Use of MALDI-TOF to characterize staphylococcal intramammary infections in dairy goats

The most common pathogens causing intramammary infections (IMI) in dairy goats are staphylococci. Gene sequencing has been the reference method for identification of staphylococcal species, but MALDI-TOF mass spectrometry could represent a rapid and cost-effective alternative method. The objectives were to evaluate the typeability and accuracy of partial gene sequencing and MALDI-TOF for identifying staphylococci isolated from caprine milk samples, and to evaluate the relationship between staphylococcal species IMI, milk somatic cell score (SCS), and milk yield (MY). A composite (goat-level) milk sample was collected from all 940 lactating goats in a single herd. Dairy Herd Information Association test-day data for parity, days in milk, SCS, and MY were retrieved from Dairy Herd Information Association records. Milk samples were cultured on Columbia blood agar, and isolates from samples that yielded a single colony type of a presumptively identified Staphylococcus spp. were identified by PCR amplification and partial sequencing of rpoB, tuf, or 16S-rRNA, and MALDI-TOF. Mixed linear models were used to evaluate the relationship between staphylococcal IMI, SCS, and MY. The goat-level prevalence of staphylococcal IMI based on isolation of a single colony type was 24.4% (213/874). Seventeen goats had a contaminated sample. Among the remaining goats (n = 857), the most common species causing single colony-type IMI were Staphylococcus simulans (7.9%), Staphylococcus xylosus (3.5%), Staphylococcus caprae (3.6%), Staphylococcus chromogenes (2.9%), and Staphylococcus epidermidis (2.2%). The typeability of staphylococcal isolates with partial housekeeping gene sequence analysis (rpoB, complemented by tuf and 16S as needed) was 97.7%. The typeability and accuracy of MALDI-TOF were 84 and 100%, respectively. Overall, only Staphylococcus chromogenes IMI was associated with a higher SCS than goats with no growth. After adjusting for parity and stage of lactation, staphylococcal IMI status was not significantly associated with MY. For the staphylococci isolated from goats in this herd, MALDI-TOF proved an accurate method of speciation with a relatively high typeability. An association between staphylococcal IMI, SCS, and MY was not defined using goat-level data with the exception of S. chromogenes IMI, which was associated with a higher SCS than goats with no growth.     

Prevalence of non-aureus staphylococci species causing intramammary infections in Canadian dairy herds

Non-aureus staphylococci (NAS), the microorganisms most frequently isolated from bovine milk worldwide, are a heterogeneous group of numerous species. To establish their importance as a group, the distribution of individual species needs to be determined. In the present study, NAS intramammary infection (IMI) was defined as a milk sample containing ≥1,000 cfu/mL in pure or mixed culture that was obtained from a cohort of cows assembled by the Canadian Bovine Mastitis Research Network. Overall, 6,213 (6.3%) of 98,233 quarter-milk samples from 5,149 cows and 20,305 udder quarters were associated with an NAS IMI. Of the 6,213 phenotypically identified NAS isolates, 5,509 (89%) were stored by the Canadian Bovine Mastitis Research Network Mastitis Pathogen Collection and characterized using partial sequencing of the rpoB housekeeping gene, confirming 5,434 isolates as NAS. Prevalence of each NAS species IMI was estimated using Bayesian models, with presence of a specific NAS species as the outcome. Overall quarter-level NAS IMI prevalence was 26%. The most prevalent species causing IMI were Staphylococcus chromogenes (13%), Staphylococcus simulans (4%), Staphylococcus haemolyticus (3%), Staphylococcus xylosus (2%), and Staphylococcus epidermidis (1%). The prevalence of NAS IMI as a group was highest in first-parity heifers and was evenly distributed throughout cows in parities ≥2. The IMI prevalence of some species such as S. chromogenes, S. simulans, and S. epidermidis differed among parities. Overall prevalence of NAS IMI was 35% at calving, decreased over the next 10 d, and then gradually increased until the end of lactation. The prevalence of S. chromogenes, Staphylococcus gallinarum, Staphylococcus cohnii, and Staphylococcus capitis was highest at calving, whereas the prevalence of S. chromogenes, S. haemolyticus, S. xylosus, and S. cohnii increased during lactation. Although the overall prevalence of NAS IMI was similar across barn types, the prevalence of S. simulans, S. xylosus, S. cohnii, Staphylococcus saprophyticus, S. capitis, and Staphylococcus arlettae IMI was higher in tiestall barns; the prevalence of S. epidermidis IMI was lowest; and the prevalence of S. chromogenes and Staphylococcus sciuri IMI was highest in bedded-pack barns. Staphylococcus simulans, S. epidermidis, S. xylosus, and S. cohnii IMI were more prevalent in herds with intermediate to high bulk milk somatic cell count (BMSCC) and S. haemolyticus IMI was more prevalent in herds with high BMSCC, whereas other common NAS species IMI were equally prevalent in all 3 BMSCC categories. Distribution of NAS species IMI differed among the 4 regions of Canada. In conclusion, distribution differed considerably among NAS species IMI; therefore, accurate identification (species level) is essential for studying NAS epidemiology.     

Quarter- and cow-level risk factors for intramammary infection with coagulase-negative staphylococci species in Swiss dairy cows

Bacteriological status, evaluation of udder symmetry, udder hygiene, and teat end scores of 92 dairy cows were assessed on 3 Swiss dairy farms in a longitudinal 1-yr study to determine risk factors for intramammary infection (IMI) with coagulase-negative staphylococci (CNS) species. Farm visits were performed monthly including sterile quarter milk sampling and udder evaluation of all lactating cows. Milk samples were evaluated for the presence of staphylococci using selective agar plates. Species identification was performed using MALDI-TOF mass spectrometry. Intramammary infection was defined as milk samples having ≥100 cfu per mL of milk according to culture results. Overall, 3,151 quarter samples were included in the statistical analysis. Staphylococcus chromogenes, Staphylococcus haemolyticus, Staphylococcus xylosus, and a Staphylococcus warneri-like species were the 4 most prevalent CNS species found. Hierarchical multivariable logistic regression models were built to evaluate risk factors for species-specific CNS IMI. Risk factors for Staph. chromogenes IMI were presence in herd B, the period from June 2014 to August 2014 and December 2014 to February 2015, and presence of udder edema. For Staph. haemolyticus, the relevant risk factor included coinfection with Staph. xylosus coinfection with other than the above-mentioned CNS species (“others”) and the period from June 2014 to November 2014. Coinfection with Staph. haemolyticus and “others,” the periods from June 2014 to August 2014 and December 2014 to February 2015, early phase of lactation (1–60 d in milk), and belonging to herd B were significantly associated with Staph. xylosus IMI. Mid and late lactation, coinfection with Staph. xylosus, and the period September 2014 to May 2015 were identified as significant risk factors for Staph. warneri-like IMI. For Staph. chromogenes, 60.6 and 26% of the variance was observed at the quarter and cow level, respectively, whereas for the other investigated species the highest variance was observed at the sample level. The predominant species within herds differed and was most pronounced for the Staph. warneri-like species.     

Quarter-level analyses of the associations among subclinical intramammary infection and milk quality,udder health,and cheesemaking traits in Holstein cows

In this study, we investigated associations among subclinical intra-mammary infection (IMI) and quarter-level milk composition, udder health indicators, and cheesemaking traits. The dataset included records from 450 Holstein cows belonging to three dairy herds. After an initial screening (T0) to identify animals infected by Streptococcus agalactiae, Streptococcus uberis, Staphylococcus aureus, and Prototheca spp., 613 quarter milk samples for 2 different sampling times (T1 and T2, 1 mo after T1) were used for analysis. Milk traits were analyzed using a hierarchical linear mixed model including the effects of days in milk, parity and herd, and bacteriological and inflammatory category [culture negative with somatic cell count (SCC) <200,000 cells/mL; culture negative with SCC ≥200,000 cells/mL; or culture positive]. All udder health indicators were associated with increased SCC and IMI at both sampling times. The largest effects were detected at T2 for milk lactose (?7% and ?5%) and milk conductivity (+9% and +8%). In contrast, the increase in differential SCC (DSCC) in samples with elevated SCC was larger at T1 (+17%). Culture-negative samples with SCC ≥200,000 cells/mL had the highest SCC and greatest numbers of polymorphonuclear-neutrophils-lymphocytes and macrophages at both T1 and T2. Regarding milk cheesemaking ability, samples with elevated SCC showed the worst pattern of curd firmness at T1 and T2. At T2, increased SCC and IMI induced large decreases in recoveries of nutrients into the curd, in particular recovered protein (?14% and ?16%) and recovered fat (?12% and ?14%). Different behaviors were observed between Strep. agalactiae and Prototheca spp., especially at T2. In particular, samples that were positive for Strep. agalactiae had higher proportions of DSCC (+19%) compared with negative samples with low SCC, whereas samples that were positive for Prototheca spp. had lower DSCC (?11%). Intramammary infection with Prototheca spp. increased milk pH compared with culture-negative samples (+3%) and negative samples that had increased SCC (+2%). The greatest impairment in curd firmness at 30 min from rennet addition was observed for samples that were positive for Prototheca spp. (?99% compared with negative samples, and ?98% compared with negative samples with high SCC). These results suggest that IMI caused by Prototheca spp. have detrimental effects on milk technological traits that deserve further investigation of the mechanisms underlying animals' responses to infection.     

Intramammary infections with different non-aureus staphylococci in dairy cows

Subclinical mastitis causes an increase in milk somatic cell count (SCC) and can lead to reduced milk production and early culling. In many countries, non-aureus staphylococci (NAS) is the most common bacterial finding in subclinical mastitis of dairy cows. New methodology makes it possible to identify NAS species, but knowledge about the epidemiology is limited. The objective of this project was to improve advisory services for mastitis control by investigating associations between NAS and SCC, milk production, and persistence of intramammary infections (IMI). Farmers who had sent milk samples to the Swedish National Veterinary Institute (Uppsala, Sweden) were asked to participate if NAS was identified in the samples. Participating farmers were asked to resample all udder quarters of the cow once within 1 mo. Regression models were used to investigate associations between NAS and cow factors, udder quarter California mastitis test and SCC, and persistence of IMI. Associations with cow composite milk yield and SCC were also investigated. In total, 671 cows from 201 herds were enrolled in the study, and 19 NAS species were identified, of which the 4 most common were Staphylococcus epidermidis, Staphylococcus simulans, Staphylococcus chromogenes, and Staphylococcus haemolyticus. Persistent IMI was more common in udder quarters with Staphylococcus hyicus and S. simulans and less common in those with Staphylococcus saprophyticus IMI. β-Lactamase production by the different NAS species varied from 0 to 100%. There was a significant association between NAS species and California mastitis test and SCC of udder quarters, and this varied depending on parity. The cow composite milk SCC at the test milking before the initial sample was taken differed significantly with NAS species, but not at the subsequent test milking. Milk yield—at the test milking before or after the initial sample—did not differ significantly for NAS species. There were no significant associations between milk yield or SCC and persistent NAS IMI. In conclusion, the NAS species affects SCC and persistent IMI differently but not milk yield.     

Genetic analysis of pathogen-specific intramammary infections in dairy cows

Mastitis is one of the most common diseases in dairy cattle, causing severe economic losses to dairy farmers. Mastitis usually occurs due to intramammary infection (IMI) caused by a variety of pathogenic bacteria. Although good progress has been made in understanding genetics of pathogen-specific clinical mastitis, studies involving genetic analysis of pathogen-specific IMI are scarce. The overall objective of this study was, therefore, to assess genetic variation of overall and pathogen-specific IMI in nonclinical primiparous and multiparous cows using bacterial culture. Data and milk samples were collected over a 2-yr interval as part of the Canadian Bovine Mastitis Research Network. The final data set contained records of 46,900 quarter milk samples from 3,382 clinically healthy primiparous and multiparous Holstein cows from 84 dairy herds. For the genetic analysis, we considered the following 7 traits: overall IMI, non-aureus staphylococci (NAS) IMI, contagious pathogen IMI, environmental pathogen IMI, major pathogen IMI, minor pathogen IMI and somatic cell score (SCS). Data were analyzed at the quarter level using a threshold-probit model via Gibbs sampling in BLUPF90. Prevalence of IMI traits at the quarter level in multiparous cow from 0 to 400 DIM ranged from 6.8 to 45.5%. Posterior mean of quarter heritability estimates (on the underlying scale, posterior SD in brackets) of overall IMI and pathogen-specific IMI traits ranged from 0.017 to 0.073 (±0.009 to 0.030). Weak to strong genetic correlations [ranging from 0.18 to 0.97 (±0.01 to 0.29)] among pathogen-specific IMI traits and with overall IMI indicated that not all of these traits were genetically similar. Weak to moderate Spearman rank correlations between estimated breeding values for overall IMI and pathogen-specific IMI traits (from 0.31 to 0.87) indicated possible substantial reranking of sires. The percentage of daughters with IMI caused by various pathogen groups ranged from 13 to 80% and from 38 to 94% for the best (10% decile) and worst sires (90% decile) according to their IMI trait-specific estimated breeding values, respectively. Pathogen-specific IMI traits and overall IMI had weak to moderate positive genetic correlations [ranging from 0.11 to 0.81 (±0.11 to 0.22)] with SCS. Therefore, selection for lower SCS will improve resistance to IMI. However, based on the observed weak to moderate rank correlations (0.04 to 0.47) between pathogen-specific IMI traits and SCS, selection for lower SCC will not improve resistance to IMI from every pathogen-specific IMI group in the same manner. Therefore, despite low heritability estimates, there was sizeable genetic variation for pathogen-specific IMI traits, indicating that long-term direct genetic selection for pathogen-specific IMI can improve pathogen-specific IMI resistance.     

In vitro antimicrobial resistance profiles of Streptococcus uberis,Lactococcus spp., and Enterococcus spp. from quarter milk samples of cows between 2015 and 2019 in Southern Germany

The objective was to describe and compare antimicrobial resistance patters of esculin-hydrolyzing streptococci and streptococcal-like organisms (Streptococcus uberis, Enterococcus faecium, Enterococcus faecalis, Lactococcus garvieae, Lactococcus lactis) from routine diagnostic samples of the udder health laboratory of the Bavarian Animal Health Services between 2015 and 2019. All routine diagnostic samples of the udder health laboratory of the Bavarian Animal Health Services, that were tested with a standard microbroth dilution, were eligible to be included in this retrospective case series. A California Mastitis Test result was available for all samples. Most Strep. uberis and L. lactis were susceptible to all antibiotics tested. Enterococcus faecium had consistently the highest minimum inhibitory concentration required to inhibit the growth of 90% of tested isolates. The resistance patterns of Lactococcus garvieae were positioned between enterococci and L. lactis. The minimum inhibitory concentration for various antibiotics and pathogens tended to decrease over the 5-yr period. Regardless of the pathogen, isolates of clinical cases were less likely to express in vitro resistance than isolates of healthy or subclinical cases. Streptococcus uberis or L. lactis showed hardly any in vitro resistance to tested antibiotic groups. Penicillin should remain the first-choice antimicrobial for the therapy of Strep. uberis and Lactococcus spp. However, a success of any antimicrobial treatment of enterococcal infections seems questionable.     

Influence of dry period bacterial intramammary infection on clinical mastitis in dairy cows

Milk samples were taken from 1920 quarters (480 cows, six herds) on four occasions to examine the relationship between quarter level intramammary infection (IMI) during the dry period and clinical mastitis in the next lactation. All quarters were sampled at drying off and within 1 wk of calving, and two quarters from each cow were sampled both 0 to 7 and 8 to 14 d before calving. Milk samples were collected from all cases of clinical mastitis during the following lactation. Logistic regression models were developed to investigate the associations between IMI present during the sampling period and clinical mastitis. The probability of a quarter succumbing to clinical mastitis increased when Streptococcus dysgalactiae, Streptococcus faecalis, Escherichia coli, or Enterobacter spp. were cultured at drying off and when Escherichia coli, coagulase-positive staphylococcus, Serratia spp., or Streptococcus faecalis were cultured in two out of three late dry and post-calving samples. Quarters from which Corynebacterium spp. were isolated at drying off were at an increased risk of clinical mastitis, whereas the presence of Corynebacterium spp. in the late dry and post-calving samples was associated with a reduction in the risk of clinical mastitis. The risk of mastitis for specific pathogens increased if the same species of bacteria that had caused mastitis was isolated at least twice in the late dry and post-calving samples. Kaplan-Meier survival plots indicated that clinical mastitis associated with dry period infections was more likely to occur earlier in lactation than clinical mastitis not associated with dry period infections. There was evidence of quarter susceptibility to IMI or the possibility that infection with one organism led to clinical mastitis with another.     

Staphylococcal intramammary infection dynamics and the relationship with milk quality parameters in dairy goats over the dry period

The objectives of this study were (1) to report the rates of new intramammary infection (IMI) and spontaneous IMI cure over the dry period in 3 dairy goat herds; (2) to evaluate the factors predicting infection dynamics over the dry period; and (3) to define milk quality parameter thresholds that predict infection dynamics over the dry period. Two consecutive udder-half milk samples were collected 10 to 14 d apart before dry-off from 288 goats in 3 herds, and 2 consecutive udder-half samples were collected 7 to 14 d apart in the following lactation, with the first sample being collected ≤10 d in milk, from 200 of the same goats. In 2 of the herds, udder-half milk samples were also collected at the same time points (n = 312 halves; 157 goats) for measurement of milk quality parameters. Standard aerobic culture of milk samples was performed for the detection of mastitis pathogens. To rule out the presence of Mycoplasma spp. IMI, milk samples were also cultured on modified Hayflick medium. Non-Mycoplasma isolates were speciated using MALDI-TOF mass spectrometry. Staphylococcal isolates, when not identified by MALDI-TOF, were speciated using partial gene sequence analysis of rpoB or tuf. When >1 sample from an udder half yielded the same species, available isolates from the first and last positive samples for that species were strain-typed using pulsed-field gel electrophoresis. Incidence of new IMI and cure rate were computed. Generalized linear mixed regression models were built to evaluate the associations between new IMI and pre-dry somatic cell score (SCS), between IMI persistence and half-level SCS, and between IMI persistence and pre-dry IMI species. Thresholds for pre-dry SCS and lactose concentration were computed to predict IMI persistence. Overall, 12.6% (48/380) of halves had a persistent IMI. Cumulative incidence of new IMI over the dry period was 13.2%, and cure rate was 52.0%. Pre-dry SCS was not associated with odds of new IMI or IMI persistence. Pre-dry IMI species was not associated with odds of persistence. Lactose concentration was not associated with odds of persistence. Regardless of culture data, the optimal pre-dry SCS threshold to detect IMI that would persist into the next lactation was 8.7, with sensitivity and specificity of 50 and 73.8%, respectively. Further studies on the effect of control measures on species-specific incidence and cure rates during the dry period are warranted.     

Study of the efficacy of a Streptococcus uberis mastitis vaccine against an experimental intramammary infection with a heterologous strain in dairy cows

Streptococcus uberis is a worldwide pathogen that causes intramammary infections in dairy cattle. Nevertheless, commercial vaccines are currently not available and measures to control S. uberis mastitis are limited to the implementation of good management practices. The aim of the present study was to evaluate the efficacy of an S. uberis subunit vaccine against bovine mastitis (Laboratorios Hipra S.A., Amer, Spain) administered precalving against an experimental intramammary challenge with a heterologous S. uberis strain in dairy cows postcalving. With this objective, 25 gestating Holstein-Friesian heifers were randomly assigned to 1 of 2 groups: group 1 (n = 13), vaccinated by intramuscular route with the vaccine, and group 2 (n = 12), vaccinated by intramuscular route with phosphate-buffered saline as a control group. Both groups were immunized 60 and 21 d before the expected parturition date (75 and 36 d before challenge). Fourteen days after calving all cows were challenged by intramammary infusion of 100 colony-forming units of a heterologous S. uberis strain in 2 quarters per cow. Then, challenged quarters were monitored for clinical signs of mastitis, bacterial count, and somatic cell count for the following 21 d. Rectal temperature and daily milk yield per cow were also assessed. Results showed that all challenged quarters developed clinical mastitis. Nevertheless, vaccination significantly reduced the clinical signs of mastitis, bacterial count, rectal temperature, and daily milk yield losses after the intramammary infection and significantly increased the number of quarters with no bacterial isolation and somatic cell count <200,000 cells/mL at the end of the study (d 19, 20, and 21 after challenge). To confirm the efficacy of this vaccine, further studies under field conditions are needed.     

Identifying the major bacteria causing intramammary infections in individual milk samples of sheep and goats using traditional bacteria culturing and real-time polymerase chain reaction

Use of DNA-based methods, such as real-time PCR, has increased the sensitivity and shortened the time for bacterial identification, compared with traditional bacteriology; however, results should be interpreted carefully because a positive PCR result does not necessarily mean that an infection exists. One hundred eight lactating dairy ewes (56 Manchega and 52 Lacaune) and 24 Murciano-Granadina dairy goats were used for identifying the main bacteria causing intramammary infections (IMI) using traditional bacterial culturing and real-time PCR and their effects on milk performance. Udder-half milk samples were taken for bacterial culturing and somatic cell count (SCC) 3 times throughout lactation. Intramammary infections were assessed based on bacteria isolated in ≥2 samplings accompanied by increased SCC. Prevalence of subclinical IMI was 42.9% in Manchega and 50.0% in Lacaune ewes and 41.7% in goats, with the estimated milk yield loss being 13.1, 17.9, and 18.0%, respectively. According to bacteriology results, 87% of the identified single bacteria species (with more than 3 colonies/plate) or culture-negative growth were identical throughout samplings, which agreed 98.9% with the PCR results. Nevertheless, the study emphasized that 1 sampling may not be sufficient to determine IMI and, therefore, other inflammatory responses such as increased SCC should be monitored to identify true infections. Moreover, when PCR methodology is used, aseptic and precise milk sampling procedures are key for avoiding false-positive amplifications. In conclusion, both PCR and bacterial culture methods proved to have similar accuracy for identifying infective bacteria in sheep and goats. The final choice will depend on their response time and cost analysis, according to the requirements and farm management strategy.     

Factors associated with intramammary infection in dairy cows caused by coagulase-negative staphylococci,Staphylococcus aureus,Streptococcus uberis,Streptococcus dysgalactiae,Corynebacterium bovis,or Escherichia coli

The aim of this study was to determine risk factors for bovine intramammary infection (IMI) associated with the most common bacterial species in Finland. Large databases of the Finnish milk-recording system and results of microbiological analyses of mastitic milk samples from Valio Ltd. (Helsinki, Finland) were analyzed. The study group comprised 29,969 cows with IMI from 4,173 dairy herds. A cow with a quarter milk sample in which DNA of target species was detected in the PathoProof Mastitis PCR Assay (Thermo Fisher Scientific, Waltham, MA) was determined to have IMI. Only cows with IMI caused by the 6 most common pathogens or groups of pathogens, coagulase-negative staphylococci (CNS), Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, Corynebacterium bovis, and Escherichia coli, were included. The control group comprised 160,176 IMI-free cows from the same herds as the study group. A multilevel logistic regression model was used to study herd- and cow-specific risk factors for incidence of IMI. Pathogen-specific results confirmed those of earlier studies, specifically that increasing parity increases prevalence of IMI regardless of causative pathogen. Holsteins were more susceptible to IMI than Nordic Reds except when the causative pathogen was CNS. Occurrence of IMI caused by C. bovis was not related to milk yield, in contrast to IMI caused by all other pathogens investigated. Organic milk production was associated with IMI only when the causative pathogen of IMI was Staph. aureus; Staph. aureus IMI was more likely to occur in conventional than in organic production. Cows in older freestall barns with parlor milking had an increased probability of contracting an IMI compared with cows in tiestall barns or in new freestall barns with automatic milking. This was the case for all IMI, except those caused by CNS, the prevalence of which was not associated with the milking system, and IMI caused by Staph. aureus, which was most common in cows housed in tiestall barns. A better breeding index for milk somatic cell count was associated with decreased occurrence of IMI, indicating that breeding for improved udder health has been successful in reducing the incidence of IMI caused by the most common pathogens in Finland. In the Finnish dairy sector, the importance of other measures to control IMI will increase as the Holstein breed progressively takes the place of the Nordic Red breed. Attention should be paid to hygiene and cleanliness, especially in old freestall barns. Based on our results, the increasing prevalence of automatic milking is not a reason for special concern.     

Ability of milk pH to predict subclinical mastitis and intramammary infection in quarters from lactating dairy cattle

Milk pH is increased in lactating dairy cattle with subclinical mastitis (SCM) and intramammary infection (IMI). We hypothesized that milk pH testing provides an accurate, low-cost, and practical on-farm method for diagnosing SCM and IMI. The main objective was to evaluate the clinical utility of measuring milk pH using 3 tests of increasing pH resolution: Multistix 10 SG Reagent Strips for Urinalysis (Multistix strips, Bayer HealthCare Inc., Elkhart, IN), pH Hydrion paper (Microessential Laboratory, Brooklyn, NY), and Piccolo plus pH meter (Hanna Instruments, Woonsocket, RI), for diagnosing SCM and IMI in dairy cattle. Quarter foremilk samples were collected from 115 dairy cows at dry off and 92 fresh cows within 4 to 7 d postcalving. Quarter somatic cell count (SCC) was measured using a DeLaval cell counter (DeLaval, Tumba, Sweden), with SCM defined as SCC >200,000 cells/mL and IMI defined as SCC >100,000 cells/mL and the presence of microorganisms at ≥10 cfu/mL of milk. Milk pH was measured at 37°C using the 3 test methods. The Hydrion pH paper performed poorly in diagnosing SCM and IMI. Receiver operating curve analysis provided optimal pH cutpoints for diagnosing SCM for the pH meter (dry off, ≥6.67; freshening, ≥6.52) and Multistix strips (dry off and freshening, ≥7.0). Test performance of the pH meter and Multistix strips was poor to fair based on area under the receiver operating curve, sensitivity, specificity, positive likelihood ratio, and kappa coefficient. The pH meter and Multistix strips performed poorly in diagnosing IMI at dry off and freshening. We concluded that milk pH does not provide a clinically useful method for diagnosing SCM or IMI in dairy cattle.     

Diagnostic accuracy of Somaticell,California Mastitis Test,and microbiological examination of composite milk to detect Streptococcus agalactiae intramammary infections

The objectives of this study were to estimate the accuracy of Somaticell (Idexx Laboratories Inc., Westbrook, ME), California Mastitis Test (CMT), and microbiological examination of composite milk (MEC) to diagnose Streptococcus agalactiae intramammary infections (IMI), and to assess the agreement between Somaticell and CMT to detect these infections. A secondary objective was to estimate quarter- and cow-level prevalence of S. agalactiae IMI in the herds included in the study. Seven farms were included in the study. The CMT was performed and aseptic milk samples were collected from all quarters of all lactating cows. Composite milk samples were produced in the laboratory by mixing milk from all quarters of each sampled cow. The Somaticell test was performed on a subset of S. agalactiae-positive (n = 167) and S. agalactiae-negative (n = 152) quarter milk samples. Microbiological examination of quarter milk samples (MEQ) was considered the reference test for diagnosing S. agalactiae IMI. The accuracy of all tests at various thresholds was estimated using Bayesian latent class models. Apparent prevalence of S. agalactiae IMI was 15.8% (n = 184/1,164) at the quarter level (based on MEQ) and 28.5% (n = 83/291) at the cow level (based on MEC). True prevalence, as determined by Bayesian models, was 13.0% [95% credible interval (CR): 6.4–24.4%] at the quarter level, and 25.6% (95% CR: 15.3–39.5%) at the cow level. At the cow level (n = 285), sensitivity and specificity of MEC were 95.6 and 99.5%, respectively. The accuracy of Somaticell (n = 319 quarters) to identify S. agalactiae-infected quarters was 75.4, 86.4, 88.9, 89.4, and 91.0% at thresholds of 98,000, 147,000, 205,000, 244,000, and 282,000 cells/mL, respectively. The accuracy of CMT was 87.6, 90.7, 90.8, and 87.4% at thresholds of trace, 1, 2, and 3, respectively. The areas under the receiver operating characteristic curve for Somaticell and CMT were 94.5% (95% confidence interval: 91.8–97.2%) and 92.0% (88.6–95.4%), respectively. At the tested thresholds, the sensitivity of Somaticell ranged from 94.9 to 99.5% to detect S. agalactiae IMI, and specificity ranged from 48.1 to 87.1%. The sensitivity of Somaticell at the lowest threshold (69,000 cells/mL; sensitivity = 99.9%; 95% CR: 98.2–100%) was higher than that of CMT at any tested threshold. Results of this study could be used at the farm level to reduce the use of antimicrobials and reach specific goals in S. agalactiae eradication programs.     

Postcalving udder health and productivity in cows approaching dry-off with intramammary infections caused by non-aureus Staphylococcus,Aerococcus, Enterococcus,Lactococcus, and Streptococcus species

The objective of this prospective cohort study was to explore associations between intramammary infection (IMI) in late-lactation cows and postcalving udder health and productivity. Cows (n = 2,763) from 74 US dairy herds were recruited as part of a previously published cross-sectional study of bedding management and IMI in late-lactation cows. Each herd was visited twice for sampling. At each visit, aseptic quarter milk samples were collected from 20 cows approaching dry-off (>180 d pregnant), which were cultured using standard bacteriological methods and MALDI-TOF for identification of isolates. Quarter-level culture results were used to establish cow-level IMI status at enrollment. Cows were followed from enrollment until 120 d in milk (DIM) in the subsequent lactation. Herd records were used to establish whether subjects experienced clinical mastitis or removal from the herd, and DHIA test-day data were used to record subclinical mastitis events (somatic cell count >200,000 cells/mL) and milk yield (kg/d) during the follow-up period. Cox regression and generalized estimating equations were used to evaluate the associations between IMI and the outcome of interest. The presence of late-lactation IMI caused by major pathogens was positively associated with postcalving clinical mastitis [hazard ratio = 1.5, 95% confidence interval (CI): 1.2, 2.0] and subclinical mastitis (risk ratio = 1.5, 95% CI: 1.3, 1.9). Species within the non-aureus Staphylococcus (NAS) group varied in their associations with postcalving udder health, with some species being associated with increases in clinical and subclinical mastitis in the subsequent lactation. Late-lactation IMI caused by Streptococcus and Streptococcus (Strep)-like organisms, other than Aerococcus spp. (i.e., Enterococcus, Lactococcus, and Streptococcus spp.) were associated with increases in postcalving clinical and subclinical mastitis. Test-day milk yield from 1 to 120 DIM was lower (?0.9 kg, 95% CI: ?1.6, ?0.3) in late-lactation cows with any IMI compared with cows without IMI. No associations were detected between IMI in late lactation and risk for postcalving removal from the herd within the first 120 DIM. Effect estimates reported in this study may be less than the underlying quarter-level effect size for IMI at dry-off and postcalving clinical and subclinical mastitis, because of the use of late-lactation IMI as a proxy for IMI at dry-off and the use of cow-level exposure and outcome measurements. Furthermore, the large number of models run in this study (n = 94) increases the chance of identifying chance associations. Therefore, confirmatory studies should be conducted. We conclude that IMI in late lactation may increase risk of clinical and subclinical mastitis in the subsequent lactation. The relationship between IMI and postcalving health and productivity is likely to vary among pathogens, with Staphylococcus aureus, Streptococcus spp., Enterococcus spp., and Lactococcus spp. being the most important pathogens identified in the current study.     

Effect of intramammary Escherichia coli endotoxin in early- vs. late-lactating dairy cows

We investigated the differences in the effect of intramammarily infused endotoxin for cows in early (EL) and late (LL) lactation. In this crossover study, nine cows were challenged twice with 100 microg of intramammarily infused Escherichia coli 0111:B4 LPS, each cow serving its own control. Systemic and local signs were recorded throughout the experiment to assess the severity of each cow's response. Daily milk yield and indicators of inflammation in blood and milk were also recorded. The response was significantly more severe in the EL period. Before the challenge, milk yield and serum free fatty acid concentration were higher and serum urea concentration lower in EL compared with LL cows. No significant differences were present in other parameters. After the challenge, milk SCC and leukocyte function, measured as chemiluminescence, increased, more markedly in EL. Blood neutrophil content varied from neutropenia to neutrophilia. Simultaneously with neutropenia, the function of neutrophils increased in LL but decreased in EL. Serum cortisol peaked 4 h after challenge in both groups. In conclusion, endotoxin challenge resulted in more severe response in EL. During that time cows also had impaired neutrophil function postchallenge.     

Relationship between postmilking standing duration and risk of intramammary infection in freestall-housed dairy cows milked 3 times per day

Recent evidence exists to suggest that the risk of subclinical mastitis, particularly those infections caused by environmental pathogens, in dairy cows is related to standing and lying patterns. The objective of this study was to determine the association between postmilking standing duration (PMSD) of dairy cows milked 3×/d and risk of intramammary infection (IMI). Four commercial freestall dairy herds in Eastern Ontario, milking 3×/d, were enrolled in a longitudinal study. Forty Holstein-Friesian cows per herd were randomly selected as focal animals from those cows in each herd that met our selection criteria of days in milk (<200 d) and somatic cell count (<100,000 cells/mL). The study consisted of three 28-d periods. The study began following a regularly scheduled Dairy Herd Improvement test with the collection of quarter-level milk samples from all focal animals. Bacteriology was used to confirm infection status at the start of the study and for determination of incidence of IMI throughout the study. A new IMI was defined as having a culture-positive quarter-level sample when the previous sample (28 d prior) had been culture negative for the pathogen of interest. Four sets of quarter-level milk samples were obtained for each focal animal. Lying behavior was recorded for 5 d after each milk sampling using data loggers. For these 5 d, individual milking times, production, and feeding times were also recorded. Postmilking standing duration was analyzed by milking event, with increased PMSD being positively associated with provision of fresh feed or freshly pushed-up feed around the time of milking, greater feed bunk space per cow, and lower freestall stocking density. Over the study period, 456 new IMI were detected, resulting in a mean herd incidence rate of 3.22 IMI per quarter year. Coagulase-negative staphylococci (CNS) and Corynebacterium spp. IMI were statistically analyzed to determine relationship with PMSD; they were the 2 predominant pathogens representing 45 and 31% of IMI, respectively. Only CNS IMI was associated with PMSD. A nonlinear relationship between PMSD and incidence of CNS IMI was found; cows with a PMSD of 90 to 120 min were at a reduced risk for CNS IMI. The risk of experiencing CNS IMI was also reduced with increased frequency of feed push-ups and provision of fresh feed 60 min before to 90 min after milking and >540 min after milking. These results indicate that management practices that promote PMSD of 90 to 120 min, such as the provision of fresh feed or freshly pushed-up feed around the time of milking, providing ample feed bunk space per cow, and keeping freestall stocking density low, should be encouraged to reduce the risk of CNS IMI in freestall-housed cows milked 3×/d.     

Blood biochemical changes upon subclinical intramammary infection and inflammation in Holstein cattle

The aim of this study was to investigate the associations between subclinical intramammary infection (IMI) from different pathogens combined with inflammation status and a set of blood biochemical traits including energy-related metabolites, indicators of liver function or hepatic damage, oxidative stress, inflammation, innate immunity, and mineral status in 349 lactating Holstein cows. Data were analyzed with a linear model including the following fixed class effects: days in milk, parity, herd, somatic cell count (SCC), bacteriological status (positive and negative), and the SCC × bacteriological status interaction. Several metabolites had significant associations with subclinical IMI or SCC. Increased SCC was associated with a linear decrease in cholesterol concentrations which ranged from −2% for the class ≥50,000 and <200,000 cells/mL to −11% for the SCC class ≥400,000 cells/mL compared with the SCC class <50,000 cells/mL. A positive bacteriological result was associated with an increase in bilirubin (+24%), paraoxonase (+11%), the ratio paraoxonase/cholesterol (+9%), and advanced oxidation protein product concentration (+23%). Increased SCC were associated with a linear decrease in ferric reducing antioxidant power concentrations ranging from −3% for the class ≥50,000 and <200,000 cells/mL to −9% for the SCC class ≥400,000 cells/mL (respect to the SCC class <50,000 cells/mL). A positive bacteriological result was associated with an increase in haptoglobin concentrations (+19%). Increased SCC were also associated with a linear increase in haptoglobin concentrations, which ranged from +24% for the class ≥50,000 and <200,000 cells/mL (0.31 g/L) to +82% for the SCC class ≥400,000 cells/mL (0.45 g/L), with respect to the SCC class <50,000 cells/mL (0.25 g/L). Increased SCC were associated with a linear increase in ceruloplasmin concentrations (+15% for SCC ≥50,000 cells/mL). The observed changes in blood biochemical markers, mainly acute phase proteins and oxidative stress markers, suggest that cows with subclinical IMI may experience a systemic involvement.     

Evaluation of test-day milk somatic cell count to predict intramammary infection in late lactation grazing dairy cows

The use of selective dry cow antimicrobial therapy requires precisely differentiating cows with an intramammary infection (IMI) from uninfected cows close to drying-off to enable treatment allocation. Milk somatic cell count (SCC) is an indicator of an inflammatory response in the mammary gland and is usually associated with IMI. However, SCC can also be influenced by cow-level variables such as milk yield, lactation number, and stage of lactation. In recent years, predictive algorithms have been developed to differentiate cows with IMI from cows without IMI based on SCC data. The objective of this observational study was to explore the association between SCC and subclinical IMI, taking cognizance of cow-level predictors on Irish seasonal spring calving, pasture-based systems. Additionally, the optimal test-day SCC cut-point (maximized sensitivity and specificity) for IMI diagnosis was determined. A total of 2,074 cows across 21 spring calving dairy herds with an average monthly milk weighted bulk tank SCC of ≤200,000 cells/mL were enrolled in the study. Quarter-level milk sampling was carried out on all cows in late lactation (interquartile range = 240–261 d in milk) for bacteriological culturing. Bacteriological results were used to define cows with IMI when ≥1 quarter sample resulted in bacterial growth. Cow-level test-day SCC records were provided by the herd owners. The ability of the average, maximum, and last test-day SCC to predict infection were compared using receiver operator curves. Predictive logistic regression models tested included parity (primiparous or multiparous), yield at last test-day, and a standardized count of high SCC test-days. In total, 18.7% of cows were classified as having an IMI, with first-parity cows having a higher proportion of IMI (29.3%) compared with multiparous cows (16.1%). Staphylococcus aureus accounted for the majority of these infections. The last test-day SCC was the best predictor of infection with the highest area under the curve. The inclusions of parity, yield at last test-day, and a standardized count of high SCC test-days as predictors did not significantly improve the ability of last test-day SCC to predict IMI. The cut-point for last test-day SCC that maximized sensitivity and specificity was 64,975 cells/mL. This study indicates that in Irish seasonal pasture-based dairy herds with low bulk tank SCC, the last test-day SCC (interquartile range days in milk = 221–240) is the best predictor of IMI in late lactation.