Microbiota of bovine milk, teat skin, and teat canal: Similarity and variation due to sampling technique and milk fraction.

The aim of this study was to evaluate the effect of sampling technique and milk fraction on bovine milk microbiota data and to compare the microbiota in milk to microbiota on the teat end and in the teat canal. Representative milk samples are highly important for assessment of bacteriological findings and microbiota in milk. Samples were obtained from 5 healthy lactating dairy cows at udder quarter level during 1 milking. Swab samples from the teat end and teat canal, and milk samples collected using different techniques and in different milk fractions were included. Milk was collected by hand stripping and through a teat canal cannula before and after machine milking, through a trans-teat wall needle aspirate after milking, and from udder quarter composite milk. The microbiota of the samples was analyzed with sequencing of the V1-V3 region of the 16S rRNA gene. In addition, somatic cell counts and bacterial cultivability were analyzed in the milk samples. Microbiota data were analyzed using multivariate methods, and differences between samples were tested using analysis of similarity (ANOSIM). Differences between samples were further explored via individual studies of the 10 most abundant genera. The microbiota on the teat end, in the teat canal, and in udder quarter composite milk, collected using a milking machine, differed in composition from the microbiota in milk collected directly from the udder quarter. No differences in milk microbiota composition were detected between hand-stripped milk samples, milk samples taken through a teat canal cannula, or milk samples taken as a trans-teat wall needle aspirate before or after milking. We conclude that for aseptic milk samples collected directly from the lactating udder quarter, sampling technique or milk fraction has minor effect on the microbiota composition.

Mild and severe udder cleft dermatitis-Prevalence and risk factors in Swedish dairy herds.

Udder cleft dermatitis (UCD) is an inflammatory skin condition affecting the anterior parts of the udder of dairy cows. The lesions may present as mild or severe skin lesions and have been associated with mastitis and digital dermatitis. The full etiology and pathogenesis are not understood and no large-scale studies have investigated prevalence and risk factors. Therefore, the main objectives of the study were to investigate the prevalence of mild and severe UCD in Swedish dairy herds and to identify risk factors associated with such lesions. We also wanted to investigate risk factors for all cases of UCD and to determine whether UCD increases the risk for mastitis and culling. A random sample of 100 freestall dairy herds were included in the study, and each herd was visited once. Cows were registered as having no, mild, or severe UCD. Additional cow and herd data were obtained via observations, interviews, and the Swedish Official Milk Recording Scheme. The data were analyzed using logistic regression models to identify risk factors for mild and severe UCD. In total, data from 3,479 cows in 99 herds were analyzed. The prevalence of mild and severe UCD was 19 and 9%, respectively. Lesions were found in 98 of 99 herds but the within-herd prevalence of mild (0-43%) and severe (0-33%) UCD varied notably between herds. Breed (Swedish Red compared with Swedish Holstein), certain udder conformation traits, and higher parity were risk factors associated with increased risk of UCD. In addition, cows with hock lesions and cows in herds with high incidence of culling due to hoof and leg diseases had a higher risk for mild UCD. More days in milk and high milk yield were cow-related risk factors associated with severe UCD. Three housing-related factors (shorter cubicles, mattress as cubicle base, and cubicles installed before 2001 compared with 2001-2005), a high incidence of veterinary-treated clinical mastitis and culling due to udder diseases, and a low incidence of culling of first-parity cows in early lactation were herd-related risk factors associated with increased risk for severe UCD. In addition, cows in herds with a high proportion of heifers older than 17 mo that were not inseminated were associated with lower risk of all UCD. Finally, UCD was not associated with the outcomes milk somatic cell count, veterinary-treated clinical mastitis, or culling in the multivariable analyses. The etiology of UCD is most likely multifactorial, involving udder conformation traits and other cow-related risk factors as well as herd-related risk factors. The high prevalence of severe UCD lesions in Swedish dairy cows emphasizes the need for preventive measures and efficient treatments.

Intramammary infections with different non-aureus staphylococci in dairy cows.

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

Udder infections with Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus uberis at calving in dairy herds with suboptimal udder health.

Udder infections with Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus uberis are common causes of bovine mastitis. To study these pathogens in early lactation, a 12-mo longitudinal, observational study was carried out in 13 herds with suboptimal udder health. The aims of the study were to investigate the occurrence of these pathogens and to identify if presence of the 3 pathogens, and of genotypes within the pathogens, differed with respect to herd, season, and parity. Quarter milk samples, collected at calving and 4 d in milk (DIM), were cultured for the 3 pathogens. Genotyping of staphylococcal and streptococcal isolates was performed using spa typing and pulsed-field gel electrophoresis, respectively. For each of the 3 pathogens, cows with an udder infection at calving or 4 DIM were allocated to 1 of 4 infection types: cleared (pathogen present only at calving), persistent (pathogen present in the same quarter at calving and 4 DIM), new (pathogen present only at 4 DIM), or cleared/new (pathogen present in 1 quarter at calving and in another quarter at 4 DIM). Associations between season or parity and overall occurrence of pathogens or infection types were determined using univariable mixed-effect logistic-regression models and the Fisher's exact test, respectively. The most commonly occurring pathogen was Staph. aureus, followed by Strep. dysgalactiae and Strep. uberis. Persistent infections were the most common infection type among Staph. aureus-infected cows, whereas cleared infections were the most common among Strep. dysgalactiae- and Strep. uberis-positive cows. The proportion of cows with persistent Staph. aureus infections and the proportion of cows having a Strep. uberis infection at calving or 4 DIM were higher in the multiparous cows than in primiparous cows. Infections with Strep. dysgalactiae were less common during the early housing season than during the late housing or pasture seasons, whereas persistent Strep. uberis infections were less common during the pasture season than during the late housing season. The relative occurrence of the 3 pathogens, infection types of each pathogen, and genotype diversity of each pathogen throughout the year or in different seasons and parities varied among the herds, indicating that underlying factors predisposing for udder infections at calving differ between herds. Genotyping of bacterial isolates gave important insight into how such infection patterns differed within and between herds. These findings emphasize the need to choose preventive strategies for each individual herd.

Diagnostic test performance of somatic cell count, lactate dehydrogenase, and N-acetyl-ß-D-glucosaminidase for detecting dairy cows with intramammary infection.

The main objective of this study was to investigate the diagnostic test performance of somatic cell count (SCC), lactate dehydrogenase (LDH), and N-acetyl-ß-D-glucosaminidase (NAGase), analyzed in composite test milking samples, for detecting dairy cows with or without intramammary infection (IMI). A second objective was to investigate whether an adjustment of these udder health indicators according to their associations with different influential factors (i.e., parity, days in milk, and season) improved their test performance. Moreover, we wanted to investigate whether test performance of SCC improved if SCC results from previous adjacent test milkings were included in the model. Such test milking data were not available for LDH or NAGase. In this cross-sectional study, quarter milk samples for bacteriological examination were taken from almost 1,000 cows from 25 dairy herds during 3 consecutive days: the day before test milking, the day of test milking, and the day after test milking. From each cow, a composite test milking sample was analyzed for milk composition, SCC, LDH, and NAGase. Among the cows sampled, 485 were IMI negative and 256 were IMI positive in one or more udder quarters according to the definitions used. The remaining cows had inconclusive IMI status. To assess the test performance of SCC, LDH, and NAGase to identify IMI-negative and IMI-positive cows, univariable generalized estimating equation models were used with the udder health indicator of interest as outcome and IMI status as explanatory variable. From these models, receiver-operator characteristic curves were created and the area under cure (AUC) was calculated. From each model, a cut-off was chosen for calculations of the sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), and accuracy (ACC) for each udder health indicator. The AUC was similar for the adjusted SCC (0.84), nonadjusted SCC (0.83) and geometric mean SCC (0.80-0.81), but much lower for LDH (0.66) and NAGase (0.62). The highest Se, Sp, PPV, NPV, and ACC were obtained using SCC. Adjustment of the udder health indicators for influential factors (e.g., parity) did not improve the test performance markedly, whereas adding information about SCC from previous adjacent test milkings improved the test performance of SCC slightly. In conclusion, of the udder health indicators investigated, SCC had the best overall ability to correctly identify IMI-negative and IMI-positive dairy cows.

CNS species and antimicrobial resistance in clinical and subclinical bovine mastitis.

Coagulase-negative staphylococci (CNS) are often associated with bovine mastitis. Knowledge about the relative importance of specific CNS species in different types of mastitis, and differences in antimicrobial resistance among CNS species is, however, scarce. Therefore, the aims of this study were to compare prevalence and antimicrobial susceptibility of CNS species in clinical and subclinical mastitis using material from two national surveys. Overall, Staphylococcus chromogenes and Staphylococcus epidermidis were the most common CNS species found followed by Staphylococcus simulans and Staphylococcus haemolyticus. S. epidermidis was significantly more prevalent in subclinical than in clinical mastitis, and a similar trend was observed for Staphylococcus saprophyticus, while Staphylococcus hyicus was significantly more common in clinical mastitis. The prevalence of ß-lactamase producing isolates varied markedly between CNS species, and was significantly higher in S. epidermidis and S. haemolyticus (∼ 40%), than in S. simulans and S. chromogenes where none or a few of the isolates produced ß-lactamase. Resistance to more than one antimicrobial substance occurred in 9% and 7% of the clinical and subclinical isolates, respectively. In conclusion, the distribution of CNS species differed between clinical and subclinical mastitis indicating inter-species variation of pathogenicity and epidemiology. Overall, the prevalence of antimicrobial resistance was low, but some variation between CNS species was observed.

Comparison of a commercialized phenotyping system, antimicrobial susceptibility testing, and tuf gene sequence-based genotyping for species-level identification of coagulase-negative staphylococci isolated from cases of bovine mastitis.

In order to evaluate the usefulness of some phenotypic and genotypic methods for species identification of coagulase-negative staphylococci (CNS), isolates were obtained from bovine cases of clinical and sub-clinical mastitis from different geographical areas in Sweden. By using the Staph-Zym test, antimicrobial susceptibility testing, and sequencing of part of the CNS tuf gene and, when needed, part of the 16S rRNA gene we characterized 82 clinical isolates and 24 reference strains of 18 different species of staphylococci. The genotypic methods identified nine different species of CNS among the 82 milk isolates. A comparison with results obtained by tuf gene sequencing showed that Staph-Zym correctly identified CNS reference strains to species level more often than bovine milk CNS isolates (83% and 61%, respectively). In addition, tests supplementary to the Staph-Zym were frequently needed in both groups of isolates (50% of reference strains and 33% of milk isolates) to obtain an identification of the strain. It is notable that Staph-Zym judged two isolates as CNS, although they belonged to other species, could not give a species name in 11% of the bovine CNS isolates, and gave 28% of the isolates an incorrect species name. The present study indicates that the studied phenotypic methods are unreliable for identification of CNS from bovine intra-mammary infections.

Metabolites and immune variables associated with somatic cell counts of primiparous dairy cows.

The main objective of this study was to investigate associations between serum concentrations of several blood variables related to metabolic and immunological status around calving, and udder health measured as milk somatic cell counts (SCC), Box-Cox transformed to bcSCC, at first test-milking in 287 primiparous cows in 20 Swedish dairy herds. Possible systematic effects of breed and age at calving on blood profiles were also investigated. Ordinary linear regression models, with robust standard errors and adjusting for clustering within herds, were used to investigate associations between blood variables and bcSCC. Hierarchical linear regression models, with herd as random factor, were used to investigate systematic effects on blood variables. The results showed that greater concentrations of beta-hydroxybutyrate (BHBA) and glucose before calving were associated with lesser bcSCC at first test-milking, whereas greater concentrations of nonesterified fatty acids (NEFA) before calving and greater delta NEFA (describing the difference in concentrations before and after calving) were associated with greater bcSCC at first test-milking. In addition, greater alpha-tocopherol concentrations in the period -5 to +5 d relative to calving were associated with lesser bcSCC at first test-milking, whereas greater concentrations of collectin of 43 kDa (CL-43) postpartum (1 to 21 d after calving) were associated with greater bcSCC. Postpartum concentrations of conglutinin and haptoglobin were also associated with bcSCC, but not independently of each other. Moreover, significant breed differences were observed for insulin, urea nitrogen, conglutinin, cholesterol, NEFA, and CL-43, the latter 3 as an interaction with period. Overall, cows of the Swedish Red breed had greater concentrations of insulin, cholesterol, urea nitrogen, and conglutinin, and lesser concentrations of NEFA and CL-43 than cows of the Swedish Holstein breed. Age at calving as main effect was significantly associated with BHBA, glucose, insulin, NEFA, urea nitrogen, and conglutinin. Heifers calving at >27 mo of age had greater BHBA and NEFA values, and lesser glucose, insulin, and urea nitrogen values compared with heifers calving at <27 mo. Heifers calving at an age <25 mo had greater conglutinin and urea nitrogen values, and lesser NEFA values compared with heifers calving at >25 mo. The results show that there are several associations among metabolites, immunological variables, and udder health of primiparous cows, but also that these variables vary between breeds and between cows of different age at first calving.

Characterization of bovine lymphocytes stimulated in vitro by Dictyocaulus viviparus homogenate.

Adult Dictyocaulus viviparus homogenate induced proliferation of lymphocytes from naïve cattle. We characterized the responding cells by carboxyfluorescein diacetate succinimidyl ester (CFSE) loading, for detection of proliferation, and antibody labelling for cell surface molecules. Lymphocytes expressing CD4, CD8 and gamma/delta TCR, rather than Ig expressing cells, proliferated after in vitro stimulation with D. viviparus homogenate. Of gamma/delta TCR expressing cells, both CD8, WC1.1 and WC1.2 co-expressing cells proliferated. Moreover, gamma/delta T cells expressing MHC class II proliferated to a higher extent than those negative for MHC class II. Of CD4 and CD8 expressing lymphocytes, both those co-expressing CD45R and CD45R0 proliferated. Among CD4 expressing lymphocytes, those that were CD45R0 positive had a larger proportion of proliferated cells than did CD45R positive cells. Compared to stimulation with Con A, the proportion of dividing cells after D. viviparus stimulation was smaller although the cells had divided more times. Furthermore, we also compared in vitro responses of peripheral blood mononuclear cells collected before and after two subsequent infections with D. viviparus, but no clear acquired responses could be detected. Overall, this suggests that most T lymphocytes are stimulated by the D. viviparus homogenate rather than any particular lymphocyte subpopulation.

Effects of two different feeding strategies during dry-off on certain health aspects of dairy cows.

With increasing milk production and short calving intervals, high daily milk yields at dry-off are rather common, making the dry-off procedure difficult and increasing the risk for health problems during the dry-off period. The objective of the following study was to compare the effects of 2 dry-off protocols, using different nutrient supplies, on health, as measured by clinical findings, intramammary infections, milk somatic cell count, and plasma concentrations of cortisol, serum amyloid A, haptoglobin, and Mg, in dairy cows. Twenty-one primi- and multiparous dairy cows were randomly assigned to 2 different feeding treatments. One group was fed ad libitum straw (straw), whereas the other group was fed 4 kg of DM silage daily and ad libitum straw (silage) during dry-off (i.e., for 5 d). All cows were milked in the morning of d 3 and 5 during this period. At the start of dry-off (d 0), the average daily milk yield was 17.1 +/- 0.8 kg. The plasma cortisol concentration increased during dry-off only in cows fed straw. There was no significant effect of treatment on plasma serum amyloid A, but the concentration increased during dry-off in both groups. The plasma Mg concentration decreased during dry-off, and the values tended to be lower in the straw group. The milk somatic cell count increased in both groups during dry-off but did not differ between groups. In both groups the heart rate decreased at dry-off, but the decrease was more pronounced in the straw group. Overall, this study (together with a previous report) shows that the common dry-off procedure of feeding straw only may give rise to metabolic disturbances. However, this might be avoided without any apparent negative effects on udder health if a limited amount of silage is added during dry-off.

Risk factors associated with the incidence of veterinary-treated clinical mastitis in Swedish dairy herds with a high milk yield and a low prevalence of subclinical mastitis.

We conducted a case-control study to investigate factors associated with the incidence rate of veterinary-treated clinical cases of mastitis (IRVTCM) in Swedish dairy herds with the overall objective to reduce the incidence of clinical mastitis (CM) and excessive use of antibiotics. Herds with documented high milk yield, low prevalence of subclinical mastitis and high (27.3-67.5 cases/100-cow years=case) or low (0.0-10.8 cases/100-cow years=control) IRVTCM were selected for this study. One hundred and fifty-eight herds participated. Logistic-regression models were used to assess statistically significant risk factors associated with a high or low IRVTCM. HI-herd farmers more often contacted a veterinarian for treatment of a cow with CM as soon as the milk appearance was altered, compared to LO-herd farmers that more often waited until the general condition of a cow was altered. HI-herd farmers also treated more cows due to high somatic-cell counts during lactation and/or at drying off. There were more high-yielding cows with dirty lower hind-legs, a higher percentage of first-parity cows and a higher incidence of veterinary-treated teat injuries in HI-herds. HI-herd farmers had their cows claw-trimmed more than once per year, compared to LO-herd farmers that had their cows claw-trimmed only once per year, and the condition of silage storage at HI-farms more often had one or more remarks. LO-herds consisted more often of cows of the Swedish Red and White breed, and their roughage diet was more often complemented with only commercial concentrates compared to HI-herds that more often fed grain in combination with commercial concentrates. The herds participating in this study were all well managed herds, and the attitude towards treatment and production seemed to influence the IRVTCM more than environmental factors.

Effects of feeding intensity during the dry period on leukocyte and lymphocyte sub-populations, neutrophil function and health in periparturient dairy cows.

The objective of this investigation was to study (1) the numbers of leukocytes, (2) the proportions of lymphocytes expressing CD4, CD8, WC1, B or IL2R and (3) neutrophil phagocytosis and oxidative burst activity in blood around parturition in three groups of dairy cows fed different levels of a total mixed ration during the last eight weeks before calving. All cows were fed ad libitum during the first eight weeks of lactation. Serum concentration of the acute phase protein serum amyloid A (SAA), the milk somatic cell count (SCC) and disease incidence were also recorded. Special emphasis was given to the weeks just before and just after calving as dairy cows are known to be immune suppressed during this period. Dry period diet had only minor effects on leukocyte numbers, and did not influence neutrophil phagocytosis and oxidative burst. In addition, no effect was observed on disease incidence or SAA concentrations. However, an increase in the proportion of B-lymphocytes and a decrease in the proportion of WC1+ T lymphocytes were observed after calving in cows fed high or low energy rations during the dry period, but not in cows fed a medium energy ration. The weeks just before and after parturition were characterised by neutrophilia, eosinopenia, lymphopenia and monocytosis, but time had no effect on neutrophil phagocytosis and oxidative burst. The proportions of CD4+, CD8+, B+ and IL-2R+ lymphocytes increased in early lactation relative to the mid dry period. In addition, the concentration of SAA increased dramatically at calving. The results emphasise the need for more studies to clarify the complex interactions between nutrition and immunity during the peripartum period in dairy cattle.

Acute phase proteins in response to Dictyocaulus viviparus infection in calves.

Three experiments were carried out to examine the acute phase response, as measured by the acute phase proteins (APP) haptoglobin, serum amyloid A (SAA) and fibrinogen, in calves infected with lungworm, Dictyocaulus vivparus. In addition, eosinophil counts were analysed. Three different dose models were used in 3 separate experiments: 1) 250 D. viviparus infective third stage larvae (L3) once daily for 2 consecutive days, II) 100 D. viviparus L3 once daily for 5 consecutive days, and III) 2000 L3 once. All 3 dose regimes induced elevated levels of haptoglobin, SAA and fibrinogen, although there was considerable variation both between and within experiments. A significant increase was observed in all 3 APP at one or several time points in experiment I and III, whereas in experiment II, the only significant elevation was observed for fibrinogen at one occasion. The eosinophil numbers were significantly elevated in all 3 experiments. The results show that lungworm infection can induce an acute phase response, which can be monitored by the selected APP. Elevated APP levels in combination with high numbers of eosinophils in an animal with respiratory disease may be used as an indicator of lung worm infection, and help the clinician to decide on treatment. However, high numbers of eosinophils and low levels of APP do not exclude a diagnosis of lungworm. Thus, lungworm infection may not be detected if measurements of APP are used to assess calf health in herds or individual animals.

Commercially available antibodies to human tumour necrosis factor-alpha tested for cross-reactivity with ovine and bovine tumour necrosis factor-alpha using flow cytometric assays.

A thorough understanding of the immune system, including the role of different cytokines, during inflammatory diseases in ruminants could lead to the development of new diagnostic methods and treatments. Tumour necrosis factor-alpha (TNF-alpha) is an important cytokine in the onset of the inflammatory responses. Unfortunately, the number of studies on cytokines, like TNF-alpha, in ruminants is limited due to a lack of species-specific reagents. As cytokines have remained rather conserved during evolution, cross-reactivity between animal species may occur. Therefore, the aim of the present study was to investigate 5 commercially available antibodies against human TNF-alpha for their ability to cross-react with ovine and/or bovine TNF-alpha, using a bead-based flow cytometric method. Two of the antibody clones (Mab 11 and 6401.1111) showed cross reactivity with ovine recombinant TNF-alpha in concentrations above 2.5 ng/ml. However, none of the antibodies detected TNF-alpha in bovine milk, or serum containing known concentrations of bovine TNF-alpha, as earlier determined with ELISA. The results could be due to inability of the antibodies to cross-react between species, but quenching of the signal by matrix proteins might also have lowered the response.

Prediction of vitamin A, vitamin E, selenium and zinc status of periparturient dairy cows using blood sampling during the mid dry period.

Vitamins A and E, and the trace elements selenium (Se) and zinc (Zn) are essential for the health and performance of dairy cows. Their concentrations often decrease around calving and extra supplementation is sometimes recommended at that time. However, the need for this varies, for example depending on quantity and quality of feedstuffs in the diet. The aim of this study was to measure the concentrations of serum vitamin A (S-vit A) and vitamin E (S-vit E), plasma Se (P-Se) and serum Zn (S-Zn) in blood samples taken at several time points from one month before to one month after calving, and to evaluate if a blood sample taken during the mid dry period can accurately predict the blood concentration at calving and early lactation. Dairy cows on 3 different feeding regimens during the dry period were included in the study. A significant decrease in the concentrations of S-vit A and S-vit E, and S-Zn, was observed at calving, and P-Se was significantly lower during the dry period and at calving than in early lactation. The blood concentrations of S-vit E and P-Se in the mid dry period significantly predicted the occurrence of values considered marginal or deficient at the time of calving. The data indicate that a mid dry period concentration of > or = 5.4 mg/l of S-vit E and > or = 0.09 mg/l of P-Se will result in a 90% chance that the cow stays above marginal levels at calving given that a feed of the same quality is offered.