Phenotypic and genotypic assessment of iron acquisition in diverse bovine-associated non-aureus staphylococcal strains.

Although the role of iron in bacterial infections has been well described for Staphylococcus (S.) aureus, iron acquisition in (bovine-associated) non-aureus staphylococci and mammaliicocci (NASM) remains insufficiently mapped. This study aimed at elucidating differences between four diverse bovine NASM field strains from two species, namely S. chromogenes and S. equorum, in regards to iron uptake (with ferritin and lactoferrin as an iron source) and siderophore production (staphyloferrin A and staphyloferrin B) by investigating the relationship between the genetic basis of iron acquisition through whole genome sequencing (WGS) with their observed phenotypic behavior. The four field strains were isolated in a previous study from composite cow milk (CCM) and bulk tank milk (BTM) in a Flemish dairy herd. Additionally, two well-studied S. chromogenes isolates originating from a persistent intramammary infection and from a teat apex were included for comparative purpose in all assays. Significant differences between species and strains were identified. In our phenotypical iron acquisition assay, while lactoferrin had no effect on growth recovery for all strains in iron deficient media, we found that ferritin served as an effective source for growth recovery in iron-deficient media for S. chromogenes CCM and BTM strains. This finding was further corroborated by analyzing potential ferritin iron acquisition genes using whole-genome sequencing data, which showed that all S. chromogenes strains contained hits for all three proposed ferritin reductive pathway genes. Furthermore, a qualitative assay indicated siderophore production by all strains, except for S. equorum. This lack of siderophore production in S. equorum was supported by a quantitative assay, which revealed significantly lower or negligible siderophore amounts compared to S. aureus and S. chromogenes. The WGS analysis showed that all tested strains, except for S. equorum, possessed complete staphyloferrin A (SA)-synthesis and export operons, which likely explains the phenotypic absence of siderophore production in S. equorum strains. While analyzing the staphyloferrin A and staphyloferrin B operon landscapes for all strains, we noticed some differences in the proteins responsible for iron acquisition between different species. However, within strains of the same species, the siderophore-related proteins remained conserved. Our findings contribute valuable insights into the genetic elements associated with bovine NASM pathogenesis.

Priming of the murine mammary gland with Staphylococcus chromogenes IM reduces bacterial growth of Streptococcus uberis: a proof-of-concept study.

Streptococcus uberis is a major causative agent of bovine mastitis, an inflammation of the mammary gland with substantial economic consequences. To reduce antibiotic use in animal agriculture, alternative strategies to treat or prevent mastitis are being investigated. Bovine-associated non-aureus staphylococci are proposed in that respect due to their capacity to inhibit the in vitro growth of S. uberis. We demonstrate that priming the murine mammary gland with Staphylococcus chromogenes IM reduces S. uberis growth in comparison with non-primed glands. The innate immune system is activated by increasing IL-8 and LCN2, which may explain this decreased growth.

Distinct behavior of bovine-associated staphylococci species in their ability to resist phagocytosis and trigger respiratory burst activity by blood and milk polymorphonuclear leukocytes in dairy cows.

Mastitis affects a high proportion of dairy cows and is still one of the greatest challenges faced by the dairy industry. Staphylococcal bacteria remain the most important cause of mastitis worldwide. We investigated how distinct staphylococcal species evade some critical host defense mechanisms, which may dictate the establishment, severity, and persistence of infection and the outcome of possible therapeutic and prevention interventions. Thus, the present study investigated variations among distinct bovine-associated staphylococci in their capability to resist phagocytosis and to trigger respiratory burst activity of blood and milk polymorphonuclear neutrophil leukocytes (PMNL) in dairy cows. To do so, PMNL of 6 primiparous and 6 multiparous dairy cows were used. A collection of 38 non-aureus staphylococci (NAS) and 12 Staphylococcus aureus were included. The phagocytosis and intracellular reactive oxygen species (ROS) production by blood and milk PMNL were analyzed by flow cytometry. Phagocytosis, by both blood and milk PMNL, did not differ between S. aureus and NAS as a group, although within-NAS species differences were observed. Staphylococcus chromogenes (a so-called milk-adapted NAS species) better resisted phagocytosis by blood PMNL than the so-called environmental (i.e., Staphylococcus fleurettii) and opportunistic (i.e., Staphylococcus haemolyticus) NAS species. Otherwise, S. haemolyticus was better phagocytosed by blood PMNL than S. aureus, S. fleurettii, and S. chromogenes. No influence of the origin of the isolates within the staphylococci species in the resistance to phagocytosis by blood and milk PMNL was found. Overall, both S. aureus and NAS did not inhibit intracellular ROS production in blood and milk PMNL. Non-aureus staphylococci induced fewer ROS by milk PMNL than S. aureus, which was not true for blood PMNL, although species-specific differences in the intensity of ROS production were observed. Staphylococcus chromogenes induced more blood PMNL ROS than S. fleurettii and S. haemolyticus, and as much as S. aureus. Conversely, S. chromogenes induced fewer milk PMNL ROS than S. aureus. The origin of the isolates within the staphylococci species did not affect the ROS production by blood and milk PMNL. In conclusion, our study showed differences in staphylococci species in evading phagocytosis and triggering ROS production, which may explain the ability of some staphylococci species (i.e., S. aureus and S. chromogenes) to cause persistent infection and induce inflammation.

Bovine-associated non-aureus staphylococci suppress Staphylococcus aureus biofilm dispersal in vitro yet not through agr regulation.

Biofilm formation is a significant virulence factor in Staphylococcus (S.) aureus strains causing subclinical mastitis in dairy cows. A role of environmental signals and communication systems in biofilm development, such as the agr system in S. aureus, is suggested. In the context of multispecies biofilm communities, the presence of non-aureus staphylococci (NAS) might influence S. aureus colonization of the bovine mammary gland, yet, such interspecies interactions have been poorly studied. We determined whether 34 S. chromogenes, 11 S. epidermidis, and 14 S. simulans isolates originating from bovine milk samples and teat apices (TA) were able to affect biofilm formation and dispersion of S. aureus, and if so, how isolate traits such as the capacity to regulate the S. aureus agr quorum sensing system are determinants in this process. The capacity of an agr-positive S. aureus strain to form biofilm was increased more in the presence of S. chromogenes than in the presence of S. simulans and S. epidermidis isolates and in the presence of NAS isolates that do not harbor biofilm related genes. On the other hand, biofilm dispersion of this particular S. aureus strain was suppressed by NAS as a group, an effect that was more pronounced by isolates from TA. Furthermore, the observed effects on biofilm formation and dispersion of the agr-positive S. aureus strain as well as of an agr-negative S. aureus strain did not depend on the capacity of NAS to repress the agr system.

Metabolites of bovine-associated non-aureus staphylococci influence expression of Staphylococcus aureus agr-related genes in vitro.

Communications via quorum sensing (QS) between non-aureus staphylococci (NAS) and Staphylococcus (S.) aureus in the bovine mammary gland remains largely unexplored. We determined whether 34 S. chromogenes, 11 S. epidermidis, and 14 S. simulans isolates originating from bovine milk samples and teat apices were able to regulate the QS of S. aureus, and if so, how in vitro growth inhibition of S. aureus by NAS, or NAS metabolites, or NAS cells themselves play a role in this process. In co-culture with S. aureus we observed that these 3 NAS species in general downregulated the expression of rnaIII, the effector molecule of the QS system, but this effect was more pronounced in S. chromogenes and S. simulans isolates than in S. epidermidis isolates. In vitro growth inhibition of S. aureus by NAS resulted in a small underestimation of the downregulating effect of NAS on rnaIII expression of S. aureus. Additionally, the culture supernatant of these NAS isolates and supernatant treated with proteinase K expressed greater regulatory activity over S. aureus virulence genes rnaIII, hla, and spa than washed NAS cells suspended in sterile water. These microbial interactions may influence S. aureus virulence and pathogenesis within the host. Isolation and identification of NAS metabolites affecting the QS system of S. aureus might help to develop alternative strategies for treatment and control of S. aureus mastitis.

Colonization and local host response following intramammary Staphylococcus chromogenes challenge in dry cows.

Although extensive research has been performed on bovine non-aureus staphylococci (NAS), several aspects such as bacteria-host interaction remain largely unstudied. Moreover, only a few mastitis pathogen challenge studies in cows have been conducted in the dry period, an important period that allows intramammary infection (IMI) to cure and new IMI to occur. We challenged 16 quarters of 4 Holstein Friesian cows at dry off with 100; 100 000 or 10 000 000 CFU of the udder-adapted S. chromogenes IM strain. Four quarters from one cow served as negative controls. Internally sealed quarters remained untouched, whereas non-sealed quarters were sampled 3 times during the dry period. After parturition, colostrum and daily milk samples were taken during the first week of lactation of all quarters. In total, 8 quarters appeared to be colonized, since S. chromogenes IM was recovered at least once during the experiment, as substantiated using Multilocus Sequence Typing. S. chromogenes IM shedding was highest in dry quarters inoculated with 10 000 000 CFU. Colonized quarters had the highest quarter somatic cell count (qSCC) in early lactation. Inoculated quarters (both colonized and non-colonized) had lower IL-6 and IL-10 concentrations in the dry period, whilst IFN-γ levels tended to be higher in colonized quarters compared to non-inoculated quarters. Also, IgG2 levels were higher in inoculated compared to non-inoculated quarters and the IgG2/IgG1 ratio was on average above 1. To conclude, we showed that dry quarters can be colonized with S. chromogenes IM, resulting in a shift towards a Th1 response in late gestation and early lactation characterised by an increased IgG2 concentration. However, further research is needed to confirm our findings.

Fecal non-aureus Staphylococci are a potential cause of bovine intramammary infection.

The presence of non-aureus staphylococci (NAS) in bovine rectal feces has recently been described. Similar to other mastitis causing pathogens, shedding of NAS in the environment could result in intramammary infection. The objective of this study was to investigate whether NAS strains present in feces can cause intramammary infection, likely via teat apex colonization. During a cross-sectional study in 5 dairy herds, samples were collected from the habitats quarter milk, teat apices, and rectal feces from 25%, 10%, and 25% of the lactating cows, respectively, with a cow serving as the source of one type of sample only. Samples from clinical mastitis cases were continuously collected during the 1-year study period as well. The 6 most prevalent NAS species, Staphylococcus (S.) chromogenes, S. cohnii, S. devriesei, S. equorum, S. haemolyticus, and S. hominis, were further subtyped by random amplification of polymorphic deoxyribonucleic acid polymerase chain reaction (RAPD-PCR), when the same NAS species was present in the same herd in the three habitats. For S. chromogenes, S. cohnii, S. devriesei, and S. haemolyticus, the same RAPD type was found in rectal feces, teat apices, and quarter milk, indicating that fecal NAS can infect the mammary gland. For S. hominis and S. equorum, we were unable to confirm the presence of the same RAPD types in the three habitats.

Method for short-term prediction of milk yield at the quarter level to improve udder health monitoring.

Udder health problems are often associated with milk losses. These losses are different between quarters, as infected quarters are affected both by systemic and pathogen-specific local effects, whereas noninfected quarters are only subject to systemic effects. To gain insight in these losses and the milk yield dynamics during disease, it is essential to have a reliable reference for quarter-level milk yield in an unperturbed state, mimicking its potential yield. We developed a novel methodology to predict this quarter milk yield per milking session, using an historical data set of 504 lactations collected on a test farm by an automated milking system from DeLaval (Tumba, Sweden). Using a linear mixed model framework in which covariates associated with the linearized Wood model and the milking interval are included, we were able to describe quarter-level yield per milking session with a proportional error below 10%. Applying this model enables us to predict the milk yield of individual quarters 1 to 50 d ahead with a mean prediction error ranging between 8 and 20%, depending on the amount of historical data available to estimate the random effect covariates for the predicted lactation. The developed methodology was illustrated using 2 examples for which quarter-level milk losses are calculated during clinical mastitis. These showed that the quarter-level mixed model allows us to gain insight in quarter lactation dynamics and enables to calculate milk losses in different situations.

Phenotypic and genotypic characterization of antimicrobial resistance profiles in Streptococcus dysgalactiae isolated from bovine clinical mastitis in 5 provinces of China.

Bovine mastitis is among the most prevalent and costly diseases of dairy animals and is caused by a variety of bacterial pathogens including Streptococcus dysgalactiae. However, comprehensive studies reporting the prevalence and antimicrobial resistance profiles of S. dysgalactiae isolated from bovine mastitis are scarce. Therefore, this study was to investigate the occurrence of S. dysgalactiae associated with bovine clinical mastitis, to assess their antimicrobial resistance profiles, and to analyze the phenotypic and genotypic profiling of resistant isolates. In total, 1,180 milk samples were collected from dairy cows with clinical mastitis belonging to 74 commercial dairy herds located in 14 provinces of China from January 2014 to May 2016. Overall S. dysgalactiae isolates were recovered from 88 (7.5%) of the mastitic milk samples. The antimicrobial susceptibility of these isolates was tested against 8 antimicrobial agents by using minimum inhibitory concentrations. Results showed that 82 (93.2%) isolates expressed resistance to more than one antimicrobial agent. Antimicrobial resistance was highest against kanamycin (89.8%), sulfonamide (83.0%), and streptomycin (58.0%), which can be attributed to the intrinsic resistance for most of Streptococcus spp. against those antimicrobial substances. Strikingly, 30 (34.1%) and 12 (13.6%) isolates were found resistant to cephalexin and ceftriaxone, respectively. BlaTEM, ermB, and tetM were the most prevalent resistance genes. All isolates carried at least one of all tested resistance genes. Also, 1.1, 12.5, 18.2, 36.4, and 31.8% of isolates were positive for at least one tested resistance gene in 1, 2, 3, 4, or 5 classes of antimicrobials. Survival analysis showed a significant association between ermB and survival of the S. dysgalactiae isolates at increasing erythromycin concentrations. No other statistically significant associations were observed between the phenotypic and genotypic resistance profiles. This study concludes a considerable prevalence of S. dysgalactiae associated with bovine mastitis in dairy herds of China and these isolates exhibited high resistance rates to tested antimicrobials, coupled with high occurrence of resistance genes. Both the prevalence of S. dysgalactiae and their antimicrobial resistance profiles strongly varied among dairy herds, demonstrating the need for antimicrobial susceptibility surveillance at the herd level to ensure optimal therapeutic results.

Impact of diversity of Mycoplasma hyopneumoniae strains on lung lesions in slaughter pigs.

The importance of diversity of Mycoplasma hyopneumoniae (M. hyopneumoniae) strains is not yet fully known. This study investigated the genetic diversity of M. hyopneumoniae strains in ten pig herds, and assessed associations between the presence of different strains of M. hyopneumoniae and lung lesions at slaughter. Within each herd, three batches of slaughter pigs were investigated. At slaughter, from each batch, 20 post mortem bronchoalveolar lavage fluid samples were collected for multiple locus variable-number tandem repeat analysis (MLVA), and lung lesions (Mycoplasma-like lesions, fissures) were examined. Multivariable analyses including potential risk factors for respiratory disease were performed to assess associations between the number of different strains per batch (three categories: one strain, two-six strains, ≥seven strains), and the lung lesions as outcome variables. In total, 135 different M. hyopneumoniae strains were found. The mean (min.-max.) number of different strains per batch were 7 (1-13). Batches with two-six strains or more than six strains had more severe Mycoplasma-like lesions (P = 0.064 and P = 0.012, respectively), a higher prevalence of pneumonia [odds ratio (OR): 1.30, P = 0.33 and OR: 2.08, P = 0.012, respectively], and fissures (OR = 1.35, P = 0.094 and OR = 1.70, P = 0.007, respectively) compared to batches with only one strain. In conclusion, many different M. hyopneumoniae strains were found, and batches of slaughter pigs with different M. hyopneumoniae strains had a higher prevalence and severity of Mycoplasma-like lung lesions at slaughter, implying that reducing the number of different strains may lead to less lung lesions at slaughter and better respiratory health of the pigs.

Local host response following an intramammary challenge with Staphylococcus fleurettii and different strains of Staphylococcus chromogenes in dairy heifers.

Coagulase-negative staphylococci (CNS) are a common cause of subclinical mastitis in dairy cattle. The CNS inhabit various ecological habitats, ranging between the environment and the host. In order to obtain a better insight into the host response, an experimental infection was carried out in eight healthy heifers in mid-lactation with three different CNS strains: a Staphylococcus fleurettii strain originating from sawdust bedding, an intramammary Staphylococcus chromogenes strain originating from a persistent intramammary infection (S. chromogenes IM) and a S. chromogenes strain isolated from a heifer's teat apex (S. chromogenes TA). Each heifer was inoculated in the mammary gland with 1.0 × 10(6) colony forming units of each bacterial strain (one strain per udder quarter), whereas the remaining quarter was infused with phosphate-buffered saline. Overall, the CNS evoked a mild local host response. The somatic cell count increased in all S. fleurettii-inoculated quarters, although the strain was eliminated within 12 h. The two S. chromogenes strains were shed in larger numbers for a longer period. Bacterial and somatic cell counts, as well as neutrophil responses, were higher after inoculation with S. chromogenes IM than with S. chromogenes TA. In conclusion, these results suggest that S. chromogenes might be better adapted to the mammary gland than S. fleurettii. Furthermore, not all S. chromogenes strains induce the same local host response.

Factors affecting mechanical nociceptive thresholds in healthy sows.

OBJECTIVE: To describe anatomical and methodological factors influencing mechanical nociceptive thresholds (MNTs) and intra-site variability in healthy sows. STUDY DESIGN: Prospective, randomized validation. ANIMALS: Eight pregnant, healthy, mixed-parity sows (176-269 kg). METHODS: Repeated MNT measurements were taken: 1) with a hand-held probe and a limb-mounted actuator connected to a digital algometer; 2) at nine landmarks on the limbs and tail; and 3) at 1 and 3 minute intervals. Data were analysed using linear mixed regression models. RESULTS: The MNTs (±SEM) of the limbs were lower with the probe (14.7 ± 1.2 N) than with the actuator (21.3 ± 1.2 N; p < 0.001), in the pelvic versus the thoracic limbs (16.7 ± 1.2 versus 19.2 ± 1.2 N; p < 0.001), and in the lateral versus the dorsal metatarsi and metacarpi (17.6 ± 1.2 versus 18.4 ± 1.2 N; p = 0.002). MNTs were higher in all subsequent measurements compared with the first (p < 0.001) and in the morning compared with the afternoon (p = 0.04). We found no evidence of MNT differences based on interval between consecutive measurements (1 versus 3 minutes). Variability was lower in the thoracic limbs [mean back-transformed log10 coefficient of variation (CV) ± SE = 25.5 ± 1.5% versus 30.6 ± 1.5% in the pelvic limbs; p < 0.001], with the actuator (22.7 ± 1.5% versus 33.4 ± 1.5% with the probe; p < 0.001), and on the left (CV = 26.9 ± 1.5% versus 29.3 ± 1.5% on the right; p = 0.01). Tail data (probe only) were analysed separately: mean MNT (± SE) was 11.7 (±1.8); MNT increased in days 3-6 of testing compared with day 1 (p < 0.001). The mean CV (±SE) was 38.9% (±1.1%). CONCLUSIONS AND CLINICAL RELEVANCE: MNTs and intra-site variability in healthy sows were affected by several factors, indicating that this methodology requires considerable attention to detail.

Individual commitment to a group effect: strengths and weaknesses of bovine embryo group culture.

Recently, new culture devices such as Corral and Primo Vision dishes have been designed for the culture of human embryos to allow the combination of group culture plus follow-up of individual embryos. Bovine inseminated oocytes were allocated to Primo Vision dishes, Corral dishes, individual culture or classical group culture. Blastocyst development in Primo Vision dishes was similar to classical group culture (34.3 and 39.0% respectively), and better than Corral dishes or individual culture (28.9 and 28.5% respectively). In Primo Vision dishes, a higher number of 'slow' embryos developed to the blastocyst stage compared with their individually cultured counterparts, while no differences were observed for 'fast' embryos. 'Slow' embryos in a 'standard drop' had a higher chance of becoming a blastocyst compared with individual culture (OR: 2.3), whereas blastulation of 'fast' embryos was less efficient in a 'delayed drop' than in individual culture (OR: 0.3). The number of non-cleaved embryos in Primo Vision dishes did not negatively influence blastocyst development. Likewise, removing non-cleaved embryos (NC removed) and regrouping the cleaved embryos afterwards (ReGR) did not affect blastocyst development and quality compared with group culture in Primo Vision dishes (CTRL, 31.6%, NC removed, 29.3% and ReGR, 29.6%). The experiments revealed that group culture of bovine embryos in Primo Vision dishes is superior to individual culture, primarily because of the higher blastocyst rate achieved by slow embryos. Non-cleaved or arrested embryos do not hamper the ability of co-cultured bovine embryos to reach the blastocyst stage in group culture.

Pathogen-specific incidence rate of clinical mastitis in Flemish dairy herds, severity, and association with herd hygiene.

A one-year survey on clinical mastitis was conducted on 50 randomly selected commercial Flemish dairy herds to estimate the pathogen-specific incidence rate of clinical mastitis (IRCM). The severity of the cases and the potential associations with herd hygiene were studied. Participating producers sampled 845 cases and 692 dairy cows. The mean and median IRCM was estimated at 7.4 and 5.3 quarter cases per 10,000 cow-days at risk, respectively. A large between-herd variation was observed (range of 0-21.3). In general, the IRCM was lower in heifers compared with multiparous cows (2.9 vs. 11.0 quarter cases per 10,000 cow-days at risk). However, the overall IRCM in the first week after calving was higher in heifers compared with cows (43.4 vs. 31.6 quarter cases per 10,000 cow-days at risk). Streptococcus uberis (18.2% of the cases) and Escherichia coli (15.5%) were the most frequently isolated pathogens and no growth was observed in 19.9% of the cases. The majority of the cases (63.1%) were mild (only clots in milk). Moderate (hard quarter without general signs) and severe symptoms (systemic illness) were observed in 29.9 and 7.0% of the cases, respectively. Isolation of E. coli (vs. any other culture result) was more likely in moderate and severe cases compared with mild cases. Overall IRCM and E. coli IRCM were higher in dirty compared with clean herds based on udder hygiene scores (9.0 and 1.7 vs. 6.0 and 0.6 quarter cases per 10,000 cow-days at risk, respectively). This study broadens the knowledge on clinical mastitis in Flemish dairy herds and underlines the high risk of CM in early-lactation heifers, the role of the so-called environmental pathogens, and herd hygiene.

Associations between CXCR1 polymorphisms and pathogen-specific incidence rate of clinical mastitis, test-day somatic cell count, and test-day milk yield.

The CXCR1 gene plays an important role in the innate immunity of the bovine mammary gland. Associations between single nucleotide polymorphisms (SNP) CXCR1c.735C>G and c.980A>G and udder health have been identified before in small populations. A fluorescent multiprobe PCR assay was designed specifically and validated to genotype both SNP simultaneously in a reliable and cost-effective manner. In total, 3,106 cows from 50 commercial Flemish dairy herds were genotyped using this assay. Associations between genotype and detailed phenotypic data, including pathogen-specific incidence rate of clinical mastitis (IRCM), test-day somatic cell count, and test-day milk yield (MY) were analyzed. Staphylococcus aureus IRCM tended to associate with SNP c.735C>G. Cows with genotype c.735GG had lower Staph. aureus IRCM compared with cows with genotype c.735CC (rate ratio = 0.35, 95% confidence interval = 0.14­0.90). Additionally, a parity-specific association between Staph. aureus IRCM and SNP c.980A>G was detected. Heifers with genotype c.980GG had a lower Staph. aureus IRCM compared with heifers with genotype c.980AG (rate ratio = 0.15, 95% confidence interval = 0.04­0.56). Differences were less pronounced in multiparous cows. Associations between CXCR1 genotype and somatic cell count were not detected. However, MY was associated with SNP c.735C>G. Cows with genotype c.735GG out-produced cows with genotype c.735CC by 0.8 kg of milk/d. Results provide a basis for further research on the relation between CXCR1 polymorphism and pathogen-specific mastitis resistance and MY.

Oral supplementation of medium-chain fatty acids during the dry period supports the neutrophil viability of peripartum dairy cows.

A randomised clinical trial was conducted to explore the effect of orally supplemented medium-chain fatty acids (MCFA) to heifers and cows starting 6-8 weeks prior to expected calving date on blood and milk polymorphonuclear neutrophilic leucocyte (PMNL) apoptosis between 1 and 3 d in milk (DIM). The effects of MCFA-supplementation on the likelihood of intramammary infections (IMI) in early lactation, and test-day somatic cell count (SCC) and average daily milk yield (MY) during the first 4 months of lactation were evaluated as well. Twenty-two animals were included of which half were orally supplemented with MCFA starting 6-8 weeks prior to calving and half served as non-supplemented controls. The PMNL viability in both blood and milk was quantified using dual-colour flow cytometry with fluorescein-labelled annexin and propidium iodide. In non-supplemented animals, % blood PMNL apoptosis significantly increased between start of supplementation and early lactation, reflecting a potential reduction in innate immune capacity, whereas this was not true in the MCFA-supplemented animals. Similar results were seen in milk PMNL apoptosis. Overall, the % apoptotic milk PMNL between 1 and 3 DIM was significantly lower in the MCFA-supplemented group compared with the non-supplemented group. There was no substantial effect of oral MCFA-supplementation on the likelihood of quarter IMI nor on the composite test-day milk SCC or average daily MY. In conclusion, oral MCFA-supplementation starting 6-8 weeks before expected calving date supported the blood and milk neutrophil viability in early lactating dairy cows. Still, this was not reflected in an improvement of udder health nor MY in early and later lactation. The results should trigger research to further unravel the mechanisms behind the observed immunomodulating effect, and the potential relevance for the cows' performances throughout lactation.

Impact of Selective Dry Cow Therapy on Antimicrobial Consumption, Udder Health, Milk Yield, and Culling Hazard in Commercial Dairy Herds.

The main objective of the study was to evaluate whether or not implementing selective dry cow therapy (SDCT) on commercial dairy farms reduces antimicrobial consumption without negatively affecting future performances when compared to blanket dry cow therapy (BDCT). Twelve commercial herds in the Flemish region of Belgium with overall good udder health management were enrolled in a randomized control trial, including 466 cows that were assigned to a BDCT (n = 244) or SDCT (n = 222) group within herds. Cows in the SDCT group were dried off with internal teat sealants combined or not with long-acting antimicrobials according to a predefined algorithm based on test-day somatic cell count (SCC) data. Total antimicrobial use for udder health between drying off and 100 days in milk was significantly lower in the SDCT group (i.e., a mean of 1.06 defined the course dose) compared to the BDCT group (i.e., a mean of 1.25 defined the course dose), although with substantial variation between herds. Test-day SCC values, milk yield, and the clinical mastitis and culling hazard in the first 100 days in milk did not differ between the BDCT and SDCT groups. SCC-based and algorithm-guided SDCT is suggested to decrease the overall use of antimicrobials without jeopardizing cows' udder health and milk yield.

In-Line Detection of Clinical Mastitis by Identifying Clots in Milk Using Images and a Neural Network Approach.

Automated milking systems (AMSs) already incorporate a variety of milk monitoring and sensing equipment, but the sensitivity, specificity, and positive predictive value of clinical mastitis (CM) detection remain low. A typical symptom of CM is the presence of clots in the milk during fore-stripping. The objective of this study was the development and evaluation of a deep learning model with image recognition capabilities, specifically a convolutional neural network (NN), capable of detecting such clots on pictures of the milk filter socks of the milking system, after the phase in which the first streams of milk have been discarded. In total, 696 pictures were taken with clots and 586 pictures without. These were randomly divided into 60/20/20 training, validation, and testing datasets, respectively, for the training and validation of the NN. A convolutional NN with residual connections was trained, and the hyperparameters were optimized based on the validation dataset using a genetic algorithm. The integrated gradients were calculated to explain the interpretation of the NN. The accuracy of the NN on the testing dataset was 100%. The integrated gradients showed that the NN identified the clots. Further field validation through integration into AMS is necessary, but the proposed deep learning method is very promising for the inline detection of CM on AMS farms.

Association between management practices and estimated mastitis incidence and milk losses on robotic dairy farms.

This study aims to describe the relation between farm-level management factors and estimated farm-level mastitis incidence and milk loss traits (MIMLT) at dairy farms with automated milking systems. In this observational study, 43 commercial dairy farms in Belgium and the Netherlands were included and 148 'management and udder health related variables' were obtained during a farm visit through a farm audit and survey. The MIMLT were estimated from milk yield data. Quarter-level milk yield perturbations that were caused by presumable mastitis cases (PMC) were selected based on quarter-level milk yield and electrical conductivity. On average, 57.6 ± 5.4% of the identified milk yield perturbations complied with our criteria. From these PMC, 3 farm-level MIMLT were calculated over a one-year period around the farm visit date: (1) the 'average number of PMC per cow per year', (2) the 'absolute milk loss per cow per day', calculated as the farm-level sum of all milk losses during PMC in one year, divided by the average number of lactating cows and the number of days, and (3) the 'relative milk loss', calculated as the farm-level sum of milk losses during PMC in one year, divided by the estimated total production in the absence of PMC. The 'average number of PMC per cow per year' was on average 1.81 ± 0.47. The PMC caused an average milk loss of 0.77 ± 0.26 kg per lactating cow per day, which corresponded to an average production loss of 2.38 ± 0.82% of the expected production in the absence of PMC. We performed a principal component regression (PCR) analysis to link the 3 MIMLT to the 'management and udder health related variables', whilst reducing the multicollinearity and the number of dimensions. The first principal component was mainly related to 'milking system brand, maintenance and settings'. The second component mainly linked to average productivity and somatic cell counts, whereas the third component mainly contained variables linked with mastitis management, treatment, and biosecurity. The 3 PCR models had R² ranging from 0.46 (for absolute milk loss per cow per day) to 0.57 (for relative milk loss). For all models, the second PC had the largest effect size. This analysis raises awareness of the impact of management factors on a factual basis and provides handles to take management actions to improve udder health.

In search for cross-reactivity to immunophenotype equine mesenchymal stromal cells by multicolor flow cytometry.

During recent years, cell-based therapies using mesenchymal stem cells (MSC) are reported in equine veterinary medicine with increasing frequency. In most cases, the isolation and in vitro differentiation of equine MSC are described, but their proper immunophenotypic characterization is rarely performed. The lack of a single marker specific for MSC and the limited availability of monoclonal antibodies (mAbs) for equine MSC in particular, strongly hamper this research. In this study, 30 commercial mAbs were screened with flow cytometry for recognizing equine epitopes using the appropriate positive controls to confirm their specificity. Cross-reactivity was found and confirmed by confocal microscopy for CD45, CD73, CD79α, CD90, CD105, MHC-II, a monocyte marker, and two clones tested for CD29 and CD44. Unfortunately, none of the evaluated CD34 clones recognized the equine epitopes on positive control endothelial cells. Subsequently, umbilical cord blood-derived undifferentiated equine MSC of the fourth passage of six horses were characterized using multicolor flow cytometry based on the selected nine-marker panel of both cell surface antigens and intracytoplasmatic proteins. In addition, appropriate positive and negative controls were included, and the viable single cell population was analyzed by excluding dead cells using 7-aminoactinomycin D. Isolated equine MSC of the fourth passage were found to be CD29, CD44, CD90 positive and CD45, CD79α, MHC-II, and a monocyte marker negative. A variable expression was found for CD73 and CD105. Successful differentiation towards the osteogenic, chondrogenic, and adipogenic lineage was used as additional validation. We suggest that this selected nine-marker panel can be used for the adequate immunophenotyping of equine MSC.