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

Bovine mastitis caused by coagulase-negative staphylococci : host response and bacterial factors

Coagulase-negative staphylococci (CNS) are the most common bacteria isolated in bovine subclinical mastitis in many countries, and also a frequent cause of clinical mastitis. The most common species isolated are Staphylococcus (S) chromogenes, S. simulans, S. epidermidis, and S. xylosus. One half of the intramammary infections (IMI) caused by CNS persist in the udder. The pathogenesis of IMI caused by CNS is poorly understood. This dissertation focuses on host response in experimental intramammary infection induced by S. chromogenes, S. epidermidis and S. simulans. Model for a mild experimental CNS infection was developed with S. chromogenes (study I). All cows were infected and most developed subclinical mastitis. In study II the innate immune response to S. epidermidis and S. simulans IMI was compared in eight cows using a crossover design. A larger dose of bacteria was used to induce clinical mastitis. All cows became infected and showed mild to moderate clinical signs of mastitis. S. simulans caused a slightly stronger innate immune response than S. epidermidis, with significantly higher concentrations of the interleukins IL-1beta and IL-8 in the milk. The spontaneous elimination rate of the 16 IMIs was 31%, with no difference between species. No significant differences were recorded between infections eliminated spontaneously or remaining persistent, although the response was stronger in IMIs eliminated spontaneously, except the concentration of TNF-α, which remained elevated in persistent infections. Lactoferrin (Lf) is a component of the humoral defence of the host and is present at low concentrations in the milk. The concentration of Lf in milk is high during the dry period, in colostrum, and in mastitic milk. The effect of an inherent, high concentration of Lf in the milk on experimental IMI induced with S. chromogenes was studied in transgenic cows that expressed recombinant human Lf in their milk. Human Lf did not prevent S. chromogenes IMI, but the host response was milder in transgenic cows than in normal cows, and the former eliminated infection faster. Biofilm production has been suggested to promote persistence of IMI. Phenotypic biofilm formation and slime producing ability of CNS isolates from bovine mastitis was investigated in vitro. One-third of mastitis isolates produced biofilm. Slime production was less frequent for isolates of the most common mastitis causing species S. chromogenes and S. simulans compared with S. epidermidis. No association was found between the phenotypic ability to form biofilm and the persistence of IMI or severity of mastitis. Slime production was associated with persistent infections, but only 8% of isolates produced slime.Koagulaasi-negatiiviset stafylokokit (KNS) ovat yleisimpiä lehmän piilevän utaretulehduksen aiheuttajabakteereita useissa maissa. Tavallisimmat utaretulehdusta aiheuttavat KNS-lajit ovat Staphylococcus (S). chromogenes, S. simulans, S. epidermidis, ja S. xylosus. Noin puolet KNS:n aiheuttamista utaretulehduksista paranee itsestään ilman antibioottihoitoa. KNS-utaretulehduksen patogeneesi tunnetaan huonosti. Tässä väitöskirjatyössä tutkittiin lehmän luontaista puolustusvastetta S. chromogenes-, S. epidermidis- ja S. simulans -bakteereiden aiheuttamassa kokeellisessa utaretulehduksessa. Lisäksi tutkittiin utaretulehdusta aiheuttavien KNS-lajien biofilmin- ja limantuottokykyä sekä niiden vaikutusta tulehduksen paranemiseen. Kokeellisessa utaretulehduksessa kaikki kolme KNS-lajia saivat aikaan lievän utaretulehduksen, joka käynnisti yleisen ja paikallisen puolustusvasteen. Kokeellinen malli kehitettiin S. chromogenes -bakteerin avulla. Toisessa tartutuksessa vertailtiin S. epidermidis- ja S. simulans -bakteerien aiheuttamaa luontaista puolustusvastetta. S. simulans sai aikaan voimakkaamman vasteen ja nosti erityisesti kahden interleukiinin (IL-1-beta ja IL-8) pitoisuutta maidossa. Lehmistä parani spontaanisti 31 % ilman lajien välisiä eroja. Lehmien luontaisessa puolustusvasteessa ei parantuneiden ja ei-parantuneiden välillä ollut tilastollisesti merkitseviä eroja, joskin parantuneilla lehmillä puolustusvaste, TNF-α -sytokiinia lukuun ottamatta, ilmeni hieman voimakkaampana. Laktoferriini on osa utareen luontaista puolustusvastetta. Se on antibakteerinen, rautaa sitova glykoproteiini. Normaalissa maidossa laktoferriiniä on vähän, mutta sen pitoisuus nousee utaretulehduksessa nopeasti. Muuntogeenisillä lehmillä, joiden maidossa oli synnynnäisesti korkea laktoferriinipitoisuus, kokeellinen S. chromogenes -tulehdus ilmeni lievempänä ja eliminoitui nopeammin kuin normaaleilla lehmillä. Se ei kuitenkaan estänyt utareen infektoitumista. Bakteereiden muodostaman biofilmin on arveltu edistävän utaretulehduksen kroonistumista. Utaretulehduksesta eristettyjen KNS-kantojen biofilmin- ja limanmuodostusta tutkittiin erityyppisillä kasvatusalustoilla. Kolmannes tutkituista kannoista tuotti biofilmiä. Verrattuna muihin KNS-lajeihin, S. epidermidis -lajin liman- ja biofilminmuodostus oli yleisempää ja voimakkaampaa, kun taas muilla yleisillä lajeilla vähäisempää. Bakteerikannan biofilmintuottokyky ei ollut yhteydessä sen aiheuttaman utaretulehduksen vakavuuteen tai paranemiseen, kun taas sen limanmuodostuskyky oli yhteydessä tulehduksen kroonistumiseen. Limaa tuottavia bakteerikantoja oli kuitenkin suhteellisen vähän, vain noin 8 % kannoista

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

Dry cow therapy and early lactation udder health problems - Associations and risk factors

Mastitis remains the most expensive disease of dairy cows, and antibiotic dry cow therapy (DCT) at dry-off is an important part of mastitis control. Regardless of the infection status, blanket DCT is administered to all quarters of all cows, which is controversial due to the worldwide problem of antimicrobial resistance. Even though selective DCT of only infected cows is a more sustainable approach, choosing animals for treatment is not always straightforward. Our aim was to evaluate whether the herd-level DCT approach is associated with early lactation udder health problems, taking into account the cow characteristics. The information source was 2015?2017 Dairy Herd Improvement data with 7461 multiparous cows from 241 Finnish dairy herds. Information on the herd-level DCT approach was obtained from farmers? questionnaire responses in 2017, and the three different approaches were selective DCT, blanket DCT, and no DCT. The statistical tool for the data analysis was a generalized linear mixed model with a random herd effect for binary outcomes and a linear mixed model with a random herd effect for a continuous outcome. The two binary outcomes were the odds of having high milk somatic cell count (SCC ? 200 000 cells/mL) on the first test-day within 5?45 days in milk (DIM) and the odds of mastitis treatment in early lactation up to 45 DIM. The third outcome was the mean milk lnSCC (? 1000 cells/ mL) within 120 DIM. Selective DCT was the prevailing treatment practice in our data. Blanket DCT was associated with lower SCC after calving. Cows more likely to have high SCC after calving were older cows, cows with high average SCC during the previous lactation, and cows with high milk yield near dry-off. A mastitis treatment in the early lactation was more likely if, during the previous lactation, the cow had high average SCC, high peak milk production, or high milk yield near dry-off. Our findings indicate that DCT is still effective in mastitis control. Cows with high milk yield, especially near dry-off, and cows with persistently high SCC require attention when considering next lactation udder health.Mastitis remains the most expensive disease of dairy cows, and antibiotic dry cow therapy (DCT) at dry-off is an important part of mastitis control. Regardless of the infection status, blanket DCT is administered to all quarters of all cows, which is controversial due to the worldwide problem of antimicrobial resistance. Even though selective DCT of only infected cows is a more sustainable approach, choosing animals for treatment is not always straightforward. Our aim was to evaluate whether the herd-level DCT approach is associated with early lactation udder health problems, taking into account the cow characteristics. The information source was 2015?2017 Dairy Herd Improvement data with 7461 multiparous cows from 241 Finnish dairy herds. Information on the herd-level DCT approach was obtained from farmers? questionnaire responses in 2017, and the three different approaches were selective DCT, blanket DCT, and no DCT. The statistical tool for the data analysis was a generalized linear mixed model with a random herd effect for binary outcomes and a linear mixed model with a random herd effect for a continuous outcome. The two binary outcomes were the odds of having high milk somatic cell count (SCC ? 200 000 cells/mL) on the first test-day within 5?45 days in milk (DIM) and the odds of mastitis treatment in early lactation up to 45 DIM. The third outcome was the mean milk lnSCC (? 1000 cells/ mL) within 120 DIM. Selective DCT was the prevailing treatment practice in our data. Blanket DCT was associated with lower SCC after calving. Cows more likely to have high SCC after calving were older cows, cows with high average SCC during the previous lactation, and cows with high milk yield near dry-off. A mastitis treatment in the early lactation was more likely if, during the previous lactation, the cow had high average SCC, high peak milk production, or high milk yield near dry-off. Our findings indicate that DCT is still effective in mastitis control. Cows with high milk yield, especially near dry-off, and cows with persistently high SCC require attention when considering next lactation udder health.Peer reviewe

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

Prevalence of subclinical mastitis in Finnish dairy cows : changes during recent decades and impact of cow and herd factors

Background: The dairy industry has undergone substantial structural changes as intensive farming has developed during recent decades. Mastitis continues to be the most common production disease of dairy cows. Nationwide surveys of mastitis prevalence are useful in monitoring udder health of dairy herds and to study the impact of structural changes on the dairy industry. This survey on bovine subclinical mastitis was the first based on cow composite milk somatic cell count (SCC) data from the Finnish national health monitoring and milk recording database. A cow with composite milk SCC = 200,000 cells/ml in at least one of the four test milkings during the year was considered to have subclinical mastitis and a cow with composite milk SCC = 200,000 cells/ml in three or in all four test milkings during the year to have chronic subclinical mastitis. The aim of the study was to determine the prevalence of subclinical mastitis and chronic subclinical mastitis in Finland in 1991, 2001 and 2010 and to investigate cow and herd factors associated with elevated SCC. Results: Prevalence of subclinical mastitis in Finland decreased over recent decades from 22.3% (1991) and 20.1% (2001) to 19.0% (2010). Prevalence of chronic subclinical mastitis was 20.4% in 1991, 15.5% in 2001 and 16.1% in 2010. The most significant cow and herd factors associated with subclinical mastitis or high milk SCC were increasing parity, Holstein breed, free- stalls with an automatic milking system and organic production. Milk SCC were highest from July to September. Main factors associated with chronic mastitis were increasing parity and Holstein breed. Conclusions: Prevalence of subclinical mastitis in Finland decreased over recent decades, the greatest change taking place during the first decade of the study. Prevalence of chronic subclinical mastitis significantly decreased from 1991. The most significant factors associated with both types of mastitis were increasing parity and Holstein breed, and for subclinical mastitis also free-stalls with automatic milking. National surveys on mastitis prevalence should be carried out at regular intervals to monitor udder health of dairy cows and to study the impact of the ongoing structural changes in the dairy industry to enable interventions related to udder health to be made when needed.Peer reviewe

Prototheca spp. naudan utaretulehduksen aiheuttajana : kirjallisuuskatsaus ja tapausselostus kolmesta karjaongelmasta

Lypsylehmien utaretulehdus heikentää eläinten hyvinvointia sekä tuotannon taloudellisuutta. Prototheca spp. -levät elävät kosteissa ympäristöissä ja kykenevät muodostamaan pinnoille biofilmejä. Prototekan aiheuttama utaretulehdus on harvinainen mutta yleistynyt viime vuosina. Useimmiten aiheuttajana on Prototheca bovis. Prototekan taudinaiheutus perustuu kudosinvaasioon. Tulehdus voi olla paikallinen, jolloin lehmällä ei ole yleisoireita, mutta maito voi muuttua vetiseksi tai utare turvonneeksi ja kovaksi. Toisinaan prototeka aiheuttaa akuutin kuumeisen utaretulehduksen. Useimmiten tulehdukset ovat lieviä tai kohtalaisia. Prototeka-utaretulehdukseen ei ole tehokasta hoitoa, joten vaihtoehdoiksi jäävät erilleen lypsy, infektoituneen neljänneksen umpeutus tai lehmän poisto. Ehkäisyssä on tärkeää huolehtia lypsyhygieniasta sekä ympäristön puhtaudesta. Osa prototeka-lajeista on zoonoottisia ja ne aiheuttavat tyypillisimmin ihmiselle iho-oireita. Kolmella suomalaisella lypsykarjatilalla prototeka-utaretulehdukset olivat muodostuneet karjaongelmaksi ja niille tehtiin ongelmanselvityskäynnit. Käynneillä otetuista ympäristönäytteistä löytyi PCR-menetelmällä Prototheca spp. useista kohteista. Tiloille annettiin toiminnan parannusehdotuksia. Kahdella tilalla prototekan aiheuttamat utaretulehdukset loppuivat ja yhdellä niiden määrä väheni.Peer reviewe

Field trial to evaluate the effect of an intranasal respiratory vaccine protocol on bovine respiratory disease incidence and growth in a commercial calf rearing unit

BackgroundBovine respiratory disease (BRD) continues to be great challenge in calf rearing units. The urgent need to decrease the use of antibiotics and increase animal welfare in beef production has forced us to introduce new preventive methods. Vaccinations could contribute to the solution, but the high incidence of BRD already at an early age has made it difficult to introduce suitable vaccination programs. Challenge studies have shown promising results in 3-14day old calves vaccinated with intranasal BRD vaccine, but very few field trials are available to assess the efficacy of the intranasal vaccines in field conditions. We evaluated the effect of one dose of commercial intranasal vaccination on calf mortality, daily gain, and treatment incidence for BRD in one calf rearing unit. In total, 497 calves (mean age 19days) were included in our study, 247 of which were vaccinated at the time of arrival to the unit and 250 served as negative controls (unvaccinated). Vaccinated and unvaccinated calves were situated in separate compartments until weaning. Daily gain, treatment incidence, and mortality were recorded until the calves were transported to the finishing unit, which averaged 154.5days from arrival.ResultsAverage daily gain over the complete study period was 1151.9g/day (SD 137.9) for the vaccinated calves and 1139.5g/day (SD 135.9) for the unvaccinated calves. Intranasal vaccination combined with older arrival age (17days or older) resulted in a higher daily gain (47.8g/day) compared with unvaccinated calves (coef. 0.0478, p=0.003). This association was not recorded in calves that were younger than 17days upon arrival. Intranasal vaccination was not significantly associated either with mortality (OR 0.976, p=0.968) or treatment incidence for BRD (OR 1.341, p=0.120). In total, six vaccinated calves (2.43%) and six unvaccinated calves (2.40%) died during the study period.ConclusionsVaccinating arriving calves with intranasal vaccine in the calf rearing unit did not decrease the mortality or treatment incidence for BRD, but it significantly improved the weight gain in calves transported to the unit at the age of 17days or older.Peer reviewe