Application of udder surface temperature by infrared thermography for diagnosis of subclinical mastitis in Holstein cows located in tropical highlands

Several reports have indicated that udder surface temperature (UST) can be a useful indicator of subclinical mastitis (SCM). The objective was to evaluate UST by infrared thermography (IRT) as a diagnostic tool for SCM and intramammary infection (IMI), and to assess the influence of environmental conditions in the potential diagnosis of this disease in dairy cows located at high-altitude tropical regions. A total of 105 cows (397 quarters) from 3 dairy farms with mechanical and manual milking methods were enrolled in the study. Subclinical mastitis was diagnosed when quarter samples had a somatic cell count (SCC) ≥200 × 10³ cells/mL, microbial growth (MG) was defined when a major pathogen (≥1 cfu/plate) or Corynebacterium spp. (≥10 cfu/plate) was isolated, and IMI was defined as the presence of MG and SCC ≥100 × 10³ cells/mL. Infrared images were taken with a thermal camera placed 1 m away from the udder, and shots of the rear and left and right lateral view were made during the morning milking, before any manipulation of the udder and employing dark cardboard on the contralateral side to avoid artifacts in the background. A multilevel mixed effects linear regression model clustered within cows and herd was performed to evaluate the associations with UST. Clinical performance was evaluated using the Youden index to establish the optimum UST thresholds, which were set at 32.6°C for any case definition when milking was by hand, at 33.7°C for MG, and at 34°C for SCM and IMI in machine-milked quarters. Sensitivity (Se), specificity (Sp), area under curve (AUC), and positive likelihood ratio (+LR) were also assessed. Test agreement was assessed by kappa coefficient (κ). The UST of healthy quarters ranged between (95% CI) 32.4 and 32.6°C, lower than SCM quarters (n = 88) at 32.9°C (95% CI: 32.7–33.1 °C), MG quarters (n = 56) at 33.5°C (95% CI: 33.3–33.7°C), and IMI quarters (n = 50) at 33.5°C (95% CI: 33.2–33.7 °C). The UST was also related to the milking method: higher temperatures were observed for hand milking (n = 90) compared with machine milking (n = 185). No relation between environmental conditions such as wind speed, atmospheric temperature, relative humidity, and temperature-humidity index and UST were observed during this study. For hand milking, the optimal UST threshold was 32.6°C; for SCM, Se = 0.53, Sp = 0.89, AUC = 0.71, κ = 0.4; for MG, Se = 0.83, Sp = 0.93, AUC = 0.88, κ = 0.77; and for IMI, Se = 0.82, Sp = 0.92, AUC = 0.87, κ = 0.74. The machine milking threshold for SCM resulted in Se = 0.42, Sp = 0.97, AUC = 0.70, κ = 0.47; for MG, Se = 0.82, Sp = 0.89, AUC = 0.85, κ = 0.60; and for IMI, Se = 0.82, Sp = 0.98, AUC = 0.90, κ = 0.79. These findings suggest that UST determined by IRT is higher in machine-milked cows and in quarters with MG and IMI than in healthy quarters; therefore, UST by IRT is a reliable, clinically useful method for MG and IMI diagnosis.     

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.     

Infection dynamics across the dry period using Dairy Herd Improvement somatic cell count data and its effect on cow performance in the subsequent lactation

In this study, we studied infection dynamics across the dry period using test-day somatic cell count (SCC) data from 739 Holstein cows from 33 randomly selected commercial dairy herds in Flanders, all of which applied blanket dry-cow therapy at dry-off. First, we determined infection dynamics, combining the last test-day SCC before dry-off and the first test-day SCC after calving. Next, we determined the effect of dry period infection dynamics, adjusting for the level of the second test-day SCC after calving, on the evolution of test-day SCC and milk yield (MY) and on clinical mastitis and culling hazard in the subsequent lactation. Using an SCC threshold of 200,000 cells/mL, 12.6% of the cows considered healthy before dry-off acquired a new intramammary infection (IMI) across the dry period, whereas 66.9% of the cows considered infected before dry-off cured from IMI. Infection dynamics across the dry period significantly affect a cow's SCC, clinical mastitis risk, and culling hazard in the subsequent lactation. Cows with a new IMI, a cured IMI, or a chronic IMI across the dry period had higher test-day SCC than healthy cows, and their test-day SCC evolved differently over time. This was not the case for test-day milk yield, for which no association with infection dynamics was detected. Furthermore, cows with a second test-day SCC <200,000 cells/mL had a lower test-day SCC in the remainder of the lactation than cows with a second test-day SCC ≥200,000 cells/mL, but this association was modified by infection dynamics across the dry period. The lowest test-day SCC in the remainder of the lactation was observed for cows that remained healthy across the dry period combined with a low (<200,000 cells/mL) second test-day SCC. Cows that cured from an IMI present at dry-off and cows with a chronic IMI across the dry period were more likely to develop clinical mastitis (hazard ratio = 2.22 and 2.89; 95% confidence interval = 1.45–3.43 and 1.60–5.20, respectively), and chronic IMI cows were more likely to be culled (hazard ratio = 3.68; 95% confidence interval = 1.64–8.20) in the subsequent lactation compared with healthy cows. This was not true for cows that became infected across the dry period. This study underlines the importance of good udder health management during lactation to prevent IMI at dry-off rather than curing infected cows during the dry period to ensure optimal udder health in the subsequent lactation.     

Diagnosing intramammary infections: evaluation of composite milk samples to detect intramammary infections

Composite milk samples, in which milk from all 4 bovine quarters is collected in a single vial, are widely used in many developed dairy industries for detection of intramammary infections (IMI). These samples are more economical for use in culturing protocols than individual quarter samples, and may be useful when considering management options at the cow and herd level. The dilution effect may be problematic, however, resulting in lower sensitivity (Se) in IMI detection on composite samples. Relative Se and specificity (Sp) in composite samples have previously been described for some major pathogens, but because the causative organism for IMI is initially unknown, it is beneficial to investigate the reliability of composite samples for detection of all types of mastitis-causing bacteria. The Canadian Bovine Mastitis Research Network has a large data collection platform—the National Cohort of Dairy Farms—containing a vast amount of data on mastitis in Canada. These data have been used to further investigate the Se and Sp of composite samples in detecting IMI caused by specific mastitis pathogens. Milk samplings of selected cows before dry-off, after calving, and during lactation (n = 48,835 samples) were employed to this end. Composite samples showed moderately high Se for Staphylococcus aureus (77.1%, 95% CI = 73.3-80.5) and Streptococcus dysgalactiae (73.4%, 95% CI = 60.9-83.7), with moderate Se for Streptococcus uberis (62.1%, 95% CI = 49.3-73.8) and coagulase-negative staphylococci (59.8%, 95% CI = 58.4-61.2). Sensitivities always increased as the number of affected quarters increased. Composite samples also showed high Sp (>97%) for most organisms. Factors such as lactation number and stage of lactation were evaluated for their influence on the Se and Sp of composite sampling, but were only found to be significant for coagulase-negative staphylococci. Predictive values using the herd prevalences found across Canada were calculated and can be useful in field scenarios when composite sampling is employed to assist mastitis management. When used to detect newly occurring IMI in pairs of samples taken before dry-off, post-calving, and also prior to and subsequent to the dry period, composite samples were shown to have lower Se but similar Sp for all pathogens investigated. Composite samples can be used to detect IMI and new IMI in dairy cows, but the Se and Sp of the procedure should be taken into account.     

Sensitivity and specificity of somatic cell count and California Mastitis Test for identifying intramammary infection in early lactation

Associations between values for the somatic cell count (SCC) or the California Mastitis Test (CMT) and intramammary infection (IMI) were studied in 131 dairy cows from three herds during the first 10 d post-calving. Intramammary infection was defined as the presence of one or two bacterial species in one or both quarter milk samples taken within 12 h of calving and at d 3 postcalving. Quarter milk samples identified IMI in 36% of glands. Values for SCC declined at a significantly faster rate over the first 10 d postcalving in non-infected quarters than in infected quarters. The usefulness of quarter milk SCC and CMT for screening was evaluated by calculating the sensitivity and specificity for various threshold values and days postcalving. A SCC threshold of 100,000 cells/ml for quarter samples evaluated on d 5 postcalving had the maximal sensitivity and specificity for detecting IMI. Evaluation of the CMT samples taken on d 3 postcalving using a threshold reaction of greater than zero had the highest sensitivity and specificity for detecting IMI. With this CMT sampling scheme, the sensitivities for detecting IMI with any pathogen, IMI with a major pathogen, and IMI with a minor pathogen were 56.7, 66.7, and 49.5, respectively. The CMT could have a useful role in dairy herd monitoring programs as a screening test to detect fresh cows with IMI caused by major pathogens.     

Effects of bovine casein hydrolysate as a dry cow therapy on prevention and cure of bovine intramammary infection,milk production,and somatic cell count in the subsequent lactation

The primary objectives were to investigate the efficacy of bovine casein hydrolysate (bCNH) as a dry cow therapy at (1) preventing new intramammary infection (IMI) postpartum of all bacteria and coagulase-negative staphylococci (CNS), and (2) curing existing subclinical infections, mainly of CNS. The secondary objective was to measure the effects of bCNH on milk yield, composition, and somatic cell count (SCC) during the lactation postcalving. The trial was conducted as a randomized, blinded controlled experiment. Israeli Holstein dairy cows (n = 170) in first or higher lactations were recruited from 4 large commercial dairy herds. Cows were enrolled following clinical examination and bacteriological sampling of each quarter, which was the experimental unit. Random allocation was implemented at the cow level. All quarters of 100 cows were treated with 1 dose of bCNH (60 mg diluted in 20 mL of sterile solution) and those of 70 control cows were treated with saline solution. Clinical assessment of each cow's general appearance, teat-end leakage, and teat morphology was performed for 0, 1, 2, 3, 7, and 14 d after treatment, together with follow-up clinical observation and clinical examination of udder quarters. Quarter aseptic milk samples were obtained for bacteriological culture 48 h pretreatment, at time of treatment, and 3 and 5 d postcalving. Multivariable analyses were conducted to study the effects of bCNH on cure and prevention of IMI, adjusting for parity, farm, average of daily milk yield for 305 d, and average of monthly SCC values for 305 d of previous lactation. The odds of preventing IMI in cows treated with bCNH at dry-off were 2.15 times higher [95% confidence interval (CI): 1.15 to 4.00] than in cows treated with saline. Prevention was mostly of CNS. The odds of preventing CNS in cows treated with bCNH at dry-off were 2.20 times higher (95% CI: 1.58 to 3.07) than in control cows. The odds of curing IMI caused by CNS in cows treated with bCNH at dry-off were 4.80 times higher (95% CI: 0.75 to 30.75) than in saline-treated cows. Log SCC, adjusted to that of the previous lactation, was lower in the bCNH group compared with controls for 305 d in milk postcalving. The average milk yield per day for 305 d, adjusted to average daily milk yield of previous lactation, was higher by 2.1 kg in the bCNH group compared with controls (95% CI: 1.21 to 3.20). Clinical assessment of udders and cows posttreatment showed no negative effects of bCNH. Incidence of stillbirth, clinical mastitis, retained placenta, endometritis (5 to 12 d postcalving), ketosis, abortions, and reproduction did not differ between the 2 groups. Results suggest that a single intramammary administration of bCNH at dry-off effectively increases milk yield and lowers SCC, prevents new IMI during the dry period, and may be a beneficial alternative for curing existing IMI at dry-off, mainly by CNS.     

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

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

Bacteriological outcomes following random allocation to quarter-level selection based on California Mastitis Test score or cow-level allocation based on somatic cell count for dry cow therapy

Intramammary infusion of antimicrobials at the end of lactation (dry cow therapy) has been a cornerstone of mastitis management for many years. However, as only a proportion of cows are infected at this time, treating only those cows likely to be infected is an important strategy to reduce antimicrobial usage and minimize risk of emergence of antimicrobial resistance. Such an approach requires the ability to discriminate between cows and quarters likely to be infected and uninfected. This study compared assignment of cows or quarters to antimicrobial treatment at the end of lactation based on cow composite somatic cell count (SCC; i.e., all quarters of cows with a maximum SCC across lactation >200,000 cells/mL received an antimicrobial; n = 891 cows, SCC-group) or assignment to quarter-level treatment based on a quarter level California Mastitis Test (CMT) score ≥ trace (n = 884 cows; CMT-group) performed immediately before drying off. All quarters of all cows also received an infusion of a bismuth-based internal teat sealant. Milk samples were collected for microbiology following the last milking, and again within 4 d of calving. Clinical mastitis records from dry off to 30 d into the subsequent lactation were collected. Multilevel, multivariable models were used to assess the effect of assignment to antimicrobial treatment. At drying off, a total of 575 (8.8%) and 147 (2.3%) of the 6,528 quarters had a minor, and a major intramammary infection (IMI), respectively. At drying off, 2089/3270 (63.9%) and 883/3311 (26.7%) of quarters were treated with dry cow therapy in the CMT and SCC-groups, respectively. Apparent bacteriological cure proportion for any IMI was higher in quarters assigned to the CMT than the SCC-group (349/368 (0.95, 95% CI 0.92–0.97) versus 313/346 (0.90, 95% CI 0.87–0.93)). New IMI proportion was lower among quarters assigned to the CMT than SCC-group [101/3,212 (0.032, 95% CI 0.025–0.038) versus 142/3,232 (0.044, 95% CI 0.036–0.051)]. The prevalence of any IMI postcalving was lower in quarters assigned to the CMT than SCC-group [119/3,243 (0.037, 95% CI: 0.030–0.044) versus 173/3,265 (0.054, 95% CI: 0.045–0.062)]. There was no difference in incidence of clinical mastitis between treatment groups. The total mass of antimicrobials used was 63% higher in the CMT-group than in the SCC-group (3.47 versus 2.12 mg/kg of liveweight). Selection of quarters for antimicrobial treatment at the end of lactation based on CMT resulted in greater proportion undergoing bacteriological cure, reduced risk of any new IMI and reduced post calving prevalence of any IMI compared with selection of cows based on SCC. However, CMT-based selection resulted in higher antimicrobial use compared with SCC-based selection, and further research is required to analyze the cost benefit and impact on risk of antimicrobial resistance of these 2 strategies.     

Evaluation of an automated milk leukocyte differential test and the California Mastitis Test for detecting intramammary infection in early- and late-lactation quarters and cows

Study objectives were to (1) describe the diagnostic test characteristics of an automated milk leukocyte differential (MLD) test and the California Mastitis Test (CMT) to identify intramammary infection (IMI) in early- (EL) and late-lactation (LL) quarters and cows when using 3 different approaches to define IMI from milk culture, and (2) describe the repeatability of MLD test results at both the quarter and cow level. Eighty-six EL and 90 LL Holstein cows were sampled from 3 Midwest herds. Quarter milk samples were collected for a cow-side CMT test, milk culture, and MLD testing. Quarter IMI status was defined by 3 methods: culture of a single milk sample, culture of duplicate samples with parallel interpretation, and culture of duplicate samples with serial interpretation. The MLD testing was completed in duplicate within 8 h of sample collection; MLD results (positive/negative) were reported at each possible threshold setting (1–18 for EL; 1–12 for LL) and CMT results (positive/negative) were reported at each possible cut-points (trace, ≥1, ≥2, or 3). We created 2 × 2 tables to compare MLD and CMT results to milk culture, at both the quarter and cow level, when using each of 3 different definitions of IMI as the referent test. Paired MLD test results were compared with evaluate repeatability. The MLD test showed excellent repeatability. The choice of definition of IMI from milk culture had minor effects on estimates of MLD and CMT test characteristics. For EL samples, when interpreting MLD and CMT results at the quarter level, and regardless of the referent test used, both tests had low sensitivity (MLD = 11.7–39.1%; CMT = 0–52.2%) but good to very good specificity (MLD = 82.1–95.2%; CMT = 68.1–100%), depending on the cut-point used. Sensitivity improved slightly if diagnosis was interpreted at the cow level (MLD = 25.6–56.4%; CMT = 0–72.2%), though specificity generally declined (MLD = 61.8–100%; CMT = 25.0–100%) depending on the cut-point used. For LL samples, when interpreted at the quarter level, both tests had variable sensitivity (MLD = 46.6–84.8%; CMT = 9.6–72.7%) and variable specificity (MLD = 59.2–79.8%; CMT = 52.5–97.3%), depending on the cut-point used. Test sensitivity improved if interpreted at the cow level (MLD = 59.6–86.4%; CMT = 19.1–86.4%), though specificity declined (MLD = 32.4–56.8%; CMT = 14.3–92.3%). Producers considering adopting either test for LL or EL screening programs will need to carefully consider the goals and priorities of the program (e.g., whether to prioritize test sensitivity or specificity) when deciding on the level of interpretation (quarter or cow) and when selecting the optimal cut-point for interpreting test results. Additional validation studies and large randomized field studies will be needed to evaluate the effect of adopting either test in selective dry cow therapy or fresh cow screening programs on udder health, antibiotic use, and economics.     

Evaluation of diagnostic procedures for subclinical mastitis in meat-producing sheep

Samples of foremilk were collected from 261 clinically normal glands of 150 ewes, and tested using the California mastitis test (CMT). Further samples were collected from 195 of these glands for determination of automated somatic cell counts (SCC), and from 60 of these glands for bacteriological assessment. The sensitivity and specificity of CMT for detecting samples with SCC above different threshold levels and for CMT and SCC in determining bacteriological status were evaluated using two-graph receiver operating characteristics (TG-ROC). Milk samples were obtained subsequently from ten CMT positive, and five CMT negative first- and second-lactation ewes. Samples were cultured using a variety of media, incubation temperatures and atmospheric conditions, immediately after collection, and 1 week after storage at 4 degrees C and -21 degrees C. Results suggested that CMT is best used as a diagnostic test for ovine subclinical mastitis (SCM) with a cut-off of 3 (distinct gel formation), and that automated SCC thresholds of > 1200 x 10(3) cells/ml are appropriate, especially where low prevalences are expected (e.g. < 5%). Additionally, this study showed that routine bacteriological methods were appropriate for isolation of most species of pathogen responsible for ovine SCM, but storage of samples prior to culture, either at 4 degrees C or -21 degrees C, was detrimental to the isolation of several of these organisms.     

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.     

Evaluation of somatic cell count thresholds to detect subclinical mastitis in Gyr cows

The objectives of this study were (1) to determine the sensitivity (Se) and specificity (Sp) of somatic cell count (SCC) thresholds to identify subclinical mastitis in Gyr cows caused by major and minor pathogens; (2) to study the effects of month of sampling, rear or front mammary quarters, herd, intramammary infection (IMI), and bacterial species on SCC at quarter level; and (3) to describe the prevalence of IMI in Gyr cows in commercial dairy herds. In total, 221 lactating Gyr cows from 3 commercial dairy farms were selected. Milk samples were collected from individual quarters once a month for 1 yr from all lactating cows for SCC and bacteriological analysis. Mammary quarters were considered the experimental units and the SCC results were log₁₀-transformed. Four SCC thresholds (100, 200, 300 and 400 × 10³ cells/mL) were used to determine Se and Sp to identify infected mammary quarters. The overall prevalence of IMI in quarter milk samples of Gyr cows was 49.8%, and the prevalence of minor pathogens was higher (31.9%) than that of major pathogens (17.8%). Quarter samples with microbial isolation presented higher SCC compared with negative samples. Sensitivity and Sp of selected SCC thresholds varied according to the group of pathogen (major and minor) involved in the IMI definition. Sensitivity increased and Sp decreased when mammary quarters with only major pathogens isolation were considered positive. The use of a single SCC analysis to classify quarters as uninfected or infected in Gyr cows may not be a useful test for this breed because Se and Sp of SCC at the studied thresholds were low. The occurrence of IMI and the bacterial species are the main factors responsible for SCC variation in mammary quarters of Gyr cows. Milk samples with major pathogens isolation elicited higher SCC than those with minor pathogens.     

Milk selenium concentration and its association with udder health in Atlantic Canadian dairy herds

Soils and plants in Atlantic Canadian provinces are known to contain low concentrations of selenium (Se). Earlier studies have indicated that dairy producers in Atlantic Canada are providing insufficient supplementary Se in the ration to meet the Se requirements of dairy cattle, as measured by herd-level milk Se concentration. The objective of this study was to evaluate the association between milk Se concentration and somatic cell count (SCC) and the risk of new intramammary infection (IMI) in the dry period, in Atlantic Canadian dairy cows. Eighteen dairy farms participating in the Canadian Bovine Mastitis Research Network cohort study were selected as a convenience sample. On each farm 15 cows to be dried off were selected. Quarter milk samples were collected at 4 and 2 wk before drying-off, within 24 h after calving, and at 7 d after calving to evaluate IMI status. Composite milk samples were analyzed for SCC and Se concentration. Mean milk Se concentration was marginal in 14% of the cows that were on pasture during the grazing season. Milk Se concentration was not associated with the overall odds of new IMI in the dry period; however, the odds of having a new Streptococcus spp. and other gram-positive pathogen IMI in the dry period increased with increasing milk Se concentration. Somatic cell count increased with milk Se concentration, even after adjusting for IMI status. The dairy population in our study had higher ranges for milk Se concentration, whereas ranges for prevalence of IMI, and SCC were lower, compared with those in studies where a negative relationship between Se status and udder health was first noted. Therefore, under the current management conditions, milk Se concentration did not appear to be a principal determinant of udder health.     

Suitability of milk lactate dehydrogenase and serum albumin for pathogen-specific mastitis detection in automatic milking systems

In response to intramammary infection (IMI), blood-derived leukocytes are transferred into milk, which can be measured as an increase of somatic cell count (SCC). Additionally, pathogen-dependent IgG increases in milk following infection. The IgG transfer into milk is associated with the opening of the blood-milk barrier, which is much more pronounced during gram-negative than gram-positive IMI. Thus, milk IgG concentration may help to predict the pathogen type causing IMI. Likewise, lactate dehydrogenase (LDH) and serum albumin (SA) cross the blood-milk barrier with IgG if its integrity is reduced. Because exact IgG analysis is complicated and difficult to automate, LDH activity and SA concentration aid as markers to predict the IgG transfer into milk in automatic milking systems (AMS). This study was conducted to test the hypothesis that LDH and SA in milk correlate with the IgG transfer, and in combination with SCC these factors allow the differentiation between gram-positive and gram-negative IMI or even more precisely the infection-causing pathogen. Further, the expression of these parameters in foremilk before (BME) and after (AME) milk ejection was tested. In the AMS, quarter milk samples (n = 686) from 48 Holstein-Friesian cows were collected manually BME and AME, followed by an aseptic sample for bacteriological culture. Mixed models were used to (1) predict the concentration of IgG transmitted from blood into milk based on LDH and SA; (2) use principal component analysis to evaluate joint patterns of SCC (cells/mL), IgG (mg/mL), LDH (U/L), and SA (mg/mL) and use the principal component scores to compare gram-positive, gram-negative, and control IMI types and BME versus AME samples; and (3) predict gram-positive and gram-negative IMI by inclusion of combined SCC-LDH and SCC-SA as predictors in the model. Overall, the SA and LDH had similar ability to predict IgG transmission from blood into milk. Comparing the areas under the curve (AUC) of the receiver operator characteristic curves, the SCC-LDH versus SCC-SA had lower gram-positive (AUC = 0.984 vs. 0.986) but similar gram-negative (AUC = 0.995 vs. 0.998) IMI prediction ability. The SCC, IgG, LDH, and SA were greater in gram-negative than in gram-positive IMI (BME and AME) in early lactation. All measured factors had higher values in milk samples taken BME than AME. In conclusion, LDH and SA could be used as replacement markers to indicate the presence of IgG transfer from blood into milk; in combination with SCC, both SA and LDH are suitable for differentiating IMI type, and BME is better for mastitis detection in AMS.     

Test characteristics of milk amyloid A ELISA,somatic cell count,and bacteriological culture for detection of intramammary pathogens that cause subclinical mastitis

Bovine mastitis is an important disease in the dairy industry, causing economic losses as a result of withheld milk and treatment costs. Several studies have suggested milk amyloid A (MAA) as a promising biomarker in the diagnosis of mastitis. In the absence of a gold standard for diagnosis of subclinical mastitis, we estimated the diagnostic test accuracy of a commercial MAA-ELISA, somatic cell count (SCC), and bacteriological culture using Bayesian latent class modeling. We divided intramammary infections into 2 classes: those caused by major pathogens (e.g., Escherichia coli, Staphylococcus aureus, streptococci, and lacto-/enterococci) and those caused by all pathogens (major pathogens plus Corynebacterium bovis, coagulase-negative staphylococci, Bacillus spp., Streptomyces spp.). We applied the 3 diagnostic tests to all samples. Of 433 composite milk samples included in this study, 275 (63.5%) contained at least 1 colony of any bacterial species; of those, 56 contained major pathogens and 219 contained minor pathogens. The remaining 158 samples (36.5%) were sterile. We determined 2 different thresholds for the MAA-ELISA using Bayesian latent class modeling: 3.9 µg/mL to detect mastitis caused by major pathogens and 1.6 µg/mL to detect mastitis caused by all pathogens. The optimal SCC threshold for identification of subclinical mastitis was 150,000 cells/mL; this threshold led to higher specificity (Sp) than 100,000 cells/mL. Test accuracy for major-pathogen intramammary infections was as follows: SCC, sensitivity (Se) 92.6% and Sp 72.9%; MAA-ELISA, Se 81.4% and Sp 93.4%; bacteriological culture, Se 23.8% and Sp 95.2%. Test accuracy for all-pathogen intramammary infections was as follows: SCC, sensitivity 90.3% and Sp 71.8%; MAA-ELISA, Se 88.0% and Sp 65.2%; bacteriological culture, Se 83.8% and Sp 54.8%. We suggest the use of SCC and MAA-ELISA as a combined screening procedure for situations such as a Staphylococcus aureus control program. With Bayesian latent class analysis, we were able to identify a more differentiated use of the 3 diagnostic tools. The MAA-ELISA is a valuable addition to existing tools for the diagnosis of subclinical mastitis.     

Cross-sectional study of the relationships among bedding materials,bedding bacteria counts,and intramammary infection in late-lactation dairy cows

Objectives of this study were to (1) describe the intramammary infection (IMI) prevalence and pathogen profiles in quarters of cows approaching dry-off in US dairy herds, (2) compare IMI prevalence in quarters of cows exposed to different bedding material types, and (3) identify associations between bedding bacteria count and IMI in cows approaching dry-off. Eighty herds using 1 of 4 common bedding materials (manure solids, organic non-manure, new sand, and recycled sand) were recruited in a multi-site cross-sectional study. 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). Samples of unused and used bedding were also collected. Aerobic culture was used to determine the IMI status of 10,448 quarters and to enumerate counts (log₁₀ cfu/mL) of all bacteria, Staphylococcus spp., Streptococcus spp. and Streptococcus-like organisms (SSLO), coliforms, Klebsiella spp., noncoliform gram-negatives, Bacillus spp., and Prototheca spp. in unused (n = 148) and used (n = 150) bedding. The association between bedding bacteria count and IMI was determined using multivariable logistic regression with mixed effects. Quarter-level prevalence of IMI was 21.1%, which was primarily caused by non-aureus Staphylococcus spp. (11.4%) and SSLO (5.6%). Only modest differences in IMI prevalence were observed between the 4 common bedding material types. Counts of all bacteria in unused bedding was positively associated with odds of IMI caused by any pathogen [ALL-IMI; odds ratio (OR) = 1.08]. A positive association was also observed for counts of SSLO in unused bedding and SSLO-IMI (OR = 1.09). These patterns of association were generally consistent across the 4 common bedding materials. In contrast, the association between counts of all bacteria in used bedding and ALL-IMI varied by bedding type, with positive associations observed in quarters exposed to manure solids (OR = 2.29) and organic non-manure (OR = 1.51) and a negative association in quarters exposed to new sand (OR = 0.47). Findings from this study suggest that quarter-level IMI prevalence in late-lactation cows is low in US dairy herds. Furthermore, bedding material type may not be an important risk factor for IMI in late lactation. Higher levels of bacteria in bedding may increase IMI prevalence at dry-off in general, but this relationship is likely to vary according to bedding material type.     

Efficacy of cabergoline in a double-blind randomized clinical trial on milk leakage reduction at drying-off and new intramammary infections across the dry period and postcalving

The abrupt cessation of milking at dry-off may induce milk leakage, which may increase the risk of new intramammary infections (IMI). This study assessed the efficacy of 1 i.m. injection of 5.6 mg of cabergoline (Velactis, Ceva Santé Animale, Libourne, France) at drying-off on milk leakage after dry-off and new IMI across the dry period and postcalving compared with a placebo (negative control) and an intramammary antibiotic treatment (positive control) under field conditions. The study was a double-blind, randomized, 3-arm, multicenter, clinical trial performed under Good Clinical Practice conditions. Data from 900 dairy cows of various breeds from 63 farms in France, Germany, and Hungary were analyzed. Only quarters with no bacterial growth at drying-off and a cow somatic cell count ≤200,000 cells/mL were included. Quarters infected with major or minor pathogens or cows with high somatic cell count at time of inclusion were excluded. Cows that qualified for the study were visited 7 times in total before and after drying-off and after calving. Presence (yes/no) of milk leakage was recorded on the day after dry-off. A new infected quarter (new IMI) was defined as one with a major pathogen present in any one of the 2 postcalving samples. Two mixed logistic regression models were fitted to the data to evaluate the efficacy of cabergoline in the reduction of milk leakage and new IMI. One i.m. injection of cabergoline at drying-off significantly reduced the incidence of milk leakage the day after dry-off compared with both placebo and antibiotic treatment. Cabergoline-treated cows significantly reduced the risk of new IMI by major pathogens across the dry period and postcalving by 21% when compared with placebo cows (20.5 vs. 26.0%, respectively). However, when milk leakage was added to the model, the significance of cabergoline was reduced. We interpreted this to show that milk leakage is an intervening variable between treatment with cabergoline and lower risk of new IMI. The antibiotic treatment significantly decreased the odds of new IMI compared with both cabergoline and placebo. However, because several countries are currently disallowing the preventive use of antibiotics at dry-off in noninfected quarters, the dry-off facilitator cabergoline may therefore be of particular value to reduce the risk of new IMI across the dry period.     

Evaluation of 4 predictive algorithms for intramammary infection status in late-lactation cows

The objective of this observational study was to compare 4 cow-level algorithms to predict cow-level intramammary infection (IMI) status (culture and MALDI-TOF) in late-lactation US dairy cows using standard measures of test performance. Secondary objectives were to estimate the likely effect of each algorithm, if used to guide selective dry cow therapy (SDCT), on dry cow antibiotic use in US dairy herds, and to investigate the importance of including clinical mastitis criteria in algorithm-guided SDCT. Cows (n = 1,594) from 56 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 farm 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, which was considered the reference test in this study. Clinical mastitis records and Dairy Herd Improvement Association test-day somatic cell count data were extracted from herd records and used to perform cow-level risk assessments (low vs. high risk) using 4 algorithms that have been proposed for SDCT in New Zealand, the Netherlands, United Kingdom, and the United States. Agreement between aerobic culture (reference test; IMI vs. no-IMI) and algorithm status (high vs. low risk) was described using Cohen's kappa, test sensitivity, specificity, negative predictive value, and positive predictive value. The proportion of cows classified as high risk among the 4 algorithms ranged from 0.31 to 0.63, indicating that these approaches to SDCT could reduce antibiotic use at dry-off by 37 to 69% in the average US herd. All algorithms had poor agreement with IMI status, with kappa values ranging from 0.05 to 0.13. Sensitivity varied by pathogen, with higher values observed when detecting IMI caused by Streptococcus uberis, Streptococcus dysgalactiae, Staphylococcus aureus, and Lactococcus lactis. Negative predictive values were high for major pathogens among all algorithms (≥0.87), which may explain why algorithm-guided SDCT programs have been successfully implemented in field trials, despite poor agreement with overall IMI status. Removal of clinical mastitis criteria for each algorithm had little effect on the algorithm classification of cows, indicating that algorithms based on SCC alone may have similar performance to those based on SCC and clinical mastitis criteria. We recommend that producers implementing algorithm-guided SDCT use algorithm criteria that matches their relative aspirations for reducing antibiotic use (high specificity, positive predictive value) or minimizing untreated IMI at dry-off (high sensitivity, negative predictive value).     

Use of an enzyme-linked immunosorbent assay to monitor the control of Staphylococcus aureus mastitis.

Conventional culture methods were used to evaluate the ability of an ELISA to identify Staphylococcus aureus IMI. The test was 96% accurate; sensitivity was 90%, and specificity was 97%. The test was used to screen preserved milk samples rapidly in 10 cooperator herds. Prevalence of IMI was greater than 10% in 6 herds at the first test. Average prevalence of cows scoring +2 (suspect) and +3 (positive) was 12.6%. Prevalence declined during the 12-mo study. Incidence of new IMI decreased from 7.9% at 6 mo to 3.6% at 12 mo. Rinsing teat cup liners with a 25-ppm iodophor or 100-ppm chlorine solution reduced the presence of S. aureus on the milking machine liners by 97%. Elevated scores were correlated with increases in lactation number. Milk antibody concentrations changed quadratically with increasing SCC. The SCC increased as milk antibody concentration increased. In 38 dairy herds, bulk tank antibody tests reflected herd prevalence of S. aureus infection. The average prevalence was 15.0% in 87 herds in which all lactating cows were tested.