Composite Milk Somatic Cell Count Research Articles

Association of Staphylococcus aureus genotypes with milk or colonization of extramammary sites in Dutch dairy cattle indicates strain variation in reservoirs for intramammary infections

Staphylococcus aureus (S. aureus) is a major mastitis pathogen with a detrimental impact on udder health in dairy cattle. Although S. aureus is generally classified as a contagious mastitis pathogen, control measures aimed at preventing contagious transmission are not always effective. Previous studies showed that various extramammary sites can be colonized with S. aureus and could be a reservoir for S. aureus intramammary infections (IMI). The aim of this study was to determine the prevalence of S. aureus extramammary site colonization in Dutch dairy cattle and to compare the spa genotypes of S. aureus isolates from IMI to spa genotypes of isolates from extramammary sites. Six herds were visited and from cows with a composite milk somatic cell count ≥200,000 cells/mL quarter milk samples and swabs from various extramammary sites (hock, groin, udder cleft, nares, and feces) were taken. Extramammary site samples were processed by a two-step high salt selective culture and presence of S. aureus was confirmed by femA PCR. S. aureus isolates from milk and extramammary sites were compared by spa typing. The cow level colonization varied from 0% to 73%, and the prevalence of IMI in the sampled cows varied from 0% to 61% between herds. The extramammary site with the highest prevalence of colonization was the hock (23%) and the lowest prevalence of colonization was found for the nares (5%) and feces (5%). Spa typing of S. aureus isolates from either extramammary sites or milk showed that in most herds there were one or two predominant S. aureus spa genotypes present. Different S. aureus spa genotypes could be categorized into three groups based on the distribution between milk or extramammary sites: i) predominantly milk associated, ii) associated with both milk and extramammary sites, and iii) associated with extramammary sites. In conclusion, we showed that the prevalence of extramammary site colonization differed significantly between herds and extramammary sites and that specific S. aureus spa genotypes were associated with milk (IMI) or extramammary site colonization. Extramammary S. aureus reservoirs could be a source for IMI that cannot be eradicated by intervention measures aimed at contagious mastitis pathogens.

Prevalence and risk factors associated with high somatic cell count in Chinese dairy herds.

This study aimed to (1) estimate the prevalence of cow-level high somatic cell count (SCC) in Chinese dairy herds and (2) identify potential factors associated with cow- and herd-level SCC variables. The monthly data on dairy herd improvement were collected from a total of 131 dairy herds in 11 provinces in China in 2019. Mixed models were constructed using the cow composite milk SCC and the variance of cow SCC as dependent variables separately and parity, seasons, days in milk (DIM), herd size, and farm types (family-owned vs. company-owned) as fixed effects, accounting for the nested random herd and cow effect. We used negative binomial regression using herd-level SCC-related variables, namely, monthly proportion of high SCC, monthly proportion of new high SCC, monthly proportion of chronic high SCC, and monthly proportion of new chronic high SCC as dependent variables separately against seasons, herd size, and farm types with the random herd effect. The overall average prevalence of high SCCs for each month per farm was 0.26 (2.5-97.5% quantile: 0-0.56). Company-owned farms performed better in herd SCC management. Seasons were significantly associated with all the aforementioned variables, and summer and autumn were the seasons associated with worse outcomes in herd SCCs. This study is the first to assess high SCC in a large number of Chinese dairy herds, which is useful for farms to tailor the on-farm mastitis control programs in China.

Species identification and cow risks of non-aureus staphylococci from South African dairy herds.

Detailed information on specific species of non-aureus staphylococci (NAS) has become a necessity for effective udder health control programs in South Africa. The main objective of this preliminary study was to identify the different NAS species and strains present in dairy herds in South Africa using a cost-effective method. A further objective was to investigate the effects of cow risk factors and farming systems on the NAS isolates identified. A total of 214 NAS, isolated from milk collected from 17 South African dairy herds, were identified using three diagnostic tests (API Staph test, MALDI-TOF and 16s rRNA). There was a good observed agreement between the MALDI-TOF and 16S rRNA sequencing (92.2%) and a poor observed agreement between the MALDI-TOF and API Staph (25.7%). The genetic relatedness within species was investigated in 128 of these isolates using random polymorphic amplified deoxyribonucleic acid (DNA) (RAPD), verified by multilocus sequence typing (MLST), and phylogenetic analysis and cow risk factors were investigated on species level. The main NAS species isolated were Staphylococcus chromogenes (75.2%), Staphylococcus epidermidis (9.4%) and Staphylococcus haemolyticus (8.9%). The RAPD test identified 34 Staphylococcus chromogenes, 13 Staphylococcus epidermidis and nine Staphylococcus haemolyticus strains, indicating genetic diversity amongst strains and herds. The presence of NAS intramammary infections was found to be significantly related to the farming systems, composite cow milk somatic cell count (SCC), parity and days in milk (DIM). Significantly more NAS were isolated from primiparous and from older cows. This knowledge could assist with the management of NAS on dairy farms.

Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited effects on selected inflammation indicators in early-lactation cows

The objective was to assess effects of experimentally induced undernutrition on responses to an intramammary lipopolysaccharide (LPS) challenge in early-lactation cows. Starting at 24 ± 3 d in milk, multiparous Holstein cows either received a ration containing 48% straw for 96 h to restrict nutrient intake (REST, n = 8) or were allowed ad libitum intake of a lactation diet (CONT, n = 9). After 72 h on diet or after an equivalent period for CONT, 50 µg of LPS (Escherichia coli 0111:B4) was injected into one healthy rear mammary quarter to induce an acute inflammation response. Blood samples were collected weekly until 7 wk of lactation, daily during feed restriction (or control), before and at 1, 2, 4, 6, 10, and 24 h relative to LPS injection. Foremilk quarter samples were collected before and at 4, 6, 10, and 24 h after LPS injection. Dry matter intake, milk yield, energy balance, plasma glucose, nonesterified fatty acids (NEFA), and β-hydroxybutyrate (BHB) concentrations did not differ between CONT and REST immediately before nutrient restriction in REST (least squares means at d -1 were 21.8, 39.0 kg/d, -2.5 MJ/d, and 3.78, 0.415, 0.66 mM, respectively) but were significantly altered at 72 h of nutrient restriction (9.8, 28.3 kg/d, -81.6 MJ/d, and 2.77, 1.672, and 2.98 mM, respectively), when the LPS challenge was performed. The rectal temperature increment from baseline values in response to LPS did not differ, but cortisol increment was greater and cortisol response area under the curve (AUC) tended to be greater [202 vs. 122 (ng/mL) × 10 h] for REST than CONT. No treatment differences were observed in foremilk IL-8, IL-1β, tumor necrosis factor-α, and chemokine (C-X-C motif) ligand 3 concentrations in response to LPS injection. Composite milk somatic cell count per milliliter (6.919 × 106 vs. 1.956 × 106 cells/mL) and total number of somatic cells secreted in milk per day were greater for REST than CONT during the day following LPS. Plasma glucose, urea, and insulin concentrations increased after the LPS challenge, suggesting establishment of insulin resistance and modifications of glucose metabolism to support acute inflammation in both CONT and REST. Nonetheless, nutrient-restricted cows had delayed plasma insulin and glucose responses to LPS, smaller insulin AUC but greater glucose AUC compared with CONT, despite the limited nutrient availability to sustain an inflammation response. Undernutrition altered peripheral metabolic responses to an intramammary LPS challenge but had limited effects on selected indicators of inflammation response in early-lactation cows.

Large-scale cross-sectional study of relationships between somatic cell count and milking-time test results in different milking systems

Milking-time testing (MTT) is a method for evaluating the vacuum conditions in the teatcup during milking. The purpose is to evaluate the possible impact of the milking and milking equipment on udder health and milk quality. The method is commonly implemented by herd health advisory services, but results are interpreted empirically due to lack of scientific documentation on relationships between MTT result variables and objective measures of udder health.The current study was conducted to increase our understanding of associations between cow-level differences in composite milk somatic cell count (CMSCC) and MTT results in dairy cows milked in 3 different milking systems; automatic milking systems (AMS), milking parlors, and pipeline milking systems. Data from 7069 cows (predominantly Norwegian Red breed) in 1009 herds were used in a cross-sectional study. Multilevel linear regression models with a random intercept at herd level were used to describe relationships between CMSCC (on logarithmic scale) and the following MTT explanatory variables: average vacuum level in the short milk tube and mouthpiece chamber in the main milking and overmilking periods, the duration of these two periods, and vacuum stability, measured by sudden vacuum drops in the short milk tube. The models were corrected for the herd effect, mastitis history and differences in milk yield, lactation stage and parity between cows. Separate models were run for AMS, milking parlors, and pipeline milking systems, because this approach allowed for comparison between systems and for evaluation of the herd effect independently of milking system.The models described 8–10 % of the variation in CMSCC, indicating that MTT could only explain a relatively small proportion of a large total variation in CMSCC. In most observations, vacuum levels in the short milk tube during main milking were within the range recommended by the International Organization for Standardization. The results from our multivariable models showed decreasing CMSCC with increasing vacuum level in the short milk tube during the main milking period in AMS and milking parlors. Similarly, decreasing CMSCC was also associated with increasing duration of the main milking period in all 3 systems. These relationships are important for the interpretation of MTT results under practical conditions; finding high vacuum levels and long milking durations in a MTT is not associated with elevated CMSCC. In AMS herds, we also found indications that the relationships were different for cows where a case of mastitis had been treated before the MTT.

Prepartum application of internal teat sealant or intramammary amoxicillin on dairy heifers: Effect on udder health, survival, and performance

Intramammary infusion of antimicrobials (IA) and application of an internal teat sealant (ITS) at the end of the lactation is a common strategy to prevent and treat intramammary infections during the dry period. In heifers, precalving IA has been reported to improve udder health and milk quality; however, the effect of ITS in heifers housed in freestall barns is still unknown. The objective of this study was to investigate the effect of prepartum application of ITS or IA on udder health of dairy heifers. A total of 886 heifers were randomly allocated into 1 of 4 treatment groups when they were 245 ± 3 d pregnant. Control heifers (CON) did not receive any treatment. Heifers in the ITS group received the application of ITS containing 2.6 g of bismuth subnitrate. Animals in the IA group received intramammary administration of 62.5 mg of amoxicillin. Heifers in IA+ITS group received intramammary administration of amoxicillin followed by ITS application. The follow-up period was 270 d in milk (DIM). Clinical mastitis was diagnosed and treated by trained farm personnel throughout the study period. Composite milk somatic cell counts (SCC) were determined monthly for the first 9 mo of lactation by Dairy Herd Improvement Association. Subclinical mastitis was defined as a cow having a SCC >200,000 cells/mL for at least 1 test day and not diagnosed with clinical mastitis during the study period. At 7 ± 3 DIM, composite milk samples were collected for bacteriological analysis for a subset of the study animals. The presence of pathogens in milk at 7 ± 3 DIM was more frequently detected in the CON heifers compared with animals enrolled in the treatment groups. Heifers in IA+ITS group had a decreased incidence of clinical mastitis compared with CON heifers (12.9 vs. 21.4%). Additionally, the incidence of subclinical mastitis was decreased for IA+ITS heifers compared with CON counterparts (20.1 vs. 43.8%). During the first 9 mo of lactation, IA and IA+ITS heifers had lower SCC linear scores compared with ITS and CON counterparts. Although IA+ITS treatment successfully improved udder health, it did not result in better milk yield, fertility, and survivability. In conclusion, ITS alone was not effective in improving udder health; however, the combination of ITS and IA decreased the incidence of clinical and subclinical mastitis during the first 9 mo of lactation. Additionally, IA+ITS had a long-term effect on the SCC linear score throughout the study period.

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.

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

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

Sensitivity and specificity of PCR analysis and bacteriological culture of milk samples for identification of intramammary infections in dairy cows using latent class analysis

Real-time PCR analysis of milk samples is a fast method to identify intramammary infections (IMI) in dairy cows, and has the potential to be used for routine analysis of test milking composite milk samples. However, the results of the PCR analysis can be difficult to interpret. The objective of this study was to compare the sensitivity (Se) and specificity (Sp) of PCR analysis of composite milk samples, and conventional bacteriological culturing (BC) of quarter milk samples, when used to identify cows with IMI. The comparisons were performed for IMI with four common udder pathogens; Staphylococcus aureus (S aureus), Streptococcus dysgalactiae (Str dysgalactiae), Str uberis and coagulase negative staphylococci (CoNS). The Se and Sp of real-time PCR (SePCR; SpPCR) and BC (SeBC; SpBC) was simultaneously estimated using latent class analysis (LCA), studying one pathogen at the time. Milk samples from 970 dairy cows from 25 herds were included. Aseptically collected quarter milk samples taken at the day before test milking (TM), at the day of TM, and at the day after TM, were analyzed using BC. Non-aseptically collected composite milk samples taken at the day of TM were analyzed using PCR. Moreover, the composite milk somatic cell count (SCC) was recorded and summarized by diagnostic test and bacterial finding. LCA was first performed using only test results from samples taken at the day of TM, but in a second analysis BC results from the three consecutive samplings, interpreted in parallel, were included. The SePCR was significantly higher than the SeBC for S aureus, Str dysgalactiae and CoNS in the first analysis, but only for CoNS in the second analysis. The SpPCR was significantly lower than the SpBC for Str dysgalactiae and CoNS. In conclusion, using PCR analysis of composite milk samples, as a diagnostic tool for identifying cows with IMI increased the Se for all the pathogens investigated (although not always significantly), while Sp in general remained on a similar level, compared to BC of quarter milk samples. The use of repeated quarter milk sampling improved the SeBC, making the results of PCR analysis and BC more similar. However, the SCC of cows with IMI according to BC was higher than for cows with IMI according to PCR, suggesting that some of the cows with IMI according to PCR did not have an active inflammation. Hence, extra caution is needed when decisions about treatment of IMI are based on PCR.

Evaluation of the composite milk somatic cell count as a predictor of intramammary infection in dairy cattle

The objectives of this study were (1) to evaluate the test characteristics and predictive values of quarter-composite milk somatic cell count (quarter-cSCC) values based on either a single observation or the geometric mean of multiple recordings as a predictor of intramammary infection (IMI) in lactating dairy cows; and (2) to explore to what extent herd prevalence of IMI and cow factors such as parity and stage of lactation affect them. A total of 780 single-quarter milk samples were collected from 195 dairy cows for bacteriologic culture at a single cross-sectional herd screening performed at 21 different dairy herds as part of different research projects. Additionally, monthly quarter-cSCC milk samples at test day were available as part of the Dairy Herd Improvement program. Sensitivity (Se), specificity (Sp), positive predictive valu (PPV), and negative predictive value (NPV) were calculated to differentiate cows infected with any pathogen and cows infected with major pathogens from uninfected cows. Different threshold values for quarter-cSCC, ranging between 50,000 and 500,000 cells/mL, were evaluated for all animals in the study, as well as for high- and low-prevalence herds, heifers and multiparous cows, and cows in early, mid, and late lactation. The overall Se and Sp at a threshold of 200,000 cells/mL for a single quarter-cSCC observation obtained closest to the time of bacteriologic culture were 44.3 and 87.3%, respectively, for cows infected with any pathogen, and 65.1 and 73.0%, respectively, for cows infected with major pathogens. The overall PPV and NPV at a threshold of 200,000 cells/mL for a single quarter-cSCC observation obtained closest to the time of bacteriologic culture were 89.9 and 38.1%, respectively, for cows infected with any pathogen, and 40.6% and 88.1%, respectively, for cows infected with major pathogens. No major differences were observed between estimates of the test characteristics and predictive values of the quarter-cSCC criteria based on a single observation and the geometric mean of multiple observations. For IMI with any pathogen, the Se and PPV were higher in high-prevalence herds than in low-prevalence herds, particularly at thresholds of 50,000 and 100,000 cells/mL. For IMI with major pathogens, Sp was substantially higher in low-prevalence herds than in high-prevalence herds. Sensitivity was higher in multiparous cows than in heifers infected with any pathogen, more specifically at a threshold of 100,000 and 200,000 cells/mL. For cows in early and mid lactation infected with any pathogen, Sp was higher than for cows in late lactation using the single observation closest to the time of bacteriologic culture. The results suggest that the quarter-cSCC threshold value to select cows for bacteriologic culture to maximize the likelihood of finding the causative pathogen of IMI should depend on the group of pathogens one is interested in, the herd prevalence of subclinical mastitis, lactation stage, and the cow's parity.

Randomised controlled trials demonstrate efficacy of a novel internal teat sealant to prevent new intramammary infections in dairy cows and heifers

AIMS: The main aim was to investigate the effectiveness of a novel internal teat sealant (ITS) formulation containing bismuth subnitrate and the antiseptic chlorhexidine, in adult cows (Trial A) and nulliparous heifers (Trial B), to reduce the incidence of new intramammary infection (IMI) between treatment and calving and reduce the prevalence of IMI within 6 days following calving.METHODS: In Trial A dairy cows (n=326) without history of clinical mastitis and with a maximum composite milk somatic cell count <200,000 cells/mL during the current lactation were enrolled from three spring-calving mainly pasture-based herds immediately following the final milking. In Trial B all heifers (n=166) were enrolled from two herds approximately 3 weeks before the start of calving. Glands were randomly assigned to controls or treatment with ITS, and mammary secretion samples were taken from all glands prior to treatment, and on two occasions 0–4 and 3–6 days post-calving to determine the incidence of new IMI and the prevalence of IMI immediately post-calving. Additionally, cows in Trial A were monitored for cases of clinical mastitis (CM) in the non-lactating period and animals in both trials were monitored for cases of CM within 30 days of calving.RESULTS: The ITS reduced the estimated incidence of new IMI with both major and any pathogens compared to control glands in cows (6.2 vs. 14.2% and 14.7 vs. 32.8%, respectively) and in heifers (3.0 vs. 13.1% and 13.4 vs. 25.3%, respectively) (p<0.001). The ITS reduced the estimated prevalence of a major or any pathogen compared to untreated controls at both 0–4 and 3–6 days post-calving, in both cows and heifers. Additionally, the ITS reduced the incidence of CM in the non-lactating period in cows (−1.0%, p=0.01) and in the 30 days following calving in heifers (−2.0%, p=0.06), and tended to reduce the estimated incidence of CM in cows post-calving (−1.7%, p=0.10).CONCLUSIONS: The novel ITS reduced the incidence of new IMI, and the prevalence of IMI at calving in both adult cows and heifers, and reduced the incidence of CM in the non-lactating period of cows and in the first 30 days after calving in heifers.CLINICAL RELEVANCE: The results of these trials demonstrate the efficacy of a novel ITS for the control of mastitis in likely uninfected dairy cows over the non-lactating period and in heifers in late pregnancy.

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.

The effect of intramammary infection with coagulase-negative staphylococci in early lactating heifers on milk yield throughout first lactation revisited

The objective of this study was to further scrutinize the previously found positive association between intramammary infection (IMI) caused by coagulase-negative staphylococci (CNS) in early lactating heifers and test-day daily milk yield (MY) throughout first lactation, with a specific focus on the effect of the heifers’ genetically determined milk production levels and the incidence of clinical mastitis. Two precise longitudinal data sets were analyzed using a series of statistical models including potential confounding and intermediate variables. The final database included the IMI status at calving, composite milk somatic cell count (SCC) and MY records at test day up to 285 d in milk (DIM), farmer-recorded clinical mastitis (CM) cases between 14 and 285 DIM, estimated new IMI incidence based on a SCC threshold of 100,000cells/mL between 14 and 285 DIM, DIM, average 305-d MY at the herd level, and the heifers’ genetic merit for MY from 240 dairy heifers from 29 dairy herds. Seventy-one (29.6%) early lactating heifers were noninfected, 108 heifers (45.0%) were CNS infected, and 61 heifers (25.4%) were infected with any major pathogen. The positive effect of CNS IMI in early lactation on test-day MY was estimated at 1.32kg/d using a first basic mixed regression model. Correcting for the confounder genetic merit for MY reduced this effect to 1.17kg. Interestingly, taking into account the confounding effect of herd resulted in an increase of the estimate from 1.32 to 2.2kg/d. The positive effect of CNS IMI in early lactation on MY after correcting the model for both confounders was estimated at 2.05kg/d. Heifers infected with CNS in the first DIM tended to have fewer CM cases throughout lactation compared with the noninfected herd mates. Including the intermediate variable CM in the model explained 0.16kg/d of the corrected effect of 2.05kg/d. Inclusion of test-day SCC, another intermediate variable, however, increased the estimate by 0.11kg/d. With an appropriate correction for several confounders and biologically understood intermediate variables such as CM, test-day SCC, and new IMI based on SCC threshold of 100,000cells/mL, an unexplained test-day MY difference between CNS-infected and noninfected heifers of 2.0kg/d remained.

Generation of an index for physiological imbalance and its use as a predictor of primary disease in dairy cows during early lactation

Physiological imbalance (PI) is a situation in which physiological parameters deviate from the normal and cows consequently have an increased risk of developing production diseases and reduced production or reproduction. The objectives of this work were (1) to generate an index for PI based on several plasma metabolites and (2) to compare the use of this index with calculated energy balance (EBAL) and individual plasma metabolites in relation to risk of disease during early lactation. We used a total of 634 lactations from 317 cows consisting of 3 breeds ranging from a parity of 1 to 4. Weekly blood samples were analyzed for selected metabolites; that is, urea nitrogen, albumin, cholesterol, nonesterified fatty acids (NEFA), glucose, and β-hydroxybutyrate (BHBA). Energy intake and EBAL were calculated; veterinary treatment records and daily composite milk somatic cell counts were used to determine incidence of disease. Data were adjusted for numerous fixed effects (e.g., parity, breed, and week around calving) before further statistical analysis. The time of disease (TOD) was recorded as the day in which the signs of disease were observed (TOD=0). The week before and after TOD was ± n wk relative to TOD=0. Each week, all plasma metabolites were individually adjusted to an overall mean (=0) and variance (=1). The normalized variables were included in regression analyses by week of lactation to identify metabolites that explain the variation in calculated EBAL, as a reflection of degree of PI. Nonesterified fatty acids, BHBA, and glucose were weighted within each week based on regression coefficients (i.e., x1–x3 below) generated from a model to predict EBAL. Data from wk −1 relative to TOD were analyzed using a mixed linear model to relate degree of PI and metabolites in blood to risk of disease. The weekly PI index was defined as PI=(x1 × [NEFA])+x2 × [BHBA] − x3 × [glucose])/3. For diseases that developed ≥2 wk after calving, no variables were associated with risk of disease. Prepartal PI and plasma NEFA were better predictors of disease (i.e., metritis, retained placenta, and milk fever) at wk 1 than EBAL and plasma BHBA and glucose. Examining the relationship between PI and milk constituents is needed for the development of an automated in-line and real-time surveillance system for early detection of risk animals on-farm.

Pathogen-group specific association betweenCXCR1polymorphisms and subclinical mastitis in dairy heifers

The chemokine (C-X-C motif) receptor 1 (CXCR1) gene encodes the homonymous receptor for interleukin 8 (IL8) on polymorphonuclear neutrophilic leucocytes (PMNL). Binding causes migration from blood to milk, activation and prolonged survival of PMNL, a crucial process in the innate immune defence of the bovine mammary gland against invading mastitis-causing pathogens. The main objective of this study was to screen the entire coding region of the CXCR1 gene for polymorphisms and to analyse their association with udder health of dairy heifers. One-hundred-and-forty Belgian Holstein heifers originating from 20 commercial dairy farms were genotyped by DNA sequencing. Detailed phenotypic data on udder health was available including quarter bacteriological culture results and somatic cell count (SCC) in early lactation and composite milk SCC during first lactation. In total, 16 polymorphisms (including 8 missense mutations) were detected. Polymorphism c.980A>G was associated with pathogen-group specific IMI: heifers with genotype AG were less likely to have an IMI due to major mastitis pathogens compared with heifers with genotype GG but did not have less IMI by coagulase-negative staphylococci, so-called minor pathogens. CXCR1 genotype was neither associated with quarter SCC in early lactation nor with composite SCC during lactation. Although mastitis susceptibility is influenced by many factors, some genetic polymorphisms potentially have major effects on udder health of heifers, as was shown here. These results trigger us to further study the relationship between CXCR1 polymorphisms and mastitis susceptibility in both observational and experimental trials.

Heifers infected with coagulase-negative staphylococci in early lactation have fewer cases of clinical mastitis and higher milk production in their first lactation than noninfected heifers

Intramammary infections (IMI) in recently calved dairy heifers are more common than was formerly believed but their relevance for future performance has been studied only rarely. In the present study, the association between the IMI status of fresh heifers and their subsequent udder health, milk production, and culling in first lactation was explored. Quarter milk samples were collected between 1 and 4 d in milk (DIM) and between 5 and 8 DIM from 191 dairy heifers in 20 dairy herds for bacteriological culturing and somatic cell count (SCC) analysis. Monthly milk recording data including composite milk SCC and test-day milk yield (MY) were obtained for the first 285 DIM or until culling. Farmer-recorded clinical mastitis cases were available. Data were analyzed using mixed models and survival analysis. Approximately 80% of the fresh heifers (79.8%) had at least one culture-positive quarter. Coagulase-negative staphylococci (CNS) were the most frequently isolated pathogens (72%), followed by esculin-positive streptococci (4.6%) and Staphylococcus aureus (3.5%). Overall geometric mean SCC at quarter level decreased between the first and second samplings from 348,000 to 116,000 cells/mL. Heifers infected with CNS had an intermediate average test-day SCC (84,000 cells/mL) during the first 285 DIM compared with noninfected heifers (53,000 cells/mL) and heifers infected with major pathogens (195,000 cells/mL). Heifers infected with major pathogens had a much lower average daily MY (18.3kg) during first lactation compared with noninfected animals (21.3kg). That CNS-infected heifers out-produced their noninfected counterparts could be at least partially explained by their significantly lower incidence of clinical mastitis (incidence risk 3.6 vs. 21.0%) during first lactation compared with noninfected heifers. We conclude that although CNS cause the majority of IMI in heifers around calving, they should not be a reason for serious concern.

Identification of potential markers in blood for the development of subclinical and clinical mastitis in dairy cattle at parturition and during early lactation

Our objective was to identify specific blood markers as risk factors for the development of mastitis during early lactation. We used a subset of cows from a larger experiment that consisted of a total of 634 lactations from 317 cows. Cows were of 3 breeds and ranged from parity 1 to 4. Blood samples were collected weekly from 56 d before expected calving date through 90 d in milk (DIM). Blood was analyzed for several hormones, metabolites, and enzymes, and energy intake and energy balance were calculated. Veterinary treatment records and daily composite milk somatic cell counts were analyzed and used to determine incidence and severity of mastitis in early lactation. Cows were separated into 2 groups: 1) WK0, consisting of cows that developed clinical mastitis (CM), cows that developed subclinical mastitis (SM), or cows that were healthy (H) during the first 7 DIM; and 2) EL, consisting of CM, SM, or H cows during wk 2 through 13 of lactation. Data were adjusted for numerous fixed effects (e.g., parity, breed, season, and DIM) before statistical analysis. The time of mastitis (TOM) was recorded as the DIM in which the first rise in somatic cell count was observed and was recorded as TOM=0. The time before and after TOM was distinguished as±n wk relative to TOM=0. Healthy cows were paired with either a SM or CM cow and the TOM for each H cow was equal to the TOM for its paired SM or CM cow. Data from wk −1 and −2 relative to TOM were analyzed for group WK0 and EL, respectively. For all parameters, SM cows did not differ from H cows from either group. The CM cows had higher nonesterified fatty acid levels and a tendency toward higher β-hydroxybutyrate levels than H cows before mastitis for both groups. For group WK0, glucose was higher −1 wk relative to calving in CM than H cows. For group EL, aspartate aminotransferase was higher −2 wk relative to mastitis in CM than H cows during 8 to 90 DIM. All other variables were similar among CM, SM, and H cows for both groups. Our results indicate that substances in blood, especially nonesterified fatty acids and aspartate aminotransferase, may be potential markers for the risk of mastitis in early lactation.

Prevalence and persistence of coagulase-negative Staphylococcus species in three dairy research herds

Coagulase-negative Staphylococcus species (CNS) were isolated from 11.3% (1407 of 12,412) of mammary quarter milk samples obtained from cows in three dairy research herds in 2005. Approximately 27% (383/1407) of CNS was identified to the species level. The species distribution among those CNS identified from all herds was Staphylococcus chromogenes (48%), Staphylococcus hyicus (26%), Staphylococcus epidermidis (10%), Staphylococcus simulans (7%), Staphylococcus warneri (2%), Staphylococcus hominis (2%), Staphylococcus saprophyticus (1%), Staphylococcus xylosus (1%), Staphylococcus haemolyticus (<1%), Staphylococcus sciuri (<1%), and Staphylococcus intermedius (<1%). Staphylococcuschromogenes was the predominant CNS isolated from all three herds; however, differences were seen in the prevalence of other CNS species. A total of 158 CNS ( S. chromogenes n = 66, S. hyicus n = 38, S. epidermidis n = 37, S. simulans n = 10, and S. warneri n = 7) were analyzed by pulsed-field gel electrophoresis (PFGE). The majority (33/41) of CNS isolated from the same mammary quarter on more than one occasion had the same PFGE pattern indicating persistence of the same infection over time. When all PFGE patterns for each CNS were analyzed, no common pulsotype was seen among the three herds indicating that CNS are quite diverse. Composite milk somatic cell count (SCC) data were obtained ±14 d of when CNS were isolated. Average milk SCC (5.32 log 10/ml) for cows in which CNS was the only bacteria isolated was significantly higher than the average milk SCC (4.90 log 10/ml) from cows with quarter milk samples that were bacteriologically negative.

Relationships Between Milk Culture Results and Composite Milk Somatic Cell Counts in Norwegian Dairy Cattle

Associations between test-day composite milk somatic cell counts (CMSCC) and results from quarter milk cultures for various pathogens associated with mastitis, including Staphylococcus aureus, Streptococcus spp., coagulase-negative staphylococci (CNS), were investigated. S. aureus was dichotomized according to sparse (≤1,500 colony forming units/mL of milk) or rich (>1,500 colony forming units/mL of milk) growth of the bacteria. Quarter milk samples were obtained on between 1 and 4 occasions from 2,714 cows in 354 Norwegian dairy herds, resulting in a total of 3,396 samples. Cows included in the study were randomly selected, without regard to current or previous udder health status. Measures of test-day CMSCC were obtained every second month, and related to 3528 microbiological diagnoses at the cow level. Mixed linear regression models incorporating a compound symmetry covariance structure accounting for repeated test-day CMSCC within cow, and a random effect variable on herd level, was used to quantify the relationship between a positive milk culture and the natural logarithm of test-day CMSCC (LnCMSCC). The material was stratified in time periods before 151 d in milk (DIM) and after 150 DIM. A positive diagnosis for any category of mastitis pathogen was significantly associated with elevated CMSCC. Pathogen positive cows sampled for microbiological diagnosis during the first 150 DIM had higher levels of CMSCC throughout lactation than cows with a positive diagnosis after 150 DIM. Streptococcus spp.-positive milk cultures were associated with steadily elevated values for CMSCC throughout lactation both when sampled before and after 150 DIM. Cows diagnosed with rich growth of S. aureus after 150 DIM experienced a characteristic and sharp increase in CMSCC, but this effect was not observed in cows with a positive diagnosis for rich growth of S. aureus during the first 150 DIM. A considerable increase in CMSCC in cows positive for CNS during the first part of the lactation period was also observed. The practicability of using CMSCC in a diagnostic test to identify cows with a positive milk culture for mastitis pathogens was also assessed. The sensitivity, specificity, and positive predictive values of the tests were regarded as low when sampling for milk culture was conducted, irrespective of cow level characteristics.

Risk factors for isolation ofStaphylococcus aureusorStreptococcus dysgalactiaefrom milk culture obtained approximately 6 days post calving

Milk culture results at approximately 6 d post calving were assessed in a 2-year retrospective single-cohort study in 178 Norwegian herds. A combined teat dipping and selective antibiotic therapy trial was performed in these herds where cows with composite milk somatic cell count (CMSCC) >100,000 cells/ml before drying-off (geometric mean of the last three CMSCC test-days) and isolation of Staphylococcus aureus or Streptococcus dysgalactiae were selected for either short-acting lactation antibiotic treatment or long-acting dry cow antibiotic treatment. Milk culture results at approximately 6 d post-calving were available from 437 treated cows and 3061 non-treated cows before drying-off and separate multivariable logistic regression models were ran for these two groups. Risk factors associated with isolation of Staph. aureus 6 d post calving for non-treated cows were CMSCC >400,000 cells/ml before drying-off v. <400,000 cells/ml (Odd ratio (OR) = 2.4) and clinical mastitis (CM) in the previous lactation v . non-treated (OR=1.5). Risk factors associated with Staph. aureus 6 d post calving for treated cows was a CMS > 200,000 cells/ml before drying-off v. <200,000 cells/ml (OR=2.3) and CM in the previous lactation verus non-treated (OR=1.7). For non-treated cows it was 1.7 times more likely to isolate Str. dysgalactiae 6 d post-calving if the CMSCC was > 50,000 cells/ml compared with <50,000 cells/ml. For treated cows it was 3.7-5.8-times more likely to isolate Str. dysgalactiae 6 d post calving if given short-acting lactation formula at quarter level compared with long-acting dry cow formula used at cow level. Regular use of iodine post-milking teat disinfection (PMTD) did not influence the isolation of Staph. aureus 6 d post calvin, but it was less likely to isolate Str. dysgalactiae 6 d post calving if iodine PMTD was used regularly rather than irregularly. The external teat sealant had no effect on either of the two bacteria. This study indicates that the CMSCC limit for sampling cows before drying-off can be reduced to 50,000 cells/ml in herds with a Str. dysgalactiae problem. Iodine PMTD should also be recommended in these herds. Cows with a CMSCC > 400,000 cells/ml prior to drying-off should receive long-acting dry cow formula irrespective of the milk result.