, and CC522), of which CC522 has previously been detected only in isolates originating from caprine and ovine mastitis, but not from bovine mastitis. For statistical analysis, we sorted the CCs into two groups. The group of CCs including CC133, CC479, and CC522 was associated with gangrenous mastitis, in contrast to the group of CCs including CC97 and CC151. The presence of virulence genes does not explain the clinical outcome of mastitis, but may be affected by allelic variation, and especially different regulation and thus expression in the virulence genes.

Antimicrobial Selection for the Treatment of Clinical Mastitis and the Efficacy of Penicillin Treatment Protocols in Large Estonian Dairy Herds.

Clinical mastitis (CM) is the most common microbial disease treated in dairy cows. We analyzed the antimicrobial usage in cows with CM (n = 11,420) in large dairy herds (n = 43) in Estonia. CM treatment data were collected during a 12-month study period. The antimicrobial usage was observed during the 21 days from the initiation of treatment, and the incidence of antimicrobial-treated CM was calculated for each study herd. The effect of intramammary (IMM), systemic, and combined (systemic and IMM) penicillin treatment of CM on the post-treatment somatic cell count (SCC) was analyzed using the treatment records of 2222 cows from 24 herds with a mixed multivariable linear regression model. The median incidence of antimicrobial-treated CM was 35.8 per 100 cow-years. Procaine benzylpenicillin and marbofloxacin were used in 6103 (35.5%, 95% CI 34.8-36.2) and 2839 (16.5%, 95% CI 16.0-17.1) CM treatments, respectively. Post-treatment SCC was higher after IMM penicillin therapy compared to systemic or combination therapy. Treatment of CM usually included first-choice antimicrobials, but different antimicrobial combinations were also widely used. The effect of procaine benzylpenicillin to post-treatment SCC was dependent on the administration route, cow parity, and days in milk. Further studies should evaluate the factors affecting veterinarians' choice of antimicrobial used in the treatment of CM.

Coagulase-negative staphylococci-emerging mastitis pathogens.

Coagulase-negative staphylococci (CNS) have become the most common bovine mastitis isolate in many countries and could therefore be described as emerging mastitis pathogens. The prevalence of CNS mastitis is higher in primiparous cows than in older cows. CNS are not as pathogenic as the other principal mastitis pathogens and infection mostly remains subclinical. However, CNS can cause persistent infections, which result in increased milk somatic cell count (SCC) and decreased milk quality. CNS infection can damage udder tissue and lead to decreased milk production. Staphylococcus simulans and Staphylococcus chromogenes are currently the predominant CNS species in bovine mastitis. S. chromogenes is the major CNS species affecting nulliparous and primiparous cows whereas S. simulans has been isolated more frequently from older cows. Multiparous cows generally become infected with CNS during later lactation whereas primiparous cows develop infection before or shortly after calving. CNS mastitis is not a therapeutic problem as cure rates after antimicrobial treatment are usually high. Based on current knowledge, it is difficult to determine whether CNS species behave as contagious or environmental pathogens. Control measures against contagious mastitis pathogens, such as post-milking teat disinfection, reduce CNS infections in the herd. Phenotypic methods for identification of CNS are not sufficiently reliable, and molecular methods may soon replace them. Knowledge of the CNS species involved in bovine mastitis is limited. The dairy industry would benefit from more research on the epidemiology of CNS mastitis and more reliable methods for species identification.

Coagulase-negative staphylococci as cause of bovine mastitis- not so different from Staphylococcus aureus?

In this review of the literature, mastitis-causing coagulase-negative staphylococci (CNS) and Staphylococcus aureus are compared. Staphylococci are the bacteria most commonly isolated from bovine mastitis, and CNS are now predominant over S. aureus in most countries. CNS include various species, but only a few prevail in bovine mastitis. S. aureus can cause clinical mastitis, but often causes subclinical mastitis, which remains persistent and increases milk somatic cell count. CNS, traditionally regarded as minor pathogens, seem to lack the ability to cause severe mastitis. CNS can, however, persist in the mammary gland and moderately increase milk somatic cell count. Resistance to various antimicrobials is more common in CNS than in S. aureus, but CNS mastitis responds much better to antimicrobial treatment than S. aureus mastitis.

Effect of bovine lactoferrin on the internalization of coagulase-negative staphylococci into bovine mammary epithelial cells under in-vitro conditions.

Coagulase-negative staphylococci (CNS) have emerged as bovine mastitis pathogens in many countries. CNS mastitis is generally mild but can persist in the udder for long periods. Pathogenesis of CNS intramammary infection is not well understood. In the present study, adhesion, invasion and intracellular replication of twenty-two CNS strains isolated from bovine mastitis and the effect of bovine lactoferrin (bLf) on the internalization were studied in vitro in a bovine mammary epithelial (BME) cell model. The CNS strains were of Staphylococcus chromogenes, Staph. simulans, Staph. epidermidis, Staph. haemolyticus and Staph. cohnii urealyticus; two strains of Staph. aureus were used as controls. Seven of the CNS strains originated from persistent and five from transient mastitis infections. The in-vitro susceptibility of the strains to bLf was also investigated. All CNS species examined had an adhesive ability equal to that of Staph. aureus, but internalization varied among staphylococcal strains. The antagonistic effect of bLf on the adhesion and invasion of CNS strains was weak, but bLf significantly decreased intracellular replication and replication rates of CNS. No correlation between the in-vitro susceptibility of the strain to bLf or internalization among clinical signs of mastitis was established. No difference between the persistent and transient CNS strains in adhesion, invasion or replication rate was recorded. This in-vitro BME cell model can be used to study the virulence potential of mastitis pathogens, although the severity and persistence of eventual infections shall be further investigated in vivo. The role of bLf in intramammary infection caused by CNS may be limited.

Comparative genome analysis of 24 bovine-associated Staphylococcus isolates with special focus on the putative virulence genes.

Non-aureus staphylococci (NAS) are most commonly isolated from subclinical mastitis. Different NAS species may, however, have diverse effects on the inflammatory response in the udder. We determined the genome sequences of 20 staphylococcal isolates from clinical or subclinical bovine mastitis, belonging to the NAS species Staphylococcus agnetis, S. chromogenes, and S. simulans, and focused on the putative virulence factor genes present in the genomes. For comparison we used our previously published genome sequences of four S. aureus isolates from bovine mastitis. The pan-genome and core genomes of the non-aureus isolates were characterized. After that, putative virulence factor orthologues were searched in silico. We compared the presence of putative virulence factors in the NAS species and S. aureus and evaluated the potential association between bacterial genotype and type of mastitis (clinical vs. subclinical). The NAS isolates had much less virulence gene orthologues than the S. aureus isolates. One third of the virulence genes were detected only in S. aureus. About 100 virulence genes were present in all S. aureus isolates, compared to about 40 to 50 in each NAS isolate. S. simulans differed the most. Several of the virulence genes detected among NAS were harbored only by S. simulans, but it also lacked a number of genes present both in S. agnetis and S. chromogenes. The type of mastitis was not associated with any specific virulence gene profile. It seems that the virulence gene profiles or cumulative number of different virulence genes are not directly associated with the type of mastitis (clinical or subclinical), indicating that host derived factors such as the immune status play a pivotal role in the manifestation of mastitis.

Bacteriological etiology and treatment of mastitis in Finnish dairy herds.

BACKGROUND: The Finnish dairy herd recording system maintains production and health records of cows and herds. Veterinarians and farmers register veterinary treatments in the system. Milk samples for microbiological analysis are routinely taken from mastitic cows. The laboratory of the largest dairy company in Finland, Valio Ltd., analyzes most samples using real-time PCR. This study addressed pathogen-specific microbiological data and treatment and culling records, in combination with cow and herd characteristics, from the Finnish dairy herd recording system during 2010-2012. RESULTS: The data derived from 240,067 quarter milk samples from 93,529 dairy cows with mastitis; 238,235 cows from the same herds served as the control group. No target pathogen DNA was detected in 12% of the samples. In 49% of the positive samples, only one target species and in 19%, two species with one dominant species were present. The most common species in the samples with a single species only were coagulase-negative staphylococci (CNS) (43%), followed by Staphylococcus aureus (21%), Streptococcus uberis (9%), Streptococcus dysgalactiae (8%), Corynebacterium bovis (7%), and Escherichia coli (5%). On average, 36% of the study cows and 6% of the control cows had recorded mastitis treatments during lactation. The corresponding proportions were 16 and 6% at drying-off. For more than 75% of the treatments during lactation, diagnosis was acute clinical mastitis. In the milk samples from cows with a recorded mastitis treatment during lactation, CNS and S. aureus were most common, followed by streptococci. Altogether, 48% of the cows were culled during the study. Mastitis was reported as the most common reason to cull; 49% of study cows and 18% of control cows were culled because of mastitis. Culling was most likely if S. aureus was detected in the milk sample submitted during the culling year. CONCLUSIONS: The PCR test has proven to be an applicable method also for large-scale use in bacterial diagnostics. In the present study, microbiological diagnosis was unequivocal in the great majority of samples where a single species or two species with one dominating were detected. Coagulase-negative staphylococci and S. aureus were the most common species. S. aureus was also the most common pathogen among the culled cows, which emphasizes the importance of preventive measures.