Bovine mastitis caused by coagulase-negative staphylococci

Bovine mastitis caused by coagulase-negative staphylococci Mastitis is the most common disease affecting dairy cows, and staphylococci are the most common cause of mastitis. According to Finnish studies, staphylococci are isolated from 36% of clinical and 60% of subclinical mastitis cases. In mastitis diagnostics, staphylococci are divided into coagulase-positive and coagulase-negative based on their ability to coagulate plasma. Majority of staphylococcal mastitis is caused by coagulase-negative staphylococci (CNS). In mastitis diagnostics they are not identified at species level but are treated as a uniform group. Usually they cause subclinical or mildly clinical mastitis, and are considered as harmless bacteria of the normal skin flora. CNS mastitis is most common on the first lactation. It is assumed to cure spontaneously and antimicrobial treatment is not recommended. Although usually subclinical or mildly clinical, CNS mastitis causes increase of milk leucocytes and may decrease the milk quality. CNS mastitis seems to be especially a problem in well managed dairy herds with good udder health. In contrast to the usual assumption that CNS mastitis cures spontaneously, it may persist in the udder throughout the entire lactation. In addition, the CNS species are not necessarily a uniform group but differences between the species in virulence and clinical characteristics may exist. In this dissertation, different aspects of bovine mastitis caused by CNS were studied. The response of CNS mastitis to antimicrobial treatment was investigated, as well as the persistence of CNS intramammary infection when left without antimicrobial treatment. The prevalence of different CNS species in mastitis and on cows skin, and possible differences in clinical characteristics and persistence between CNS species were studied. Species identification was performed based on bacterial phenotype and genotype. Identification using different methods was compared. Majority of CNS mastitis was caused by S. chromogenes or S. simulans. Many other CNS species were isolated from mastitis sporadically. Molecular genetic identification methods based on bacterial genotype proved more reliable than biochemical methods based on bacterial phenotype. In some cases the identification results of genotypic and phenotypic methods disagreed. About half of CNS mastitis detected during lactation persisted without antimicrobial treatment, commonly until the end of lactation. Mastitis caused by penicillin-susceptible CNS cured well, about 90% cured. Of mastitis caused by penicillin-resistant CNS, 60 to 70% cured, although the causing bacteria were susceptible to the antimicrobial used. Most often CNS mastitis was subclinical or mildly clinical. Statistically significant differences in clinical characteristics or persistence between the two most common CNS species were not detected.Bovine mastitis caused by coagulase-negative staphylococci Utaretulehdus on lypsylehmien yleisin sairaus ja stafylokokit yleisimpiä utaretulehduksen aiheuttajabakteereita. Kliinisistä eli näkyviä oireita aiheuttavista utaretulehduksista stafylokokit aiheuttavat suomalaisten tutkimusten mukaan noin 36 % ja subkliinisistä eli piilevistä noin 60 %. Utaretulehdusdiagnostiikassa stafylokokit jaetaan koagulaasipositiivisiin ja negatiivisiin ns. koagulaasitestin perusteella. Staphylococcus (S.) aureus on tunnettu koagulaasipositiivinen utaretulehduksen aiheuttaja, joka voi aiheuttaa vakavaoireisen, mutta myös oireettoman ja helposti kroonistuvan utaretulehduksen. Muut utaretulehduksesta eristetyt stafylokokit ovat yleensä koagulaasinegatiivisia stafylokokkeja (KNS), joita ei utaretulehdusdiagnostiikassa tunnisteta lajitasolle. Ne aiheuttavat useimmiten piilevän tai lieväoireisen utaretulehduksen, ja niitä onkin pidetty harmittomina ihon normaaliflooraan kuuluvina bakteereina. KNS-utaretulehdus on yleisin ensimmäisellä maidontuotantokaudella. Sen on oletettu paranevan itsestään poikimisen jälkeen, eikä antibioottihoitoa ole suositeltu. Valtaosa stafylokokkien aiheuttamista utaretulehduksista johtuu koagulaasinegatiivisista stafylokokeista. Vaikka tulehdukset ovat piileviä tai lieviä, ne aiheuttavat kuitenkin maidon tulehdussolujen lisääntymistä ja maidon laadun heikkenemistä. Ne tuntuvat olevan ongelma etenkin hyvin hoidetuissa karjoissa, joissa muuten on hyvä utareterveys. Vastoin aiempia oletuksia KNS-utaretulehdus ei aina parane itsestään, vaan voi säilyä eli persistoida utareessa läpi koko lypsykauden. Eri KNS-lajit eivät välttämättä ole homogeeninen ryhmä, vaan eri lajien välillä saattaa olla eroja taudinaiheutuskyvyssä. Tässä väitöskirjatutkimuksessa selvitettiin KNS-utaretulehduksen persistoimiskykyä sekä vastetta antibioottihoitoon. Lisäksi selvitettiin eri KNS-lajien yleisyyttä utaretulehduksessa ja lehmien iholla sekä mahdollisia KNS-lajien välisiä eroja. KNS-lajien tunnistamista eri menetelmillä vertailtiin. Valtaosan KNS-utaretuhduksista aiheuttivat S. chromogenes ja S. simulans. Monia muita KNS-lajeja eristettiin utaretulehduksista satunnaisesti. Bakteerin genotyyppiin perustuvat, molekyyligeneettiset lajitunnistusmenetelmät osoittautuivat luotettavammiksi kuin fenotyyppiin perustuvat biokemialliset menetelmät. Eri menetelmillä saatiin joskus toisistaan poikkeava lajitunnistustulos. Noin puolet lypsykaudella todetuista ja ilman antibioottihoitoa jätetyistä KNS-utaretulehduksista pesiytyi utareeseen, useimmiten lypsykauden loppuun saakka. Penisilliiniherkän KNS-bakteerin aiheuttama tulehdus parani antibioottihoidolla hyvin, noin 90 %:sesti. Penisilliiniresistentin KNS-bakteerin aiheuttamat tulehdukset paranivat hieman huonommin, vaikka bakteeri oli herkkä käytetylle antibiootille. Valtaosa utaretulehduksista oli piileviä tai lieväoireisia. Yleisimpien KNS-lajien välillä ei todettu taudinaiheuttamiskyvyssä merkitseviä eroja

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 signifcantly 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 signifcantly 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.Peer reviewe

Species distribution and in vitro antimicrobial susceptibility of coagulase-negative staphylococci isolated from bovine mastitic milk

Background: Coagulase-negative staphylococci (CoNS) are the most common bovine mastitis causing bacteria in many countries. It is known that resistance for antimicrobials is in general more common in CoNS than in Staphylococcus aureus but little is known about the antimicrobial resistance of specific CoNS species. In this study, 400 CoNS isolates from bovine mastitic milk samples were identified to species level using ribotyping and MALDI-TOF MS, and their antimicrobial susceptibility was determined using a commercially available microdilution system. The results were interpreted according to the epidemiological cut-off values by the European Committee on Antimicrobial Susceptibility testing. Results: The most common CoNS species were S. simulans, S. epidermidis, S. chromogenes and S. haemolyticus. Penicillin resistance was the most common type of antimicrobial resistance. Staphylococcus epidermidis was the most resistant among the four major species. Almost one-third of our S. epidermidis isolates were resistant to >2 antimicrobials and close to 7 % were multidrug resistant. The majority of S. epidermidis isolates were resistant to benzylpenicillin. On the contrary, only few S. simulans isolates were penicillin-resistant. Phenotypic oxacillin resistance was found in all four main species, and 34 % of the isolates were oxacillin resistant. However, only 21 isolates (5 %) were positive for the mecA gene. Of these, 20 were S. epidermidis and one S. sciuri. mecC positive isolates were not found. Conclusion: Staphylococcus epidermidis differed from the three other major CoNS species as resistance to the tested antimicrobials was common, several isolates were multidrug resistant, and 19 % of the isolates carried the mecA gene encoding methicillin resistance.Peer reviewe

Bacteriological etiology and treatment of mastitis in Finnish dairy herds

Abstract 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

More testing, less antibiotics - future trends in mastitis treatment?

Poster201

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-β-D-glucosaminidase (NAGase) activity, milk amyloid A (MAA), serum amyloid A (SAA) and proinflammatory cytokines interleukin (IL)-1β, 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-1β 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.Peer reviewe

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

Bovine milk microbiome : a more complex issue than expected

Abstract 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