Association of conception rate with pattern and level of somatic cell count elevation relative to time of insemination in dairy cows

The aim was to evaluate the effects of mastitis, determined by the pattern and level of somatic cell count (SCC) around first artificial insemination (AI), on conception rate (CR). Data from 287,192 first AI and milk records covering a 7-yr period were obtained from the Israeli Herd Book. Analyses examined the association of probability of conception with SCC elevation relative to timing of AI, using generalized linear mixed models. A SCC threshold of 150,000 cells/mL of milk was set to distinguish between uninfected cows and cows with mastitis. Accordingly, cows with high SCC before and low SCC after AI were designated cured, those with low SCC before and high SCC after AI were designated newly infected, and cows with high SCC before and after AI were designated chronic (likely subclinical) mastitic cows. Compared with uninfected cows, the cured, newly infected, and chronic subgroups showed reduced CR (39.4 ± 0.1, 36.6 ± 0.2, 32.9 ± 0.3, and 31.5 ± 0.2, respectively). In the chronic, subclinical group, probability of conception was lowered by 14.5% in the mild and moderately elevated SCC subgroups and by 20.5% in cows with high SCC elevation compared with the uninfected group (CR of 29.7 vs. 39.4%, respectively). A single high elevation of SCC (>10⁶ cells/mL on only 1 milk test day) lowered the probability of conception by 23.6% when it occurred during the 10 d immediately before AI, but not when it occurred earlier. For 30 d after AI, probability of conception was lowered by about 23%, as reflected in a CR of about 27% compared with the uninfected group. Probability of conception was lowered in cows with uterine and foot health problems (33.9%), in multiparous cows (34.1%), and in cows in the summer (29.1%), but no interactions with mastitis were detected. Results indicate that SCC elevation around AI, typical for subclinical mastitis, was associated with a significant reduction in probability of conception, and that even mild SCC elevation reduced CR. Severe elevation of SCC before AI, typical for clinical intramammary infection, reduced the probability of conception.     

Detection of mastitis pathogens by analysis of volatile bacterial metabolites

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

Effects of pathogen-specific clinical mastitis on probability of conception in Holstein dairy cows

The objective of this study was to estimate the effects of pathogen-specific clinical mastitis (CM), occurring in different weekly intervals before or after artificial insemination (AI), on the probability of conception in Holstein cows. Clinical mastitis occurring in weekly intervals from 6 wk before until 6 wk after AI was modeled. The first 4 AI in a cow’s lactation were included. The following categories of pathogens were studied: Streptococcus spp. (comprising Streptococcus dysgalactiae, Streptococcus uberis, and other Streptococcus spp.); Staphylococcus aureus; coagulase-negative staphylococci (CNS); Escherichia coli; Klebsiella spp.; cases with CM signs but no bacterial growth (above the level that can be detected from our microbiological procedures) observed in the culture sample and cases with contamination (≥3 pathogens in the sample); and other pathogens [including Citrobacter, yeasts, Trueperella pyogenes, gram-negative bacilli (i.e., gram-negative organisms other than E. coli, Klebsiella spp., Enterobacter, and Citrobacter), Corynebacterium bovis, Corynebacterium spp., Pasteurella, Enterococcus, Pseudomonas, Mycoplasma, Prototheca, and others]. Other factors included in the model were parity (1, 2, 3, 4 and higher), season of AI (winter, spring, summer, autumn), day in lactation of first AI, farm, and other non-CM diseases (retained placenta, metritis, ketosis, displaced abomasum). Data from 90,271 AI in 39,361 lactations in 20,328 cows collected from 2003/2004 to 2011 from 5 New York State dairy farms were analyzed in a generalized linear mixed model with a Poisson distribution. The largest reductions in probability of conception were associated with CM occurring in the week before AI or in the 2 wk following AI. Escherichia coli and Klebsiella spp. had the greatest adverse effects on probability of conception. The probability of conception for a cow with any combination of characteristics may be calculated based on the parameter estimates. These findings may be helpful to farmers in assessing reproduction in their dairy cows for more effective cow management.     

Acute-phase inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) levels in serum and milk of cows with subclinical mastitis caused by Streptococcus species and coagulase-negative Staphylococcus species

The aim of the study was to investigate the concentrations of acute-phase inter-α-trypsin inhibitor heavy chain 4 (ITIH4) in serum and milk of cows with subclinical mastitis caused by Streptococcus spp. (STR) and coagulase-negative Staphylococcus spp. (CNS) and healthy cows. The blood and milk samples were obtained from 60 mid-lactation, multiparous Holstein-Friesian cows from 7 herds in the Lublin region of Poland. In the milk samples from 40 cows with subclinical mastitis, Streptococcus spp. and CNS were isolated. The ITIH4 was significantly higher in serum of cows with subclinical mastitis caused both by STR and CNS compared with healthy cows. One hundred percent of animals infected with Streptococcus spp. and 89% of animals infected with Staphylococcus spp. showed ITIH4 concentration in sera higher than 0.5 mg/mL. The concentration of ITIH4 in milk also was significantly higher in cows with subclinical mastitis caused by Streptococcus spp. and Staphylococcus spp. compared with the control group. Seventy percent of cows infected by STR and CNS showed ITIH4 concentration in milk higher than 2.5 μg/mL. Milk ITIH4 concentration higher than 5 µg/mL was found in 55% of animals infected with Streptococcus spp. and in 40% of animals infected with Staphylococcus spp. No statistically significant differences were observed in ITIH4 concentrations both in serum and in milk between the studied unhealthy animal groups. These results suggest that ITIH4 may be used in the future as a novel diagnostic marker in serum and in milk of subclinical mastitis in cows.     

Milk production change following clinical mastitis and reproductive performance compared among J5 vaccinated and control dairy cattle

Naturally occurring cases of bovine clinical mastitis (CM) were studied among J5 vaccinates and controls on 3 commercial dairy farms. Milk production change and reproductive performance following CM were compared between the 2 groups. Among 306 controls and 251 vaccinates, there were 221 new cases of CM affecting 120 cows; 437 lactations never had a case of CM. Environmental pathogens made up 90% (159/176) of etiologic agents isolated. Change in daily milk production following CM was associated with J5 vaccination, days in milk (DIM) at onset of CM, and herd effect as well as each 2-way interaction between the 3 factors. The adjusted daily milk for 21 d following CM was 7.6 kg greater among J5 vaccinates than controls; however, this protective effect of vaccination waned with increasing DIM at onset of CM. A mixed linear model with autoregressive order 1 [AR(1)] correlation structure estimated the daily milk production of any cow (whether or not she had CM) on a given DIM. Cows with CM caused by nonagalactiae streptococci, Staphylococcus aureus, Escherichia coli, or Klebsiella lost significant daily milk production for the entire lactation relative to nonmastitic cows. Another mixed linear model for only coliform CM cases (E. coli, Klebsiella, and Enterobacter) within the first 50 DIM showed milk loss for 21 d following coliform CM to be significantly less for J5 vaccinates than for controls, by 6 to 15 kg per day. Cows were significantly less likely to become pregnant if they had CM caused by E. coli (42% pregnant) or Streptococcus spp. (38% pregnant), whereas 78% (342/437) of cows with no mastitis conceived. Days open (number of days from calving until pregnancy) averaged 131 d for cows with no CM and 162 d for cows that had at least one case of CM. Days until conception, days until last breeding, days open, times bred, and percentage of cows pregnant by 200 DIM were not changed with J5 vaccination. Nonetheless, an important benefit of the use of J5 bacterin appears to be reduction of the loss of daily milk production following CM, whether all cases or only those caused by coliform bacteria were considered.     

Bayesian estimation of sensitivity and specificity of a rapid mastitis test kit,bacterial culture,and PCR for detection of Staphylococcus aureus,Streptococcus species,and coliforms in bovine milk samples

Our objectives were to evaluate the diagnostic accuracy of a rapid and novel immunochromatography-based mastitis kit that includes 3 independent tests to detect coliforms (Escherichia coli or Klebsiella pneumoniae), Streptococcus spp., and Staphylococcus aureus. The kit was developed to facilitate diagnostic-based mastitis treatment. Validation of the kit was based on 154 aseptically collected mastitis samples from 2 clinical herds (clinical population) and 120 milk samples from 3 nonclinical herds (nonclinical population) without clinical cases at the time of enrollment. One herd sampled at different times was common to both populations. A 3-test in 2-population Bayesian latent class model with uniform priors for all test parameters except specificity of culture, which was modeled informatively, was used to estimate sensitivity (Se) and specificity (Sp) of the test kit, culture, and PCR at the cow level. The mastitis test kit's 96.9% Sp for Streptococcus spp. had a low false positive percentage (3.1%), which, together with the kit's rapid turnaround time for results, makes it a suitable initial screening test that producers can use to identify clinical cows to treat based on Streptococcus spp. mastitis in kit-positive results. Due to the 60.4% kit Se, producers should follow up on Streptococcus spp. kit-negative cows using a confirmatory test such as PCR (Sp of 98.4%) or culture (Sp of 99.6%). In contrast, aerobic culture had Se of 76.5% and Sp of 99.6% for Streptococcus spp. Similarly, the Sp of the kit (98.2%) and culture (99.8%) for Staph. aureus were particularly high, and even though the kit's Se (61.0%) was lower than culture (88.4%; posterior probability of difference 98%), the kit could be beneficial before use of a confirmatory test for kit-negative samples due to its ease and rapid turnaround time. Mostly, quantitative real-time (q)PCR outperformed the kit's Se (37.7%) and Sp (92.9%) for coliforms, as well as the kit's Se (60.4%) for Streptococcus spp. However, qPCR may require more technical skills and turnaround time for final results. Use of the on-farm mastitis test kit evaluated in the present study could enhance sustainable antimicrobial drug use by rapidly identifying Streptococcus mastitis for targeted treatment. Furthermore, the kit may be used in a Staph. aureus outbreak where cows can be rapidly screened to identify cases for segregation or culling during an outbreak and kit-negative cows further confirmed by milk culture or qPCR. However, the cost-effectiveness of such an approach has not been investigated.     

Behavioral and patho-physiological response as possible signs of pain in dairy cows during Escherichia coli mastitis: A pilot study

Bovine mastitis is one of the most common diseases in the dairy industry and it is a major welfare problem. Pain during mastitis is generally assessed through behavior but a combination of indicators would increase the chances of detecting pain and assessing its intensity. The aim of this study was to assess behavioral and patho-physiological responses as possible signs of pain experienced by cows after experimental intramammary challenge (mastitis) with Escherichia coli. Six Holstein-Friesian cows received an inoculation of E. coli P4 in one healthy quarter. Evolution of the disease was assessed using bacteriological growth and somatic cell counts (SCC). Cows' response to the challenge was monitored by direct behavioral and clinical observations, data loggers, rumen temperature sensors, and indicators of inflammation, stress, and oxidative status. From all data recorded, the variables that contributed most to the discrimination of mastitis phases were obtained by factorial discriminant analysis. Baseline levels of all indicators corresponded to values before challenge. Specifically, we weighted data relating to lying behavior by the observations at the same hour of the day before challenge to eliminate the circadian rhythm effect. We identified 3 phases that were discriminated by factorial discriminant analysis with good performance. Nine indicators varied according to the phase of the disease: cows' attitude toward their surroundings, tail position, clinical signs, ear position, variation of postural changes, concentrations of haptoglobin and serum amyloid A (SAA), cortisol blood levels, and rumen temperature (as a surrogate for body temperature). In phase 1 (4 to 8 h postinoculation), E. coli proliferated exponentially in milk but inflammation indicators remained at baseline levels. Cows were less attentive toward their surroundings (median score, 0.63), and postural changes (lying/standing) were less frequent (0.75 times from baseline). In phase 2 (12 to 24 h postinoculation), bacterial concentrations peaked around 12 h and then began to decrease concomitantly with a sharp SCC increase. Cows were less attentive toward their surroundings (score, 0.54), had high plasma cortisol (31.3 ng/mL) and SAA (100.3 µg/mL) concentrations, and rumen temperature was increased (40.3°C). In phase 3 (32 to 80 h postinoculation), bacterial concentrations decreased concomitantly with high SCC levels. Cows had high levels of haptoglobin (0.57 mg/mL) and SAA (269 µg/mL) but showed no behavioral changes. Dairy cows displayed changes of behavioral, inflammatory, and stress parameters after E. coli mammary inoculation. Our results suggest that cows may have experienced discomfort in the preclinical phase (phase 1) and pain in the acute phase (phase 2) but neither discomfort nor pain in the remission phase (phase 3). Although larger controlled studies are needed to confirm our findings, this knowledge could be useful for early detection of E. coli mastitis and for decision-making regarding the initiation of pain-relief treatment during mastitis in dairy cows. This would improve animal welfare and potentially faster disease remission.     

Prevalence of periparturient diseases and effects on fertility of seasonally calving grazing dairy cows supplemented with concentrates

The objectives were to characterize the prevalence of periparturient diseases and their effects on reproductive performance of dairy cows in seasonal grazing farms. A total of 957 multiparous cows in 2 farms (555 in farm A and 402 in farm B) were evaluated and diseases characterized. At calving, dystocia, twin birth, stillbirth, and retained fetal membranes were recorded and grouped as calving problems. On d 7 ± 3 and 14 ± 3 postpartum, cows were evaluated for metritis and on d 28 ± 3 for clinical endometritis based on scoring of the vaginal discharge. From parturition to 30 d after artificial insemination (AI), prevalence of mastitis, lameness, and digestive and respiratory problems were recorded. For subclinical diseases, diagnosis was based on blood samples collected from 771 cows and analyzed for concentrations of Ca, nonesterified fatty acids (NEFA), and β-hydroxybutyrate. Cows were considered as having elevated NEFA concentration if the concentration was ≥0.70 mM, subclinical ketosis if the β-hydroxybutyrate concentration was ≥0.96 mM, and subclinical hypocalcemia if the Ca concentration was ≤2.14 mM. Ovaries were scanned on d 35 ± 3 and 49 ± 3 postpartum for determination of estrous cyclicity. All cows were enrolled in a timed AI program and inseminated on the first day of the breeding season: on average, 86 d postpartum. Overall, 37.5% (359/957) of the cows presented at least 1 clinical disease and 59.0% (455/771) had at least 1 subclinical health problem. Prevalence of individual diseases was 8.5% for calving problems, 5.3% for metritis, 15.0% for clinical endometritis, 13.4% for subclinical endometritis, 15.3% for mastitis, 2.5% for respiratory problems, 4.0% for digestive problems, 3.2% for lameness, 20.0% for elevated NEFA concentration, 35.4% for subclinical ketosis, and 43.3% for subclinical hypocalcemia. Clinical and subclinical diseases had additive negative effects on reproduction, delaying resumption of estrous cyclicity and reducing pregnancy per AI (P/AI). Occurrence of multiple diseases further reduced reproductive efficiency compared with a single disease. Individually, subclinical hypocalcemia, elevated NEFA concentration, metritis, and respiratory and digestive problems reduced estrous cyclicity by d 49 postpartum. Elevated NEFA concentration, calving problem, metritis, clinical and subclinical endometritis, and digestive problems reduced P/AI on d 65 after AI. Moreover, calving problems and clinical endometritis increased the risk of pregnancy loss between gestation d 30 and 65. Serum concentrations of Ca and NEFA were negatively correlated, and both were associated with prevalence of uterine diseases. In conclusion, periparturient diseases were highly prevalent in seasonally calving grazing dairies and affected cows had delayed resumption of estrous cyclicity, reduced P/AI, and increased risk of pregnancy loss.     

Antimicrobial resistance profiles of 5 common bovine mastitis pathogens in large Chinese dairy herds

The prevalence of antimicrobial resistance (AMR) is increasing in human and animal pathogens, becoming a concern worldwide. However, prevalence and characteristics of AMR of bovine mastitis pathogens in large Chinese dairy herds are still unclear. Therefore, our objective was to determine the AMR profile of bacteria isolated from clinical mastitis in large (>500 cows) Chinese dairy herds. A total of 541 isolates of the 5 most common species, Staphylococcus aureus (n = 103), non-aureus staphylococci (NAS; n = 107), Streptococcus species (n = 101), Klebsiella species (n = 130), and Escherichia coli (n = 100), isolated from bovine clinical mastitis on 45 dairy farms located in 10 provinces of China were included. Presence of AMR was determined by minimum inhibitory concentrations using the microdilution method. Prevalence of multidrug resistance (resistance to >2 antimicrobials) was 27% (148/541). A very wide distribution of minimum inhibitory concentrations was screened in all isolates, including Staph. aureus isolates, which were resistant to penicillin (66%). In addition, NAS (30%) were more resistant than Staph. aureus to oxacillin (84%), penicillin (62%), tetracycline (34%), and clindamycin (33%). Prevalence of resistance to tetracycline was high (59%) in Streptococcus spp. Additionally, prevalence of resistance of both E. coli and Klebsiella spp. was high to amoxicillin/clavulanate potassium (81 and 38%, respectively), followed by tetracycline (only Klebsiella spp. 32%). A high proportion (27%) of isolates were multidrug resistant; the most frequent combinations were clindamycin-cefalexin-tetracycline or enrofloxacin-cefalexin-penicillin patterns for Staph. aureus; enrofloxacin-oxacillin-penicillin-tetracycline patterns for NAS; clindamycin-enrofloxacin-tetracycline patterns for Streptococcus spp.; amoxicillin/clavulanate potassium-ceftiofur-polymyxin B patterns for Klebsiella spp.; and amoxicillin/clavulanate potassium-ceftiofur-polymyxin B patterns for E. coli. Resistance for 4 kinds of antimicrobials highly critical for human medicine, including daptomycin, vancomycin, imipenem, and polymyxin B, ranged from 0 to 24%. In conclusion, prevalence of AMR in mastitis pathogens was high on large Chinese dairy farms, potentially jeopardizing both antimicrobial efficacy and public health. Results of this study highlighted the need for improvements in antimicrobial stewardship and infection control programs in large Chinese dairy farms to reduce emergence of AMR.     

Prevalence and risk factors for multidrug-resistant Escherichia coli isolated from subclinical mastitis in the western Chitwan region of Nepal

Subclinical mastitis (SCM) represents a significant burden and challenge to modern dairy management. Multidrug-resistant Escherichia coli (MDR E. coli) in milk poses a public health threat to humans especially via the consumption of unpasteurized dairy products. This study aimed to determine the occurrence of MDR E. coli in cows and buffalo in the households of the western part of the Chitwan district of Nepal. A total of 243 lactating cows and buffalo were included in this study. Milk samples (n = 972) were screened using the California Mastitis Test (CMT). The E. coli was isolated from milk samples that were positive for CMT using standard bacteriological protocols. A semi-structured questionnaire was administered to farmers to identify the risk factors associated with the occurrence of SCM in cows and buffalo. Of the 243 dairy animals screened, 42.8% (n = 104/243) showed positive CMT results. However, of the 972 quarters sampled, only 19.3% (n = 188/972) were positive for SCM. The prevalence of E. coli in these animals was found to be 16.5% in animals (n = 40/243). However, E. coli was isolated from only 5% (n = 49/972) of the quarters. Of the 49 E. coli isolated, the resistance to ceftriaxone (38.8%, n = 19/49) and ciprofloxacin (37.7%, n = 17/49) were the most prevalent. Animals with a history of mastitis were 3.57 times more likely to have SCM than other animals. Similarly, lactating animals with previous teat abrasions were 3.22 times more likely to develop SCM than animals without teat injuries. As expected, cleaning the barn once in 2 to 3 d was associated with an increased occurrence of SCM in lactating cows. This study reports the occurrence of MDR E. coli in SCM, which poses a public health threat. Creating awareness of milk pasteurization, and food safety practices are necessary among the farmers.     

Short communication: Longitudinal study of quarter-level somatic cell responses after naturally occurring,nonsevere clinical mastitis diagnosed as culture negative,or caused by Escherichia coli or Klebsiella pneumoniae,and randomly assigned to a no-treatment group or to receive intramammary ceftiofur

The objective of this study was to describe weekly quarter-level somatic cell count (QSCC) after occurrence of nonsevere clinical mastitis (CM) that was diagnosed as culture negative, or caused by Escherichia coli or Klebsiella pneumoniae. All cases occurred in cows enrolled in negatively, controlled randomized clinical trials. We hypothesized that after occurrence of CM, QSCC patterns would vary among etiologies and this effect would not be mitigated by treatment using intramammary (IMM) ceftiofur. Data from two previously published randomized clinical trials performed on 3 Wisconsin dairy farms were used. Only cases confirmed as culture negative (NG) or E. coli or Kleb. pneumoniae (GRAMNEG) were used for analysis. In NG, cows were assigned to no antimicrobial treatment (negative control, n = 44) or 5 d of once daily IMM (n = 41) infusions with an approved product containing ceftiofur hydrochloride. In GRAMNEG, cows were assigned to IMM treatment with the same ceftiofur product for 2 different durations (2 d, n = 36; or 8 d, n = 38) or no antimicrobial treatment (negative control, n = 36). For quarters enrolled in NG, no significant differences were identified for weekly QSCC between quarters in the treated or negative control groups (5.4 log₁₀SCC for both groups). For quarters enrolled in GRAMNEG, no significant differences were identified for QSCC between quarters that received the 2-d (6.2 log₁₀SCC) or 8-d (6.3 log₁₀SCC) IMM treatment or were in the negative control group (6.0 log₁₀SCC). At the pathogen level, regardless of treatment, QSCC varied among pathogens and log₁₀SCC were 5.4 (culture negative), 5.8 (E. coli), and 6.2 (Kleb. pneumoniae). Patterns of QSCC of CM diagnosed as culture negative and E. coli were similar in magnitude and time to resolution of the inflammatory response. In conclusion, as compared to CM diagnosed as culture negative or caused by E. coli, CM caused by Kleb. pneumoniae was associated with poorer outcomes. Regardless of IMM ceftiofur treatment, the immune response of the cow resulted in rapid reduction of SCC of quarters diagnosed as culture negative and quarters with CM caused by E. coli. In contrast, the SCC remained elevated for quarters with CM caused by Kleb. pneumoniae and a greater proportion of those cases remained chronically infected.     

The microbiome of Escherichia coli and culture-negative nonsevere clinical mastitis: Characterization and associations with linear score and milk production

Culture-negative and Escherichia coli cases are uncommonly treated in pathogen-based protocols for nonsevere mastitis. High-throughput 16S rRNA sequencing might reveal the presence of other pathogens and can provide information on microbial diversity. The objective was to explore the milk microbiome at the time of the mastitis event (enrollment) and its association with survival in the herd, milk production, and postevent linear score (LS) for cows with clinical mastitis characterized as negative or E. coli by culture. Fifty E. coli-positive and 35 culture-negative samples from cases were enrolled. No cases were treated with antimicrobials. All E. coli-positive quarters were characterized as transient; microbiological culture of samples taken 15 d postmastitis were negative for this organism. However, a difference in α-diversity (Shannon index) was present between enrollment and follow-up samples (3.8 vs. 5.1). When α-diversity was explored for enrollment E. coli samples, no relationship was observed between the Shannon indices of these samples and postmastitis LS. Alpha-diversity of the enrollment samples was lower for E. coli-positive cows that subsequently had greater losses in milk production. This difference was explained by a greater relative abundance of the family Enterobacteriaceae (67.8 vs. 38.4%) for cows that dropped in production. Analysis of composition of the microbiome identified one phylum, Proteobacteria, that differed between E. coli-positive cows that dropped in production and those that did not. Evaluation of β -diversity found no statistical relationship between postmastitis LS and the microbiome. When evaluating α- and β-diversities and composition of the microbiomes for culture-negative quarters, no associations were found for milk production changes and postmastitis LS. Three cows did not remain in the herd, limiting the ability to analyze survival. The findings suggest that a contributing factor to negative outcomes in E. coli-positive cows is relative abundance of this pathogen, and that no single or collective group of bacterial families is associated with milk production changes or postmastitis LS in culture-negative quarters. Although additional studies should be performed, the absence of associations between outcomes explored and microbial profiles in this study suggests that we are not missing opportunities by not treating nonsevere E. coli or culture-negative mastitis cases.     

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

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

Curli production and genetic relationships among Escherichia coli from cases of bovine mastitis

Curli are adhesive surface structures produced by some Escherichia coli and Salmonella strains that bind host proteins and activate inflammatory mediators. In this study, 61 E. coli isolates from 36 clinical cases of bovine mastitis were characterized using enterobacterial repetitive intergenic consensus-PCR and screened for their ability to produce curli. Effect of curli production on case recovery, based on a return to precase milk yield, was investigated for a subset of 43 isolates from 20 quarters of 19 cows. Thirty-five (57%) of 61 isolates were curli positive. Fifty-eight of the 61 isolates clustered into 2 clonal groups at 52% genetic similarity. Genetically diverse E. coli isolates were simultaneously cultured from individual cases. Twenty-three isolates from 13 cows were clustered in clonal group I, of which 5 cases (38%) were curli positive; 35 isolates from 22 cows were clustered in clonal group II, of which 15 cases (68%) were curli positive. No association was found between genetic similarity and phenotypic curli expression of isolates from cows with clinical E. coli mastitis cases. Phenotypic curli expression in isolates did not affect recovery of cows’ milk yield to premastitis production levels.     

Cell function in the bovine mammary gland: a preliminary study on interdependence of healthy and infected udder quarters

Udder defence mechanisms are not completely explained by current mastitis research. The anatomical construction of the udder implies that infection of one udder quarter does not influence the immune status of neighbouring quarters. To test this hypothesis, we compared the immune reactions of individual udder quarters in response to microbial attacks. In the course of immune reactions, polymorphonuclear leucocytes (PMN) release oxygen radicals, which can be determined by chemiluminescence (CL). Milk from 140 udder quarters of 36 cows was analysed for somatic cell count (SCC), differential cell count, viability and CL activity. Quarters with an SCC < 100,000 cells/ml and free of pathogens were defined as uninfected, all other quarters were categorized as infected. Three groups of cows were classified cytologically: group A (healthy, 11 animals, SCC limit < 100,000 cells/ml); group B (moderate mastitis, 8 cows, SCC > or = 100,000 and < 400,000 cells/ml in at least one quarter); and group C (severe mastitis, 17 cows, SCC > or = 400,000 cells/ml in at least one quarter). Infected and uninfected quarters in groups B and C were analysed separately. Viability of PMN leucocytes was significantly (P=0.0012) lower in group A (72.6%) than in healthy quarters of group C (84.0%). Lowering the SCC limit of healthy quarters to <50,000 cells/ml (group A: all quarters within the udder) revealed striking differences between samples of groups B and C: in addition to varying differential cell counts and viabilities, CL activity of group B<50 (2929 CL units/million PMN) was markedly lower than that of the other groups (5616 in group A<50 and 6445 CL units/million PMN in group C<50). These results allow the conclusion that the infection of one udder quarter influences the cell activity of neighbouring quarters. When the SCC threshold for healthy quarters was reduced to 50,000 cells/ml, greater differences in cell activities were detected between healthy udders and healthy quarters of infected udders.     

Negatively controlled,randomized clinical trial to evaluate intramammary treatment of nonsevere,gram-negative clinical mastitis

The objective of this negatively controlled, randomized clinical trial was to examine clinical outcomes of 2-d or 8-d treatment using an approved intramammary (IMM) product containing ceftiofur hydrochloride compared with no antimicrobial treatment of nonsevere, gram-negative cases of clinical mastitis (CM). Additionally, we contrasted clinical outcomes of cases caused by Escherichia coli (n = 56) or Klebsiella pneumoniae (n = 54). Cases (n = 168) of nonsevere (abnormal milk or abnormal milk and udder) CM were randomly assigned to receive 2 d (n = 56) or 8 d (n = 56) of IMM ceftiofur or assigned to a negative control group (n = 56). At enrollment, quarter milk samples were collected and used for on-farm culture, somatic cell count (SCC), and confirmatory microbiological analysis. Quarter milk samples were collected weekly from 7 to 28 d after enrollment for microbiological and SCC analysis. Clinical outcomes were followed for 90 d or until the end of lactation (follow-up period, FUP). Overall, no significant differences in quarter-level recurrence of CM (32% for negative control, 34% for the 2-d treatment, and 32% for the 8-d treatment), culling (18% for negative control, 12% for 2-d treatment, and 11% for 8-d treatment), voluntary dry-off of affected quarters (20% for negative control, 30% for 2-d treatment, and 27% for 8-d treatment), days until return to normal milk (4.2 days for negative control, 4.8 days for 2-d treatment, 4.5 days for 8-d treatment), weekly quarter-SCC during the FUP (6.1, 6.3, and 6.0 log₁₀SCC for the negative control, 2-d, and 8-d treatments, respectively), or daily milk yield during the FUP (37.1, 36.3, and 37.6 kg/cow per day for the negative control, 2-d, and 8-d treatments, respectively) were observed among experimental groups. Days of discarded milk were greater for cows assigned to 8-d IMM ceftiofur (11.1 d) than for cows assigned to 2-d (6.9 d) or cows assigned to negative control (5.6 d). Bacteriological cure (BC) at 14 and 21 d after enrollment was greater in cows assigned to 8-d (89%) and 2-d (84%) treatment than in cows assigned to negative control (67%), but this outcome was confounded by pathogen. For CM caused by Kleb. pneumoniae, BC was greater for quarters assigned to receive treatment (combined 2-d and 8-d groups; 74% BC) than for quarters assigned to negative control (18%). In contrast, no differences in BC were observed for CM caused by E. coli (97–98%). Culling and voluntary dry-off of affected quarters were significantly greater for cows with quarters affected by Kleb. pneumoniae (22% culled, 39% voluntary dry-off of quarters) than for cows with quarters affected with E. coli (7% culled, 11% voluntary dry-off of quarters). Overall, use of IMM ceftiofur did not result in improvement of most clinical outcomes, but differences between E. coli and Kleb. pneumoniae were evident. In contrast to E. coli, Kleb. pneumoniae caused chronic intramammary infection and induced worse clinical outcomes. Intramammary antibiotic treatment of most mild and moderate cases of CM caused by E. coli is not necessary, but more research is needed to identify which quarters affected by Kleb. pneumoniae may benefit from antimicrobial therapy.     

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

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

Efficacy of a high free iodine barrier teat disinfectant for the prevention of naturally occurring new intramammary infections and clinical mastitis in dairy cows

Using a natural exposure trial design, the goal of our study was to evaluate the clinical efficacy of an iodine teat disinfectant with barrier properties and a high level of free iodine relative to a conventional iodine teat disinfectant with no barrier properties and low levels of free iodine. During the 18 wk of the trial, quarter milk samples were collected every 2 wk from 385 dairy cows from 2 herds. Cows on both farms were assigned in a balanced way according to milk yield, number of lactation, days in milk, somatic cell count (SCC) and microbiology culture pretrial into one of following groups: nonbarrier post milking teat disinfectant (NBAR; n = 195 cows; 747 quarters) or barrier postmilking teat disinfectant (BAR; n = 190 cows; 728 quarters). Afterward, at each scoring date every 2 wk, milk SCC was quantified in samples from all mammary quarters and microbiologic culture was only performed on milk samples with SCC >200,000 cells/mL for multiparous cows and SCC >100,000 cells/mL for primiparous cows. A new intramammary infection (NIMI) was defined when a quarter had milk SCC <200,000 cells/mL for multiparous cows and <100,000 cells/mL for primiparous without microorganism isolation, and in a subsequent sampling visit had milk SCC >200,000 cells/mL for multiparous cows and >100,000 cells/mL for primiparous cows, and positive microorganism isolation. A quarter could have several NIMI, but only 1 case per specific pathogen was considered. The most frequently isolated microorganism group on both farms was Streptococcus spp. (6.25% of total mammary quarters), followed by coagulase-negative staphylococci (3.6%) and Corynebacterium spp. (1.5%). In the present study, an interaction occurred between treatment and week of trial on the incidence risk of NIMI. Quarters disinfected with BAR had 54 and 37% lower odds of NIMI than quarters disinfected with NBAR at 8 and 16 wk of the trial, respectively; whereas at other weeks of the study both products had similar incidence risks of NIMI. Overall, teats disinfected with BAR had 46% lower odds of acquiring a clinical mastitis than those disinfected with NBAR. We concluded that the postmilking teat disinfectant with barrier properties and higher free iodine content reduced the risk of clinical mastitis, although differences in new infections were detected at only weekly time points.     

Using dairy herd improvement records and clinical mastitis history to identify subclinical mastitis infections at dry-off

Interest in selective dry cow therapy (SDCT) has been increasing owing to concerns over development of antimicrobial resistance. Implementation of SDCT, however, requires a quick and cost-effective on-farm method for identifying cows for treatment and cows that can be left without treatment. The objective of the present study was to evaluate the use of clinical mastitis (CM) history and somatic cell counts (SCC) from monthly Dairy Herd Improvement (DHI) records in identification of infected and uninfected cows at dry-off. A total of 647 Holstein cows were classified as uninfected or infected at dry-off based on CM history and varying number of monthly SCC records (with three different SCC cut-offs). Cows were considered uninfected based on the following criteria: (1) SCC <100,000 cells/ml and no CM during the lactation; (2) SCC <200,000 cells/ml and no CM during the lactation; (3) as criterion two, but additionally a cow was also considered uninfected if it experienced a case of CM during the first 3 months of the lactation and the SCC was <100,000 cells/ml for the rest of the lactation; (4) SCC <300,000 cells/ml and no CM during the lactation; otherwise they were considered infected. Infected and uninfected cows at dry-off were most efficiently identified using three months' SCC records with a threshold of 200,000 cells/ml for cows without CM during the lactation and a threshold of 100,000 cells/ml during the rest of lactation for cows with CM during the first 90 days in milk. Moreover, this criterion also most efficiently identified cows infected with major pathogens only at dry-off. The success of the criteria used for identifying infected and uninfected cows will, however, depend on herd characteristics, such as prevalence of infection and type of pathogens present in the herd.     

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.