The effect of season on somatic cell count and the incidence of clinical mastitis

Bulk milk somatic cell count (BMSCC), individual cow somatic cell count (ICSCC), and incidence rate of clinical mastitis (IRCM) are all udder health parameters. So far, no studies have been reported on the effect of season on BMSCC, IRCM, and ICSCC in the same herds and period over multiple years. The objectives of this study were to determine the seasonal pattern over a 4-yr period of 1) BMSCC, 2) elevated ICSCC, 3) IRCM, and 4) pathogen-specific IRCM. Bulk milk somatic cell count, ICSCC, and pathogen-specific clinical mastitis data were recorded in 300 Dutch dairy farms. For the analyses of BMSCC, ICSCC, and IRCM, a mixed, a transitional, and a discrete time survival analysis model were used, respectively. Sine and cosine were included in the models to investigate seasonal patterns in the data. For all parameters, a seasonal effect was present. Bulk milk somatic cell count peaked in August to September in all 4 years. The probability of cows getting or maintaining a high ICSCC was highest in August and May, respectively. Older and late-lactation cows were more likely to develop or maintain a high ICSCC. Incidence rate of clinical mastitis was highest in December to January, except for Streptococcus uberis IRCM, which was highest in August. Totally confined herds had a higher Escherichia coli IRCM in summer than in winter. Compared with the major mastitis pathogens, the seasonal differences in IRCM were smaller for the minor pathogens. Distinguishing between Strep. uberis, Streptococcus dysgalactiae, Streptococcus agalactiae, and other streptococci is essential when identifying Streptococcus spp. because each of them has a unique epidemiology. Streptococcus uberis IRCM seemed to be associated with being on pasture, whereas E. coli IRCM was more housing-related.     

Genetic correlations between pathogen-specific mastitis and somatic cell count in Danish Holsteins

The aim of this study was to estimate genetic correlations (r_a) between 2 lactation average somatic cell count (LASCC) traits and 6 different mastitis traits in 226,482 first-parity Danish Holstein cows that calved between 1998 and 2008. The LASCC traits were defined from 5 to either 170 d (LASCC_170) or 300 d (LASCC_300) after calving, and the mastitis traits were unspecific mastitis (all mastitis treatments, both clinical and subclinical, regardless of the causative pathogen) and mastitis caused by either Streptococcus dysgalactiae, Escherichia coli, coagulase-negative staphylococci (CNS), Staphylococcus aureus, or Streptococcus uberis. Variance components were estimated using bivariate threshold-Gaussian models via Gibbs sampling. The posterior means of r_a between LASCC_170 and the mastitis traits were greatest for unspecific mastitis (r_a = 0.71), followed by CNS, Strep. dysgalactiae, Strep. uberis, and E. coli (r_a = 0.54 to 0.69) and were lowest for Staph. aureus mastitis (r_a = 0.44). The genetic correlation between LASCC_300 and the mastitis traits were generally smaller (r_a = 0.47 to 0.69). Caution should be taken when interpreting the results, however, because some posterior density intervals for r_a were large (between 0.14 and 0.47 units). Phenotypically, Staph. aureus is known to be associated with high SCC and especially with subclinical mastitis through chronic infections, so the low r_a between Staph. aureus mastitis and LASCC, compared with r_a for the other pathogens, was not expected. Subclinical cases are usually submitted to dry cow therapy (not included in the present study), not treated at all, or wrongly recorded as clinical cases. Thus, the incidence of Staph. aureus mastitis is likely too low, and the genetic correlation between Staph. aureus mastitis and LASCC may therefore be underestimated in the present study. The results for the remaining pathogens were as expected, smallest for E. coli and larger but similar for Strep. dysgalactiae, Strep. uberis, and CNS. Selection for lower LASCC is expected to decrease the incidence of pathogen-specific mastitis, especially for Strep. uberis, Strep. dysgalactiae, and CNS and, to a lesser extent, for Staph. aureus and E. coli. Data recording should preferably be improved, and economic weights for the pathogen-specific mastitis traits should be estimated before implementing an udder health index that includes pathogen-specific mastitis traits.     

Detection of mastitis pathogens by analysis of volatile bacterial metabolites

The ability to detect mastitis pathogens based on their volatile metabolites was studied. Milk samples from cows with clinical mastitis, caused by Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus uberis, Streptococcus dysgalactiae, and Escherichia coli were collected. In addition, samples from cows without clinical mastitis and with low somatic cell count (SCC) were collected for comparison. All mastitis samples were examined by using classical microbiological methods, followed by headspace analysis for volatile metabolites. Milk from culture-negative samples contained a lower number and amount of volatile components compared with cows with clinical mastitis. Because of variability between samples within a group, comparisons between pathogens were not sufficient for classification of the samples by univariate statistics. Therefore, an artificial neural network was trained to classify the pathogen in the milk samples based on the bacterial metabolites. The trained network differentiated milk from uninfected and infected quarters very well. When comparing pathogens, Staph. aureus produced a very different pattern of volatile metabolites compared with the other samples. Samples with coagulase-negative staphylococci and E. coli had enough dissimilarity with the other pathogens, making it possible to separate these 2 pathogens from each other and from the other samples. The 2 streptococcus species did not show significant differences between each other but could be identified as a different group from the other pathogens. Five groups can thus be identified based on the volatile bacterial metabolites: Staph. aureus, coagulase-negative staphylococci, streptococci (Strep. uberis and Strep. dysgalactiae as one group), E. coli, and uninfected quarters.     

Pathogen-specific effects of quantitative trait loci affecting clinical mastitis and somatic cell count in Danish Holstein cattle

The aim of this study was to investigate whether quantitative trait loci (QTL) affecting the risk of clinical mastitis (CM) and QTL affecting somatic cell score (SCS) exhibit pathogen-specific effects on the incidence of mastitis. Bacteriological data on mastitis pathogens were used to investigate pathogen specificity of QTL affecting treatments of mastitis in first parity (CM1), second parity (CM2), and third parity (CM3), and QTL affecting SCS. The 5 most common mastitis pathogens in the Danish dairy population were analyzed: Streptococcus dysgalactiae, Escherichia coli, coagulase-negative staphylococci, Staphylococcus aureus, and Streptococcus uberis. Data were analyzed using 2 approaches: an independence test and a generalized linear mixed model. Three different data sets were used to investigate the effect of data sampling: all samples, only samples that were followed by antibiotic treatment, and samples from first-crop daughters only. The results showed with high certainty that 2 QTL affecting SCS exhibited pathogen specificity against Staph. aureus and E. coli, respectively. The latter result might be explained by a pleiotropic QTL that also affects CM2 and CM3. Less certain results were found for QTL affecting CM. A QTL affecting CM1 was found to be specific against Strep. dysgalactiae and Staph. aureus, a QTL affecting CM2 was found to be specific against E. coli, and finally a QTL affecting CM3 was found to be specific against Staph. aureus. None of the QTL analyzed was found to be specific against coagulase-negative staphylococci and Strep. uberis. Our results show that particular mastitis QTL are highly likely to exhibit pathogen-specificity. However, the results should be interpreted carefully because the results are sensitive to the sampling method and method of analysis. Field data were used in this study. These kind of data may be heavily biased because there is no standard procedure for collecting milk samples for bacteriological analysis in Denmark. Furthermore, using only the mean SCS from d 10 to 180 after parturition may lead to truncated effects of SCS-QTL when samples collected after d 180 are used. Additionally, repeated samples were used, which could boost the difference in incidence of pathogens between daughters of sires inheriting the positive and negative QTL allele, respectively. However, the magnitude of these effects in this study is unclear.     

Pathogen-specific production losses in bovine mastitis

Reduction in long-term milk yields represents a notable share of the economic losses caused by bovine mastitis. Efficient, economic, and safe measures to prevent these losses require knowledge of the causal agent of the disease. The aim of this study was to investigate pathogen-specific impacts of mastitis on milk production of dairy cows. The materials consisted of milk and health recording data and microbiological diagnoses of mastitic quarter milk samples of 20,234 Finnish dairy cows during 2010, 2011, and 2012. The 6 most common udder pathogens were included in the study: Staphylococcus aureus, non-aureus staphylococci (NAS), Escherichia coli, Corynebacterium bovis, Streptococcus uberis, and Streptococcus dysgalactiae. We used a 2-level multilevel model to estimate curves for lactations with and without mastitis. The data on lactation periods to be compared were collected from the same cow. To enable comparison among lactations representing diverse parities, the estimated lactation curves were adjusted to describe the cow's third lactation. Mastitis caused by each pathogen resulted in milk production loss. The extent of the reduction depended on the pathogen, the timing of mastitis during lactation, and the type of mastitis (clinical vs. subclinical). The 2 most commonly detected pathogens were NAS and Staph. aureus. Escherichia coli clinical mastitis diagnosed before peak lactation caused the largest loss, 10.6% of the 305-d milk yield (3.5 kg/d). The corresponding loss for Staph. aureus mastitis was 7.1% (2.3 kg/d). In Staph. aureus mastitis diagnosed between 54 and 120 d in milk, the loss was 4.3% (1.4 kg/d). The loss was almost equal in both clinical and subclinical mastitis caused by Staph. aureus. Mastitis caused by Strep. uberis and Strep. dysgalactiae resulted in losses ranging from 3.7% (1.2 kg/d) to 6.6% (2.1 kg/d) depending on type and timing of mastitis. Clinical mastitis caused by the minor pathogens C. bovis and NAS also had a negative effect on milk production: 7.4% (2.4 kg/d) in C. bovis and 5.7% (1.8 kg/d) in NAS when both were diagnosed before peak lactation. In conclusion, minor pathogens should not be underestimated as a cause of milk yield reduction. On single dairy farms, control of E. coli mastitis would bring about a significant increase in milk production. Reducing Staph. aureus mastitis is the greatest challenge for the Finnish dairy sector.     

Economic values and expected effect of selection index for pathogen-specific mastitis under Danish conditions

The objectives of this study were 1) to estimate costs related to 5 different pathogen-specific mastitis traits (susceptibility to different pathogens causing mastitis in dairy cattle) and unspecific mastitis, and 2) to compare selection differentials for an udder health index consisting of 5 different pathogen-specific mastitis traits and lactation average somatic cell count from 5 to 170 d after first calving (LASCC170) with another index consisting of 1 unspecific mastitis trait and LASCC170. Economic values were estimated for mastitis caused by Staphylococcus aureus, Streptococcus dysgalactiae, Escherichia coli, coagulase-negative staphylococci, and Streptococcus uberis using a stochastic simulation model (SimHerd IV). Mastitis incidences for SimHerd IV were from incidences of mastitis treatments in primiparous Danish Holstein cows calving in 2007. Estimated costs ranged from €149 to €570 per mastitis case and were highest for contagious pathogens such as Staph. aureus and coagulase-negative staphylococci and lowest for Strep. dysgalactiae and Strep. uberis. The value for unspecific mastitis was €231 per case. Selection differentials (in €) were estimated for 4 different selection indices, including 1) unspecific mastitis, 2) unspecific mastitis and LASCC170, 3) 5 pathogen-specific mastitis traits and unspecific residual mastitis (unspecific mastitis treatments minus mastitis treatments caused by the 5 pathogens), and 4) as index 3 including LASCC170. The breeding goal was identical to selection index 3. Mastitis data from primiparous cows calving from 1998 to 2008 were used to estimate genetic parameters of the mastitis traits using linear models and AI-REML algorithm. These parameters were used for construction of the selection index equations. For the selection indices, information sources were measurements of mastitis treatments and LASCC170 from 50, 80, or 130 daughters of a bull as well as measurements of mastitis treatments from 1,000 progeny of the bull's sire and 1,000 daughters of his maternal grandsire. Differences in selection differentials were marginal among the 4 indices. Without considering LASCC170, the selection differential of an unspecific mastitis index was €0.4 (<1%) better than that of a pathogen-specific index. On the other hand, the selection differential of the pathogen-specific index was €0.3 (<1%) better than that of an unspecific index when LASCC170 was included in the indices. Reliabilities of the selection indices were 0.62 to 0.67 (80 daughters) and were proportional to the selection differential. Changing the number of daughters to 50 or 130 did not change ranking of the indices. Heritabilities of the pathogen-specific traits were very low (h² = 0.005-0.021) compared with unspecific mastitis (h² = 0.062), which may limit the selection differential of the pathogen-specific index.     

Strain diversity and infection durations of Staphylococcus spp. and Streptococcus spp. causing intramammary infections in dairy cows

To effectively prevent and control bovine mastitis, farmers and their advisors need to take infection pathways and durations into account. Still, studies exploring both aspects through molecular epidemiology with sampling of entire dairy cow herds over longer periods are scarce. Therefore, quarter foremilk samples were collected at 14-d intervals from all lactating dairy cows (n = 263) over 18 wk in one commercial dairy herd. Quarters were considered infected with Staphylococcus aureus, Streptococcus uberis, or Streptococcus dysgalactiae when ≥100 cfu/mL of the respective pathogen was detected, or with Staphylococcus epidermidis or Staphylococcus haemolyticus when ≥500 cfu/mL of the respective pathogen was detected. All isolates of the mentioned species underwent randomly amplified polymorphic DNA (RAPD)-PCR to explore strain diversity and to distinguish ongoing from new infections. Survival analysis was used to estimate infection durations. Five different strains of Staph. aureus were isolated, and the most prevalent strain caused more than 80% of all Staph. aureus infections (n = 46). In contrast, 46 Staph. epidermidis and 69 Staph. haemolyticus strains were isolated, and none of these caused infections in more than 2 different quarters. The 3 most dominant strains of Strep. dysgalactiae (7 strains) and Strep. uberis (18 strains) caused 81% of 33 and 49% of 37 infections in total, respectively. The estimated median infection duration for Staph. aureus was 80 d, and that for Staph. epidermidis and Staph. haemolyticus was 28 and 22 d, respectively. The probability of remaining infected with Strep. dysgalactiae or Strep. uberis for more than 84 and 70 d was 58.7 and 53.5%, respectively. Staphylococcus epidermidis and Staph. haemolyticus were not transmitted contagiously and the average infection durations were short, which brings into question whether antimicrobial treatment of intramammary infections with these organisms is justified. In contrast, infections with the other 3 pathogens lasted longer and largely originated from contagious transmission.     

A survey of mastitis pathogens including antimicrobial susceptibility in southeastern Australian dairy herds

The objectives for this study were to (1) describe the pathogen profile in quarters from cows with clinical mastitis and in cows with subclinical mastitis in southeastern Australia; and (2) describe antimicrobial susceptibility among isolated pathogens. As a secondary objective, we aimed to compare antimicrobial resistance prevalence in pathogens isolated from clinical and subclinical mastitis samples. A convenience sample of dairy herds (n = 65) from 4 regions in southeastern Australia (Gippsland, Northern Victoria, Tasmania, Western Victoria) were invited to submit milk samples from cows with clinical and subclinical mastitis over a 14-mo period (January 2011 to March 2012). Farmers were instructed to collect aseptic quarter milk samples from the first 10 cases of clinical mastitis for each month of the study. In addition, farmers submitted composite milk samples from cows with subclinical mastitis at 1 or 2 sampling occasions during the study period. Aerobic culture and biochemical tests were used to identify isolates. Isolates were classified as susceptible, intermediate, or resistant to a panel of antimicrobial agents based on the zone of growth inhibition around antimicrobial-impregnated disks, with antimicrobial resistance (AMR) classified as nonsusceptibility by combining intermediate and resistant groups into a single category. Generalized linear mixed models were used to compare the prevalence of AMR between clinical and subclinical mastitis isolates. For clinical mastitis samples (n = 3,044), 472 samples (15.5%) were excluded for contamination. Of the remaining samples (n = 2,572), the most common results were Streptococcus uberis (39.2%), no growth (27.5%), Staphylococcus aureus (10.6%), Escherichia coli (8.4%), and Streptococcus dysgalactiae (6.4%). For subclinical mastitis samples (n = 1,072), 425 (39.6%) were excluded due to contamination. Of the remaining samples (n = 647), the most common results were no growth (29.1%), Staph. aureus (29.1%), and Strep. uberis (21.6%). The prevalence of AMR among common isolates was low for the majority of antimicrobial agents. Exploratory analysis found that the probability of Staph. aureus demonstrating resistance to penicillin was 5.16 times higher (95% confidence interval: 1.68, 15.88) in subclinical isolates relative to clinical Staph. aureus isolates. A similar association was observed for amoxicillin with subclinical Staph. aureus isolates being 4.70 times (95% confidence interval: 1.49, 14.75) more likely to be resistant than clinical Staph. aureus isolates. We concluded that the most common bacteria causing clinical mastitis in dairy herds in Australia is likely to be Strep. uberis, whereas Staph. aureus is likely to be the most common cause of subclinical mastitis. Despite decades of antimicrobial use to control these organisms, AMR appears to be uncommon.     

Incidence of clinical mastitis and distribution of pathogens on large Chinese dairy farms

Knowledge of the incidence of clinical mastitis (CM) and the distribution of pathogens involved is essential for development of prevention and control programs as well as treatment protocols. No country-wide study on the incidence of CM and the distribution of pathogens involved has been conducted in China. Core objectives of this study were, therefore, to determine the cumulative incidence of CM and the distribution of pathogens causing CM on large Chinese (>500 cows) dairy farms. In addition, associations between the distribution of CM pathogens and bedding materials and seasonal factors were also investigated. Bacterial culture was done on a total of 3,288 CM quarter milk samples from 161 dairy herds (located in 21 provinces) between March 2014 and September 2016. Additional data, including geographical region of herds, herd size, bedding types, and number of CM cases during the last month, were also recorded. Mean cumulative incidence of CM was 3.3 cases per 100 cows per month (range = 1.7 to 8.1). The most frequently isolated pathogens were Escherichia coli (14.4%), Klebsiella spp. (13.0%), coagulase-negative staphylococci (11.3%), Streptococcus dysgalactiae (10.5%), and Staphylococcus aureus (10.2%). Streptococcus agalactiae was isolated from 2.8% of CM samples, whereas Streptococcus uberis were isolated from 2.1% of samples, and 15.8% of 3,288 samples were culture-negative. Coagulase-negative staphylococci, E. coli, and other Enterobacter spp. were more frequently isolated in the northwest than the northeast or south of China. Streptococcus dysgalactiae, other streptococci, and Strep. agalactiae were more frequently isolated in winter (October–March), whereas E. coli and Klebsiella spp. were mostly isolated in summer (April–September). Streptococcus dysgalactiae was more often isolated from CM cases of herds using sand bedding, whereas Klebsiella spp. and other streptococci were more common in herds using organic bedding. The incidence of CM and distribution of pathogens differed among herds and better mastitis management is needed. Furthermore, geography, bedding materials, and season should be included when designing mastitis control and prevention schemes for Chinese dairies.     

Antimicrobial activity of crude extracts from actinomycetes against mastitis pathogens

The emergence of antimicrobial resistance to commonly used antibiotics has necessitated the development of new antimicrobial products. Crude extracts produced by actinomycetes contain antimicrobial metabolites that can inhibit bacterial growth. The objective of our study was to evaluate the antimicrobial activity of crude extracts (Caat1–54 and CaatP5–8) produced by actinomycetes against isolates of Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae, and Streptococcus uberis, which were obtained from the milk of cows affected by mastitis in 23 dairy herds. Twenty isolates of each bacterial species were used to define minimum inhibitory concentrations (MIC) of both crude extracts and ceftiofur (positive control). The MIC₅₀ and MIC₉₀ were defined at the concentration required to inhibit the growth of 50 and 90% of bacterial isolates tested, respectively. The MIC results were evaluated by survival analysis. Staphylococcus aureus isolates presented MIC₉₀ of Caat 1–54 ≥6.25 µg/mL, ceftiofur ≥12.5 µg/mL, and Caat P5–8 ≥100 µg/mL. Streptococcus uberis presented MIC₉₀ of ceftiofur ≥0.39 µg/mL, Caat 1–54 ≥50 µg/mL, and Caat P5–8 ≥100 µg/mL. Staphylococcus chromogenes isolated from subclinical mastitis presented MIC₉₀ of Caat 1–54 ≥0.78 µg/mL and ceftiofur and Caat P5–8 of ≥6.25 and ≥100 µg/mL, respectively. Streptococcus dysgalactiae isolated from clinical mastitis presented similar MIC₉₀ values between antimicrobials tested (ceftiofur, Caat 1–54, and Caat P-58), but these values (≥100 µg/mL) were higher than the values obtained from other pathogens evaluated in the present study. Our results indicate that Caat 1–54 and Caat P5–8 crude extracts present in vitro antimicrobial activity against isolates of Staph. aureus, Staph. chromogenes, Strep. dysgalactiae, and Strep. uberis isolated from clinical and subclinical mastitis.     

Relationship between milk lactoferrin and etiological agent in the mastitic bovine mammary gland

Bovine mastitis is one of the most deleterious diseases for dairy herds and is mainly caused by contagious and environmental bacterial pathogens. Among contagious bacteria, Staphylococcus aureus is the most prevalent, whereas the main environmental mastitis pathogens are Streptococcus uberis and Escherichia coli. Bovine lactoferrin (bLF) is an approximately 80-kDa glycoprotein present in milk that participates in the innate response of the mammary gland against bacterial infection. The objectives of the current study were to analyze potential changes in bLF milk concentration, which would constitute a response of the mammary gland toward mastitis induced by different etiologic agents, and to evaluate a possible relation between this response and pathogen susceptibility to bLF. Microbiology analysis and bLF quantification in milk from different bovine mammary gland quarters were performed. Infected quarters presented greater concentrations of bLF compared with those from microbiologically negative quarters. Analysis of individual pathogen contributions showed that most of this increase was attributable to Strep. uberis intra-mammary infection. The ability of mammary gland cells to synthesize bLF in response to Strep. uberis challenge was demonstrated by immunodetection of the protein in in vitro infection experiments. Susceptibility of Strep. uberis, E. coli, and Staph. aureus to the antimicrobial activity of bLF was determined by growth inhibition assays conducted with 4 different isolates of each species. Whereas Staph. aureus and E. coli were shown to be susceptible to this protein, Strep. uberis appeared to be resistant to the antimicrobial activity of bLF. Molecular typing of the 4 Strep. uberis isolates used throughout this study showed that this result was representative of the species and not exclusive of a particular strain. Results presented herein suggest that different bacteria species may elicit different mammary gland responses mediated by bLF secretion and that Strep. uberis has probably adapted to this immune reaction by developing resistance to bLF inhibitory action.     

Non-culture-based identification of mastitis-causing bacteria by MALDI-TOF mass spectrometry

The purpose of this study was to evaluate the detection limit of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for direct identification, without previous microbiological culture, of bovine mastitis-causing bacteria from milk samples. Milk samples (n = 15) were experimentally contaminated with Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae, and Escherichia coli to have bacterial counts ranging from 10³ to 10⁹ cfu/mL. These contaminated milk samples were subjected to a preparation protocol for bacterial ribosomal protein extraction using the MALDI Sepsityper kit (Bruker Daltonik, Bremen, Germany), which allowed MALDI-TOF MS coupled with Biotyper software (Bruker Daltonik) to identify bacterial fingerprints based on intact ribosomal proteins. The ability of MALDI-TOF MS to correctly identify bacterial strains from experimentally contaminated milk (without previous microbiological culture) depended on the bacterial count of the samples and on the species of the bacteria evaluated. Adequate identification at the bacterial species level (score ≥2.0) directly from milk samples required bacterial counts in the following ranges: ≥10⁶ cfu/mL of Staph. aureus, ≥10⁷ cfu/mL of E. coli, and ≥10⁸ cfu/mL of Strep. agalactiae, Strep. dysgalactiae, and Strep. uberis. We concluded that direct identification of mastitis-causing pathogens is possible for Staph. aureus, E. coli, Strep. agalactiae, Strep. dysgalactiae, and Strep. uberis, but correct identification depended on the bacterial count in the milk samples.     

Evaluation of chromogenic culture media for rapid identification of microorganisms isolated from cows with clinical and subclinical mastitis

This study aimed to evaluate the diagnostic performance (specificity, Sp; sensitivity, Se; accuracy; positive predictive value; negative predictive value; and Cohen's kappa coefficient, κ, of agreement) of chromogenic culture media for rapid identification of microorganisms isolated from cows with clinical (CM) and subclinical mastitis (SCM). For this, 2 experiments were carried out: evaluation of (1) biplate, and (2) triplate of chromogenic culture media for rapid identification of mastitis-causing microorganisms. For the evaluation of diagnostic performance, identification of microorganisms by MALDI-TOF mass spectrometry was considered the standard methodology. In experiment 1, 476 milk samples collected from cows with CM and 660 from cows with SCM were evaluated by inoculation in 2 selective chromogenic culture media (CHROMagar) for gram-positive bacteria and another for gram-negative bacteria. In experiment 2, 476 milk samples from cows with CM and 500 from cows with SCM were evaluated by inoculation in triplate chromogenic culture media (Smartcolor2, Onfarm), selective for Streptococcus and Strep-like organisms, Staphylococcus, and gram-negative bacteria. In experiment 1 for the CM samples, the use of biplates with gram-positive and gram-negative culture media showed Se that ranged from 0.56 (0.32–0.81; Staphylococcus aureus) to 0.90 (0.80–0.99 Streptococcus uberis), Sp varied from 0.94 (0.92–0.96; Strep. uberis) to 1.00 (Prototheca spp. or yeast), and κ ranged from 0.47 (0.26–0.67; Staph. aureus) to 0.84 (0.78–0.9; Escherichia coli). The Se of biplates for SCM samples ranged from 0.50 (0.15–0.85; E. coli) to 0.94 (0.87–1.00; Staph. aureus), Sp varied from 0.95 (0.93–0.97; Strep. uberis) to 0.99 (0.98–1.00; Staph. aureus and Strep. Agalactiae or dysgalactiae), and κ ranged from 0.18 (0.00–0.40; Escherichia coli) to 0.88 (0.80–0.95; Staph. aureus). In experiment 2, the Se of the triplate chromogenic media in CM samples ranged from 0.09 (0.00–0.26; Serratia spp.) to 0.94 (0.85–1.00; Klebsiella spp. and Enterobacter spp.), Sp varied from 0.94 (0.92–0.96; Strep. agalactiae and Strep. dysgalactiae) to 1.00 (Serratia spp.) and κ ranged from 0.07 (0.00–0.24; Serratia spp.) to 0.85 (0.75–0.94; Klebsiella spp. and Enterobacter spp.). For SCM samples, the use of the triplate with the chromogenic culture media showed Se that varied from 0.25 (0.10–0.40; Lactococcus spp.) to 1.00 (Strep. Agalactiae or dysgalactiae), Sp ranged from 0.92 (0.90–0.94; Strep. Agalactiae and Strep. dysgalactiae) to 0.99 (0.98–1.00; Klebsiella spp. and Enterobacter spp.), and κ varied from 0.28 (0.00–0.72; E. coli) to 0.72 (0.60–0.82; Staph. aureus). Our results suggest that the diagnostic accuracy of the biplate and triplate of chromogenic culture media varies according to pathogen, and the results of chromogenic culture media may be useful for rapid decision-making on mastitis treatment protocols of the main mastitis-causing microorganisms, but their use for implementation of mastitis control measures will depend on each farm specific needs.     

Short communication: Antimicrobial efficacy of intramammary treatment with a novel biphenomycin compound against Staphylococcus aureus, Streptococcus uberis, and Escherichia coli-induced mouse mastitis

Bovine mastitis undermines udder health, jeopardizes milk production, and entails prohibitive costs, estimated at $2 billion per year in the dairy industry of the United States. Despite intensive research, the dairy industry has not managed to eradicate the 3 major bovine mastitis-inducing pathogens: Staphylococcus aureus, Streptococcus uberis, and Escherichia coli. In this study, the antimicrobial efficacy of a newly formulated biphenomycin compound (AIC102827) was assessed against intramammary Staph. aureus, Strep. uberis, and E. coli infections, using an experimental mouse mastitis model. Based on its effective and protective doses, AIC102827 applied into the mammary gland was most efficient to treat Staph. aureus, but also adequately reduced growth of Strep. uberis or E. coli, indicating its potential as a broad-spectrum candidate to treat staphylococcal, streptococcal, and coliform mastitis in dairy cattle.     

Milk culture results in a large Norwegian survey--effects of season, parity, days in milk, resistance, and clustering

A nationwide random computerized assignment survey that included 3,538 sets of 4 quarter milk samples from 2,834 dairy cows was conducted during 2000. Every fifth cow from every 50th herd was randomly selected for sampling and culture during each quarter of the year. Milk culture results of pathogens known to be related to mastitis were recorded regardless of whether mastitis had been indicated by any inflammatory measure or not. Farmers were blinded to all test results to minimize any potential interventions that might be prompted by the results. The most prevalent isolate was Staphylococcus aureus, which was identified in 8.2% of the quarter milk samples. More than 15 colony-forming units/0.01 mL of Staph. aureus were found in 4.3% of the quarter milk samples, whereas 3.5% had only 1 to 3 colony-forming units/0.01 mL. Streptococcus dysgalactiae, coagulase-negative staphylococci (CNS), and Streptococcus uberis were isolated from 1.2, 3.3, and 0.4% of quarter milk samples, respectively. No isolates were found in 76.6% of the quarter milk samples tested. Among individual cows, 22.2% had an isolate of Staph. aureus in ≥ 1 quarter. Only Strep. dysgalactiae exhibited a higher prevalence with increased parity. Prevalence of Staph. aureus decreased throughout days in milk, but prevalence of Strep. dysgalactiae increased. There was a strong seasonal effect; the highest prevalence of Strep. dysgalactiae and CNS was observed during April and May (late indoor season), and the highest prevalence of Staph. aureus and Strep. uberis was observed during June and July (the outdoor season). A substantial within-cow clustering effect was found for Strep. dysgalactiae, Staph. aureus, and CNS. Additionally, a within-herd effect was found for Strep. uberis, penicillin-resistant Staph. aureus, total Staph. aureus, and CNS. No within-county cluster effect was found. Lastly, both Staph. aureus and CNS exhibited a surprisingly high seasonal effect regarding the prevalence of resistance to penicillin G. Penicillin resistance of Staph. aureus was likely due to higher prevalence of Staph. aureus as a whole, but for CNS, there was also an additional increase caused by a higher proportional rate of penicillin resistance during the late indoor season.     

Antimicrobial activity of bovine β-lactoglobulin against mastitis-causing bacteria

Bovine mastitis is one of the most economically deleterious diseases affecting dairy herds and results from an infection of the udder by pathogenic microorganisms such as Staphylococcus aureus, Streptococcus uberis, and Escherichia coli. The mammary gland is capable of preventing and combating bacterial infection by means of a complex network of innate and adaptive immune mechanisms. Lactoferrin is an 86-kDa protein with antibacterial activity that plays a role in the mammary gland's defense against infection. β-Lactoglobulin (β-LG) is an 18-kDa protein that is present in most mammals but is notably absent in humans, rodents, and lagomorphs. Different genetic variants of this protein exist, with β-LG A and β-LG B being the most common. In spite of being well studied, the biological function of β-LG is not thoroughly understood, and most noticeably, no reports exist on the effects of the native protein on bacterial growth. Hence, the objective of this study was to assess the potential antibacterial activity of β-LG against mastitis agents. To do this, we purified β-LG from normal bovine milk using a mild, nondenaturing method and performed in vitro growth inhibition assays with Staph. aureus, E. coli, and Strep. uberis. β-Lactoglobulin inhibited the growth of Staph. aureus and Strep. uberis but had no effect on E. coli. The antimicrobial activity against Staph. aureus and Strep. uberis was concentration dependent and was elicited by the intact protein because Tricine-sodium dodecyl sulfate-PAGE and analytical gel filtration chromatography did not reveal the presence of short degradation peptides. Analysis of the genetic variants of β-LG showed that β-LG A has higher inhibitory activity against Staph. aureus and Strep. uberis than β-LG B. Coincubation of β-LG and lactoferrin resulted in an augmented antibacterial activity against Staph. aureus, suggesting an additive effect of the proteins. This result, along with the proteins’ complementary spectrum of action, suggests that β-LG and lactoferrin may complement each other in the mammary gland's defenses against bacterial infection.     

In vitro susceptibility of bovine mastitis pathogens to a combination of penicillin and framycetin: Development of interpretive criteria for testing by broth microdilution and disk diffusion

Dry cow therapy is an important part of mastitis control. This therapy typically consists of an antibiotic or antibiotics administered at a single dose by intramammary infusion at dry off to treat or prevent infection by prevalent mastitis pathogens. A combination dry cow therapy consisting of the active components penicillin and framycetin is currently used in several countries. Despite its use, standardized methods for the susceptibility testing of this combination against mastitis pathogens have not been established. In this study, which used Clinical and Laboratory Standards Institute methodology, preliminary interpretive criteria for the broth microdilution minimum inhibitory concentration (MIC) testing of mastitis pathogens to penicillin combined with framycetin (2:1 wt/wt) were established based on the amount of drug achieved and maintained postadministration in the udder. Based on resulting MIC distributions of recent veterinary field isolates and a subset of isolates preselected for resistance to β-lactams or aminoglycosides and concentrations achieved postadministration, criteria for broth microdilution testing of the combination (susceptible, intermediate, resistant in micrograms per milliliter) were set as follows: Escherichia coli ≤8/4, 16/8, ≥32/16; Staphylococcus spp. ≤2/1, 4/2–8/4, >16/8; Streptococcus uberis and Streptococcus dysgalactiae <0.25/0.12, 0.5/0.25–2/1, >4/2. A disk diffusion test using disks containing 100 μg of framycetin and 10 IU of penicillin was also developed, and preliminary interpretive criteria (susceptible, intermediate, resistant in millimeters) were set based on correlation to broth MIC values and the minimization of interpretive errors between isolates tested concurrently by broth microdilution and disk diffusion as follows: E. coli ≥18, 16–17, ≤15; Staphylococcus spp. ≥21, 18–20, ≤17; Strep. uberis and Strep. dysgalactiae ≥21, 19–20, ≤18. In addition, ranges for the quality control of the testing of this combination by both broth microdilution and disk diffusion are provided. Based on these criteria and recent veterinary mastitis isolates, 96.0/96.8% of E. coli, 93.7/89.1% of Staph. aureus, 94.6/96.4% coagulase-negative staphylococci, 94.5/97.0% of Strep. uberis, and 96.7/100.0% Strep. dysgalactiae were susceptible to the combination by broth microdilution or disk diffusion, respectively. The availability of these methods will allow for the susceptibility testing of clinical isolates in the field and will also provide a way to monitor for resistance development as this combination is used going forward.     

Antibacterial effect of plant-derived antimicrobials on major bacterial mastitis pathogens in vitro

The objective of this study was to investigate the antimicrobial effect of plant-derived antimicrobials including trans-cinnamaldehyde (TC), eugenol, carvacrol, and thymol on major bacterial mastitis pathogens in milk. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the aforementioned compounds on Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Staphylococcus aureus, and Escherichia coli were determined. In addition, the bactericidal kinetics of TC on the aforementioned pathogens and the persistence of the antimicrobial activity of TC in milk over a period of 2 wk were investigated. All 4 plant-derived molecules exhibited antimicrobial activity against the 5 mastitis pathogens tested, but TC was most effective in killing the bacteria. The MIC and MBC of TC on Staph. aureus, E. coli, and Strep. uberis were 0.1 and 0.45%, respectively, whereas that on Strep. agalactiae and Strep. dysgalactiae were 0.05 and 0.4%, respectively. The MIC and MBC of the other 3 molecules ranged from 0.4 to 0.8% and 0.8 to 1.5%, respectively. In time-kill assays, TC at the MBC reduced the bacterial pathogens in milk by 4.0 to 5.0 log₁₀ cfu/mL and to undetectable levels within 12 and 24 h, respectively. The antimicrobial effect of TC persisted for the duration of the experiment (14 d) without any loss of activity. Results of this study suggest that TC has the potential to be evaluated as an alternative or adjunct to antibiotics as intramammary infusion to treat bovine mastitis.     

Antimicrobial susceptibilities in dairy herds that differ in dry cow therapy usage

Intramammary infusion of antimicrobials at the end of lactation (dry cow therapy; DCT) is a central part of mastitis control programs and is one of the major indications for antimicrobial use in dairy cows. However, with increasing focus on prudent use of antimicrobials and concerns about emergence of antimicrobial resistance, the practice of treating every cow at the end of lactation with DCT is in question. This cross-sectional, observational study determined the minimum inhibitory concentrations (MIC) of 10 antimicrobials for coagulase-negative staphylococci (CNS), Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus uberis isolates from milk samples from dairy cows with somatic cell counts >200,000 cells/mL in herds that had been organic for >3 yr (n = 7), or had used either ampicillin-cloxacillin DCT (n = 11) or cephalonium DCT (n = 8) in the preceding 3 yr. The organic herds were certified under the United States Department of Agriculture National Organic Program, meaning that there was no blanket DCT, and minimal use of antimicrobials in general, with a loss of organic status of the animal if treated with antimicrobials. Breakpoints (where available) were used to categorize isolates as resistant, intermediate, or susceptible to antimicrobials. The MIC distributions of isolates from different herd types were compared using binomial or multinomial logistic regression. Of 240 CNS isolates, 12.9, 0.8, 7.1, 32.6, and 1.2%, were intermediate or resistant to ampicillin, cephalothin, erythromycin, penicillin, and tetracycline, respectively. Of 320 Staph. aureus isolates, 29.0, 2.5, 1.2, and 34.9% were intermediately resistant or resistant to ampicillin, penicillin, erythromycin, and oxacillin, respectively. Of 184 Strep. uberis isolates, 1.1, 25.0, 1.6, and 1.6% were intermediately resistant or resistant to erythromycin, penicillin, pirlimycin, and tetracycline, respectively. Generally, the MIC of CNS and streptococcal isolates from organic herds were lower than isolates from herds using DCT. However, the differences in MIC distributions occurred at MIC below clinical breakpoints, so that the bacteriological cure rates may not differ between isolates of differing MIC. Bimodal distributions of MIC for ampicillin and penicillin were found in Staph. aureus isolates from organic herds, suggesting that isolates with a higher MIC are a natural part of the bacterial population of the bovine mammary gland, or that isolates with higher MIC have persisted within these organic herds from a time when antimicrobials had been used. Given these observations, further work is required to determine if exposure to DCT is causally associated with the risk of elevated MIC, and whether reduction or removal of DCT from herds would reduce the risk of elevated MIC of mastitis pathogens.     

Short communication: Lack of intramammary niche recolonization during a sanitation program for the contagious mastitis pathogen Staphylococcus aureus genotype B

In Switzerland, sanitation programs of dairy herds infected with the contagious mastitis pathogen Staphylococcus aureus genotype B (GTB) have been established for several years. In recent years, Streptococcus uberis and non-aureus staphylococci have emerged as the bacteria most frequently isolated from bovine milk samples. The latter cause subclinical mastitis, and some species are more persistent or pathogenic than others. The present study aimed to investigate the developments in the intramammary colonization spectrum of 5 dairy herds undergoing a sanitation program for Staph. aureus GTB. We collected single-quarter milk samples aseptically from all lactating cows at 3-mo intervals during the sanitation period; after classical bacteriological analysis, MALDI-TOF mass spectrometry was used to identify the isolates to the species level. Non-aureus staphylococci were found to be the bacterial group most frequently occurring on the selected farms, with Staphylococcus chromogenes and Staphylococcus xylosus being predominant. The present study demonstrated that GTB-infected cows treated with antibiotics lacked systematic recolonization with other bacteria during herd sanitation for the contagious Staph. aureus GTB.