Piret Kalmus

Udder pathogens and their resistance to antimicrobial agents in dairy cows in Estonia

BackgroundThe goal of this study was to estimate the distribution of udder pathogens and their antibiotic resistance in Estonia during the years 2007-2009.MethodsThe bacteriological findings reported in this study originate from quarter milk samples collected from cows on Estonian dairy farms that had clinical or subclinical mastitis. The samples were submitted by local veterinarians to the Estonian Veterinary and Food Laboratory during 2007-2009. Milk samples were examined by conventional bacteriology. In vitro antimicrobial susceptibility testing was performed with the disc diffusion test. Logistic regression with a random herd effect to control for clustering was used for statistical analysis.ResultsDuring the study period, 3058 clinical mastitis samples from 190 farms and 5146 subclinical mastitis samples from 274 farms were investigated. Positive results were found in 57% of the samples (4680 out of 8204), and the proportion did not differ according to year (p > 0.05). The proportion of bacteriologically negative samples was 22.3% and that of mixed growth was 20.6%. Streptococcus uberis (Str. uberis) was the bacterium isolated most frequently (18.4%) from cases of clinical mastitis, followed by Escherichia coli (E. coli) (15.9%) and Streptococcus agalactiae (Str. agalactiae) (11.9%). The bacteria that caused subclinical mastitis were mainly Staphylococcus aureus (S. aureus) (20%) and coagulase-negative staphylococci (CNS) (15.4%). The probability of isolating S. aureus from milk samples was significantly higher on farms that had fewer than 30 cows, when compared with farms that had more than 100 cows (p < 0.005). A significantly higher risk of Str. agalactiae infection was found on farms with more than 600 cows (p = 0.034) compared with smaller farms. The proportion of S. aureus and CNS isolates that were resistant to penicillin was 61.4% and 38.5%, respectively. Among the E. coli isolates, ampicillin, streptomycin and tetracycline resistance were observed in 24.3%, 15.6% and 13.5%, respectively.ConclusionsThis study showed that the main pathogens associated with clinical mastitis were Str. uberis and E. coli. Subclinical mastitis was caused mainly by S. aureus and CNS. The number of S. aureus and Str. agalactiae isolates depended on herd size. Antimicrobial resistance was highly prevalent, especially penicillin resistance in S. aureus and CNS.

Occurrence of clinical mastitis in primiparous Estonian dairy cows in different housing conditions

BackgroundObjectives of the study were to document the impact of some management factors on the occurrence of clinical mastitis in primiparous dairy cows and to identify common udder pathogens of clinical mastitis in freshly calved heifers and multiparous cows on the day of calving.MethodsA one-year study was conducted during 2004 and 2005 in 11 selected Estonian dairy herds. Data consisted of 68 heifers with clinical mastitis and 995 heifers without clinical mastitis on the day of calving. Multivariable logistic regression with a random herd effect was used to investigate any association between housing system or the time interval from movement of heifers to the calving facility and day of calving on occurrence of clinical mastitis. Milk samples for bacteriological analysis were collected from affected heifers and multiparous cows on the day of calvingResultsClinical mastitis occurrence in the study population of freshly calved heifers equalled 6.1 %. Housing system was not a significant risk factor for clinical mastitis of freshly calved heifers.Moving heifers to the cowbarn less than two weeks before calving in tiestall farms increased risk (OR = 5.9 p = 0.001) for clinical mastitis at parturition. The most frequently isolated udder pathogens among heifers were Escherichia coli (22.1%), Streptococcus uberis (19.1%) and coagulase-negative staphylococci (8.8%). In comparison, the main pathogen in multiparous cows with clinical mastitis at parturition was Staphylococcus aureus (11.2%).ConclusionMoving heifers to the calving facilities too late in tiestall farms increased risk for clinical mastitis at parturition. The isolated udder pathogens did not differ significantly in tiestall farms compared to freestall farms in heifers, but differences were found between heifers and multiparous cows at parturition.

Milk haptoglobin, milk amyloid A, and N-acetyl-β-D-glucosaminidase activity in bovines with naturally occurring clinical mastitis diagnosed with a quantitative PCR test.

The associations between quantitative bacteriological results from a real-time PCR test and concentrations of acute-phase proteins (APP) and N-acetyl-β-d-glucosaminidase (NAGase) activity in milk in naturally occurring clinical mastitis were investigated. Milk APP concentrations and NAGase activity in clinical mastitis caused by different udder pathogens were studied. The associations between the severity of the clinical signs and concentrations of APP and NAGase activity were estimated. Milk samples from 281 cases of clinical mastitis were collected from 3 Estonian dairy farms and analyzed by PCR to identify pathogens. Twenty-seven samples out of 281 (9.6%) were PCR negative. Milk samples containing 4 or more bacterial species (n=28) were considered possibly contaminated and excluded from all further analyses. In total, 443 bacterial identifications were made from the remaining 226 milk samples. A single bacterial species was detected in 68 samples (30.1%), 2 species were detected in 99 samples (43.8%), and 3 species were detected in 59 (26.1%) samples. To determine the inflammatory response in the udder, the concentrations of milk amyloid A (MAA) and haptoglobin (Hp) and NAGase activity in the milk were analyzed. A significant positive association was found between the severity of the clinical signs and inflammatory markers in the milk. Milk amyloid A and Hp concentrations and NAGase activity were significantly higher in samples with large quantities of bacterial DNA from Escherichia coli or Streptococcus dysgalactiae compared with milk samples not containing those species. Large quantities of bacterial DNA from Trueperella pyogenes or Streptococcus uberis in the milk were associated with elevated concentrations of Hp and high NAGase activity, but not with increased MAA concentrations. Milk samples containing Corynebacterium bovis and coagulase-negative staphylococci had significantly lower concentrations of MAA and Hp and lower NAGase activity compared with samples where these species were not detected. It can be concluded that concentrations of APP and NAGase activity in the milk were associated with the quantity of bacterial DNA in the milk samples.

Plasmid with Colistin Resistance Gene mcr-1 in Extended-Spectrum-β-Lactamase-Producing Escherichia coli Strains Isolated from Pig Slurry in Estonia

ABSTRACT A plasmid carrying the colistin resistance gene mcr-1 was isolated from a pig slurry sample in Estonia. The gene was present on a 33,311-bp plasmid of the IncX4 group. mcr-1 is the only antibiotic resistance gene on the plasmid, with the other genes mainly coding for proteins involved in conjugative DNA transfer (taxA, taxB, taxC, trbM, and the pilX operon). The plasmid pESTMCR was present in three phylogenetically very different Escherichia coli strains, suggesting that it has high potential for horizontal transfer.

Effect of yeast culture on milk production and metabolic and reproductive performance of early lactation dairy cows.

BackgroundThe main objective of this study was to estimate the effect of supplementation with Saccaromyces cerevisiae (SC) (Yea-Sacc® 1026) on milk production, metabolic parameters and the resumption of ovarian activity in early lactation dairy cows.MethodsThe experiment was conducted during 2005/2006 in a commercial tied-house farm with an average of 200 milking Estonian Holstein Friesian cows. The late pregnant multiparous cows (n = 46) were randomly divided into two groups; one group received 10 g yeast culture from two weeks before to 14 weeks after calving. The groups were fed a total mixed ration with silages and concentrates. Milk recording data and blood samples for plasma metabolites were taken. Resumption of luteal activity was determined using milk progesterone (P4) measurements. Uterine bacteriology and ovarian ultrasonography (US) were performed and body condition scores (BCS) and clinical disease occurrences were recorded. For analysis, the statistical software Stata 9.2 and R were used to compute Cox proportional hazard and linear mixed models.ResultsThe average milk production per cow did not differ between the groups (32.7 ± 6.4 vs 30.7 ± 5.3 kg/day in the SC and control groups respectively), but the production of milk fat (P < 0.001) and milk protein (P < 0.001) were higher in the SC group. There was no effect of treatment on BCS. The analysis of energy-related metabolites in early lactation showed no significant differences between the groups. In both groups higher levels of β-hydroxybutyrate (BHB) appeared from days 14 to 28 after parturition and the concentration of non-esterfied fatty acid (NEFA) was higher from days 1–7 post partum (PP). According to US and P4 results, all cows in both groups ovulated during the experimental period. The resumption of ovarian activity (first ovulations) and time required for elimination of bacteria from the uterus did not differ between the groups.ConclusionSupplementation with SC had an effect on milk protein and fat production, but did not influence the milk yield. No effects on PP metabolic status, bacterial elimination from the uterus nor the resumption of ovarian activity were found.

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.

Efficacy of 5-day parenteral versus intramammary benzylpenicillin for treatment of clinical mastitis caused by gram-positive bacteria susceptible to penicillin in vitro

The efficacy of parenteral (intramuscular) or intramammary (IMM) benzylpenicillin treatment for clinical mastitis caused by gram-positive bacteria susceptible to penicillin in vitro was investigated. Cows with clinical mastitis in 1 udder quarter were randomly placed into 2 treatment groups. The preliminary bacteriological diagnosis of intramammary infection (IMI) was based on on-farm culturing, and the bacteriological diagnoses were later confirmed by a quantitative PCR assay. Clinical mastitis caused by gram-positive bacteria susceptible to benzylpenicillin was treated with penicillin via either the parenteral route (20mg/kg) or IMM route (600mg) once per day for 5d. The outcome of the treatment was evaluated 3 to 4wk after the onset of the treatment. The affected quarter was examined to assess the clinical cure, and milk samples were collected from the affected quarter to determine the bacteriological cure and milk N-acetyl-β-d-glucosaminidase activity. The survival and the composite milk somatic cell counts of the treated cows were followed up for 6 and 3mo after treatment, respectively. A total of 140 cows with clinical mastitis were included in the study, 61 being treated with benzylpenicillin parenterally and 79 via the IMM route. From all quarters treated, 108 of 140 (77.1%) were cured clinically and 77 of 140 (55.0%) were cured bacteriologically. The route of treatment did not significantly affect the outcome of the treatment; 80.3% of the quarters with parenteral treatment and 74.7% of the quarters with IMM treatment showed a clinical cure, and 54.1 and 55.7% a bacteriological cure, respectively. The milk N-acetyl-β-d-glucosaminidase activity was significantly lower in the quarters with a clinical or bacteriological cure than in the quarters with no cure. The 6-mo survival and the proportion of cows with composite milk somatic cell counts <200,000/mL among the treated cows during the 3-mo follow-up period did not significantly differ between the treatment groups. In conclusion, the outcome of either parenteral or IMM benzylpenicillin treatment of clinical mastitis caused by penicillin-susceptible bacteria was similar.

N-acetyl -β-D-glucosaminidase activity in cow milk as an indicator of mastitis.

Activity of lysosomal N-acetyl-β-d-glucosaminidase (NAGase) in milk has been used as an indicator of bovine mastitis. We studied NAGase activity of 808 milk samples from healthy quarters and quarters of cows with spontaneous subclinical and clinical mastitis. Associations between milk NAGase activity and milk somatic cell count (SCC), mastitis causing pathogen, quarter, parity, days in milk (DIM) and season were studied. In addition, the performance of NAGase activity in detecting clinical and subclinical mastitis and distinguishing infections caused by minor and major bacteria was investigated. Our results indicate that NAGase activity can be used to detect both subclinical and clinical mastitis with a high level of accuracy (0·85 and 0·99). Incomplete correlation between NAGase activity and SCC suggests that a substantial proportion of NAGase activity comes from damaged epithelial cells of the udder in addition to somatic cells. We therefore recommend determination of NAGase activity from quarter foremilk after at least six hours from the last milking using the method described. Samples should be frozen before analysis. NAGase activity should be interpreted according to DIM, at least during the first month of lactation. Based on the results of the present study, a reference value for normal milk NAGase activity of 0·1-1·04 pmoles 4-MU/min/μl for cows with ≥30 DIM (196 samples) could be proposed. We consider milk NAGase activity to be an accurate indicator of subclinical and clinical mastitis.

Within-herd prevalence of intramammary infection caused by Mycoplasma bovis and associations between cow udder health, milk yield, and composition

Subclinical mastitis is one of the major health problems in dairy herds due to decreased milk production and reduced milk quality. The aim of this study was to examine the within-herd prevalence of subclinical intramammary infection caused by Mycoplasma bovis and to evaluate associations between M. bovis and cow daily milk yield, udder health, and milk composition. Individual cow composite milk samples (n = 522) were collected from all lactating dairy cows in 1 Estonian dairy farm in November 2014. Daily milk yield, days in milk, and parity were recorded. Collected milk samples were analyzed for somatic cell count, milk protein, fat, and urea content. The presence of M. bovis, Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus uberis in the milk samples was confirmed by quantitative PCR analysis. The within-herd prevalence of M. bovis was 17.2% in the study herd. No association was observed between days in milk and parity to the presence of M. bovis in milk. According to linear regression analysis, the daily milk yield from cows positive for M. bovis was on average 3.0 kg lower compared with cows negative for M. bovis. In addition, the presence of M. bovis in milk samples was significantly associated with higher somatic cell count and lower fat and urea content compared with milk samples negative for M. bovis. In conclusion, subclinical M. bovis intramammary infection is associated with decreased milk yield and lower milk quality.

Elimination of selected mastitis pathogens during the dry period

We aimed to evaluate the elimination of 4 different mastitis pathogens, Streptococcus agalactiae, Mycoplasma bovis, Staphylococcus aureus, and Streptococcus uberis, from infected udder quarters during the dry period using quantitative PCR. The second purpose of this study was to evaluate the association between milk haptoglobin (Hp) concentration and the presence of udder pathogens (Strep. agalactiae, Staph. aureus, M. bovis, and Strep. uberis) in udder quarter milk samples before and after dry period. Aseptic udder quarter milk samples (n = 1,001) were collected from 133 dairy cows at dry off and at the first milking after calving from 1 large dairy herd. Bacterial DNA of Strep. agalactiae, Staph. aureus, Strep. uberis, and M. bovis in the udder quarter milk samples was identified with commercial quantitative PCR analysis Mastitis 4B (DNA Diagnostic A/S, Risskov, Denmark). Milk Hp concentration (mg/L) was measured from udder quarter milk samples. The elimination rates during the dry period for M. bovis, Staph. aureus, Strep. agalactiae, and Strep. uberis were 86.7, 93.6, 96.2, and 100.0%, respectively. The new IMI rate was 3.0% for M. bovis, 2.9% for Staph. aureus, 2.4% for Strep. agalactiae, and 3.1% for Strep. uberis. The milk Hp concentration was significantly higher in udder quarter milk samples with blood and in samples positive for Strep. agalactiae at dry off and for Staph. aureus postcalving. Elevated milk Hp concentration was not associated with the presence of M. bovis in the udder quarter milk samples. In conclusion, elimination of Staph. aureus, Strep. agalactiae, and Strep. uberis during the dry period was high; the elimination of M. bovis from infected udder quarters was lower, but probably spontaneous. Additionally, milk Hp concentration may be used as a marker for udder inflammation when combined with the bacteriological results at dry off and postpartum.