Lactobacillus rhamnosus GR-1 Prevents Escherichia coli-Induced Apoptosis Through PINK1/Parkin-Mediated Mitophagy in Bovine Mastitis.

Escherichia coli is one of the most important pathogens that cause clinical mastitis in dairy cattle worldwide and lead to severe economic losses. Antibiotics are often used to treat this inflammatory disease; however, antimicrobial resistance and environmental pollution cannot be ignored. Probiotic is the best alternative; however, its mechanisms of action to prevent mastitis remain unclear. Moreover, the role of probiotics in regulating mitophagy, a selective autophagy that maintains mitochondrial quality, needs to be explored. E. coli infection induced NOD-like receptor family member pyrin domain-containing protein 3 (NLRP3) inflammasome assembly, Caspase-1 activation, and apoptosis in MAC-T cells. Infection also resulted in mitochondrial damage and subsequent increase in reactive oxygen species (ROS) production. Moreover, inhibition of ROS release by scavenger N-acetyl-L-cysteine (NAC) abrogated the importance of ROS in NLRP3 assembly and apoptosis in MAC-T cells. Pretreatment with Lactobacillus rhamnosus GR-1 (LGR-1), a probiotic, alleviated E. coli-induced NLRP3 inflammasome activation and apoptosis via ROS inhibition. Besides, E. coli infection inhibited mitophagy while LGR-1 pretreatment augmented PINK1/Parkin-mediated mitophagy activation, which further blocked ROS generation. To explore the effect of LGR-1 in vivo, a mouse mastitis model was established. The results showed that LGR-1 pretreatment had preventive and protective effects on E. coli induced mastitis, and could reduce cytokines levels such as IL-1ß and TNF-α. In accordance with the results in vitro, E. coli can inhibit mitophagy and activate NLRP3 inflammasome and apoptosis, while LGR-1 can weaken the effect of E. coli. Taken together, our data indicated that LGR-1 pretreatment induced PINK1/Parkin-mediated mitophagy that eliminated damaged mitochondria and reduced ROS production and NLRP3 inflammasome activation, which subsequently decreased E. coli-induced apoptosis. To conclude, our study suggests that therapeutic strategies aiming at the upregulation of mitophagy under E. coli-induced mastitis may preserve mitochondrial function and provide theoretical support for the application of probiotics in bovine mastitis.

Study on the regulation mechanism of lipopolysaccharide on oxidative stress and lipid metabolism of bovine mammary epithelial cells.

The long-term feeding of a high-concentrate diet (the concentrate ratio is greater than 60 %) leads to mammary gland inflammatory response in ruminants and decreased quality in dairy cows and affects the robust development of the dairy industry. The main reason is closely related to elevated lipopolysaccharide (LPS) in the body. In this experiment, a bovine mammary epithelial cell line (MAC-T) was used as a model, and LPS at different concentrations (0 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, 1000 ng/ml, 10000 ng/ml) was added to the cells. The cell survival rate, oxidative stress indicators, total lipid droplet area, triglyceride content and key genes regulating lipid metabolism were detected by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT), assay kit, microscope observation and RT-PCR methods to explore the regulatory mechanism of mammary health and milk fat synthesis. The results showed that compared with those of the control group, the survival rates of cells were significantly decreased after 9 h of stimulation with 1000 ng/ml and 10000 ng/ml LPS (P<0.01). The contents of superoxide dismutase (SOD), catalase (CAT) and total antioxidant capacity (T-AOC) in cells were significantly decreased (P<0.05). Compared with that of the control group, the content of malondialdehyde (MDA) in cells was significantly increased (P<0.05) after stimulation with 10000 ng/ml LPS for 9 h. After 9 h of stimulation with 100 ng/ml, 1000 ng/ml and 10000 ng/ml LPS, the total lipid drop area and triglyceride (TG) content of MAC-T cells were significantly decreased (P<0.05). The expression levels of fatty acid synthesis-related genes Acetyl-CoA carboxylase (ACC) and Stearoyl-CoA desaturase 1 (SCD-1) were significantly decreased after 9 h of stimulation with 100 ng/ml, 1000 ng/ml and 10000 ng/ml LPS (P<0.05), while the expression levels of Fatty Acid synthetase (FAS) were significantly decreased after stimulation with 1000 ng/ml and 10000 ng/ml LPS (P<0.05). TG synthesis by the related gene Diacylglycerol acyltransferase-1 (DGAT1) was significantly lower than that of the control group after stimulation with 1000 ng/ml and 10000 ng/ml LPS for 9 h (P<0.05), and Diacylglycerol acyltransferase-2 (DGAT2) also showed a significant decrease after 10000 ng/ml LPS stimulation (P<0.05). In conclusion, adding different concentrations of LPS to MAC-T cells not only led to a decrease in cell activity, resulting in oxidative damage, but also affected fatty acid and TG synthesis, which may ultimately be closely related to the decrease in milk fat synthesis.

Genome-Wide Analysis of LncRNA in Bovine Mammary Epithelial Cell Injuries Induced by Escherichia Coli and Staphylococcus Aureus.

Escherichia coli and Staphylococcus aureus are two common pathogenic microorganisms that cause mastitis in dairy cows. They can cause clinical mastitis and subclinical mastitis. In recent studies, lncRNAs have been found to play an important role in the immune responses triggered by microbial inducers. However, the actions of lncRNAs in bovine mastitis remain unclear. The purpose of this study was to investigate the effects of bovine mammary epithelial cell injuries induced by treatment with E. coli and S. aureus, and to explore the lncRNA profile on cell injuries. The lncRNA transcriptome analysis showed a total of 2597 lncRNAs. There were 2234 lncRNAs differentially expressed in the E. coli group and 2334 in the S. aureus group. Moreover, we found that the E. coli and S. aureus groups of maternal genes targeted signaling pathways with similar functions according to KEGG and GO analyses. Two lncRNA-miRNA-mRNA interaction networks were constructed in order to predict the potential molecular mechanisms of regulation in the cell injuries. We believe that this is the first report demonstrating the dysregulation of lncRNAs in cells upon E. coli and S. aureus infections, suggesting that they have the potential to become important diagnostic markers and to provide novel insights into controlling and preventing mastitis.

Prevalence, aetiology and risk factors of mastitis of dairy cows kept under extensive management system in west Wollega, western Oromia, Ethiopia.

INTRODUCTION: Mastitis is an inflammation of the mammary gland that could either be clinical or subclinical, and can be caused by infectious agents. It has different aetiological pathogens such as Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli that pose different economic and health impacts. In Ethiopia, previous studies on mastitis have been focused on semi-intensive and intensive dairy production system. The objectives of this study were therefore to estimate prevalence, aetiology of causative pathogens and identify different risk factors of mastitis in extensive management systems. A total of 532 lactating cows were randomly selected for a cross-sectional study carried out in four districts of west Wollega, western Oromia, Ethiopia. RESULT: Based on the clinical examination and California mastitis test, 39.67% overall prevalence of mastitis on cow level was recorded. Of them, 16.70% and 22.70% were clinical and subclinical mastitis, respectively. Among 2,128 quarters examined 210 were blind teats. The associated risk factors age ≥8 years (16.35%), parity 1-3 calving (27.63%), milk yield 1-2 litres (21.98%), lactation stage <4 months (18.04%) and tick infestation (26.70%) were significantly associated in the occurrence of mastitis. Staphylococcus spp. (15.60%) were the dominant bacteria isolated from collected mastitic milk. CONCLUSION: Mastitis is an important disease for dairy cows kept under an extensive management system. Its occurrence is determined by risk factors like age, parity, milk yield, lactation stage and tick infestation. This study is indicated Staphylococcus species as the major bacteria isolated from mastitic milk. This study warrants further study on the prevalence, aetiology, economic impact and potential risk factors of mastitis in dairy cows kept in extensive production system.

The relationship of excessive energy deficit with milk somatic cell score and clinical mastitis.

Periparturient cows go through a period of immune suppression often marked by immune cell dysfunction. Further exacerbation of this dysfunction through early-lactation excessive energy deficit (EED) has been associated with increased susceptibility to infectious conditions such as mastitis. Our objective was to explore the association of milk somatic cell score (SCS) and clinical mastitis (CM) diagnosis in cows identified with EED, diagnosed using each of the following: blood and milk ß-hydroxybutyrate (BHB), milk predicted blood nonesterified fatty acid (mpbNEFA) concentrations, or milk de novo fatty acid (FA) relative percentages (rel %). We analyzed data collected from 396 multiparous Holstein cows from 2 New York farms in a prospective cohort study. Coccygeal vessel blood samples and composite milk samples were collected twice weekly from 3 to 18 days in milk (DIM) for a total of 4 time points per cow (T1, T2, T3, T4). Blood was analyzed using a hand-held meter, and milk was analyzed using Fourier-transform mid-infrared spectrometry for milk BHB and mpbNEFA concentrations, milk de novo FA rel %, and somatic cell count. Excessive energy deficit was diagnosed as blood BHB ≥ 1.2 mmol/L, milk BHB ≥ 0.14 mmol/L, mpbNEFA ≥ 0.55 mmol/L, or de novo FA ≤ 22.7 rel %, depending on the model. Clinical mastitis cultures were collected from 4 to 60 DIM by on-farm personnel. Incidence of hyperketonemia as determined by blood BHB was 13.4%, and incidence of CM was 23.9%. Separate repeated-measures ANOVA models were developed for each EED diagnostic analyte for parity groups 2, 3, and ≥4 to assess differences in SCS; t-test analyses were similarly used to assess the association of each diagnostic analyte with CM at each time point. For all diagnostic analytes, apart from milk BHB, cows diagnosed with EED tended to have lower SCS than their non-EED counterparts. This was especially apparent at T1 for all parity groups, and at T2, T3, and T4 for blood BHB and mpbNEFA. For EED diagnosis via mpbNEFA, mean SCS were lower in parity ≥4, with a difference in mean SCS between EED and non-EED animals of 0.7 SCS units, equating to a somatic cell count in EED animals approaching half that of non-EED (EED = 67,000 cells/mL, non-EED = 107,000 cell/mL). No important relationships were observed between CM diagnosis and blood BHB, milk BHB, or mpbNEFA. For de novo FA rel %, reductions in this analyte were noted before CM diagnosis at all time points. Although the relationship between EED and CM is still unclear, our findings suggest that cows in EED, diagnosed using blood BHB or mpbNEFA during the first 18 DIM, have a tendency toward lower SCS compared with their non-EED counterparts.

Mycoplasma bovis infection in dairy herds-Risk factors and effect of control measures.

As Mycoplasma bovis spreads to new countries and becomes increasingly recognized as a disease with major welfare and economic effects, control measures on dairy farms are needed. To minimize the risk of infection spread to naive herds, all possible risk factors for M. bovis infection should be identified and controlled. Mycoplasma bovis was first diagnosed in dairy cattle in Finland in 2012, and by January 2020, 86 Finnish dairy farms (<1.5%) supporting M. bovis infections were identified. We evaluated risk factors for M. bovis infection using a questionnaire provided to 40 infected and 30 control dairy farms. Control measures were advised for 19 of the infected dairy farms during visits by a veterinarian. The course of the infection on those farms was followed by analyzing calf nasal swabs with PCR for presence of M. bovis 4 times at 6-mo intervals. Control measures included culling of M. bovis mastitic cows, isolation of new calves from older animals after initial M. bovis mastitic cows had been culled, prevention of nose-to-nose contact with infected animals, early detection of mastitis cases using M. bovis PCR, and hygiene measures mainly related to milking, calf pens, feeding buckets, and teats. Farms implemented the control measures related to the isolation of calves or avoidance of nose-to-nose contact in various ways, according to farm structures and financial circumstances.In our study, the control measures recommended to the dairy farms appeared effective, such that 13 of 19 farms reached a low risk level during at least 3 consecutive negative samplings from calves, with no M. bovis mastitis detected subsequently. Among risk factors, insemination with an M. bovis-positive bull indicated a trend of increasing the odds of M. bovis infection on the farm in a multivariable logistic model. In contrast, higher herd average milk yield had an association with lower odds for M. bovis infection. Occurrence of other infectious diseases affecting several animals on the dairy farm in the previous 6 mo before M. bovis infection were more frequent on M. bovis-infected farms.

Associations of freestall design and cleanliness with cow lying behavior, hygiene, lameness, and risk of high somatic cell count.

The objective of this study was to investigate associations of freestall design and cleanliness with cow lying behavior, hygiene, lameness, and risk of new high somatic cell count (SCC). Cows from 18 commercial freestall dairy herds (22 ± 15 cows/farm; mean ± SD) in Ontario, Canada, were enrolled in a longitudinal study. Four hundred focal cows that were <120 d in milk, had no mastitis treatment in the last 3 mo, and had an SCC <100,000 cells/mL at their most recent milk test were selected for the study. Data on SCC were collected through Dairy Herd Improvement Association milk testing (at ~5-wk intervals). Each farm was visited 5 ± 3 d (mean ± SD) after each milk test until 3 tests were completed (~105 d), for a total of 3 observation periods per cow. Elevated SCC was used as an indicator of subclinical mastitis. An incident of new high SCC was defined as a cow having SCC >200,000 cells/mL at the end of an observation period, when SCC was <100,000 cells/mL at the beginning of that period. Lying behavior was recorded for 6 d after each milk sampling, using electronic data loggers. Cows were scored during each period for lameness (5-point scale, with scores ≥3 = lame), body condition score (BCS; 5-point scale; 1 = thin to 5 = fat), and hygiene (4-point scale). Stall cleanliness was assessed during each period with a 1.20 × 1.65-m metal grid, containing 88 squares. The grid was centered between stall partitions of every tenth stall on each farm, and the squares containing visible urine or fecal matter (or both) were counted. Cow lying time averaged 10.9 ± 1.9 h/d. On average, cows with low BCS (≤2.5) spent 37 ± 16.6 min/d less time lying down than high-BCS cows (≥4.0). On average, cows tended to spend 36 ± 18.3 min/d more time lying down in deep-bedded versus mattress-based stalls. Mean proportion of soiled squares per stall was 20.1 ± 0.50%. Across farms, cow lying time decreased as the proportion of soiled squares per stall increased. A difference in daily lying time of ~80 more min/d was modeled for cows housed in barns with the cleanest stalls compared with those with the dirtiest stalls. Higher neck rail height [for every 1 SD (10 cm) increase] increased the odds (odds ratio = 1.5) of cows having a dirty upper leg-flank and udder. The odds of a cow having a dirty upper leg-flank, udder, and lower legs were 1.5, 2.0, and 1.9 times greater, respectively, for cows housed with dirtier stalls. Also, cows housed on farms with dirtier stalls had 1.3 times greater odds of being lame at the time of observation. Over the study period, 50 new high-SCC cases were detected, resulting in an incidence rate of 0.45 cases of new high SCC per cow-year at risk. No measured factors were detected to be associated with risk of a new high SCC. Overall, our results confirm that cows lie down longer in cleaner and more comfortable environments. Further, these results highlight the need for improved stall cleanliness to optimize lying time and potentially reduce lameness.

Prototheca from bovine milk and associated minimal algaecide concentration of chlorhexidine and povidone-iodine in Taiwan.

OBJECTIVE: Protothecal mastitis has emerged and affected the dairy industries globally in last decades; however, little is known about the occurrence of Prototheca in Taiwan. The present study identified the Prototheca isolated from bovine milk and revealed the algaecide efficacy of 2 disinfectants. MATERIAL AND METHODS: From 2011 to 2014, a total of 176 Prototheca isolates were obtained from 10 625 milk samples collected from dairy cows in 24 Taiwanese dairy farms. Of these, 149 isolates were identified by biochemical tests and genotype differentiation with a specific PCR protocol. Minimal algaecide concentration (MAC) of chlorhexidine and povidone-iodine were obtained from 16 isolates by microdilution method. RESULTS: Almost all (n = 147) isolates were P. zopfii, and remaining 2 isolates were P. wickerhamii and unidentified, respectively. Based on molecular genotyping, all P. zopfii isolates belonged to genotype 2. The MAC of 2 commonly used disinfectants, chlorhexidine and povidone-iodine, against 16 P. zopfii isolates was 1.56-3.13 and 48.83-390.63 µg/ml, respectively. CONCLUSION: This report revealed for the first time the prevalence and the predominant P. zopfii genotype 2 of bovine protothecal infection in Taiwan. In vitro algaecide efficacy indicated that both tested disinfectants were effective against P. zopfii.

Basal Levels of CD18 Antigen Presenting Cells in Cow Milk Associate with Copy Number Variation of Fc Gamma Receptors.

Differentiation of cells by flow cytometry provides informative somatic cell counts (SCCs) that allow analyzing leukocyte population patterns in udder infections of different etiologies. Postulating that this approach also enhances the statistical power to detect genetic variants linked to cell levels in milk of healthy mammary glands, we used monoclonal antibodies anti-CD18, anti-CD4, anti--CD14, and anti-PMN to count cells presenting these surface antigens, and performed a genome-wide association study of these counts in 125 Israeli Holsteins genotyped using SNP BeadChips. We identified an informative haplotype of 15 SNPs in the centromeric end of BTA3 that was strongly associated with CD18 cells (p < 2.3 × 10-9). Within this region, examination of the network of genes interacting with ITGB2 (CD18) indicated an Fc-γ-receptor gene cluster, including FCGR2A (CD32). Sanger-sequence analysis of FCGR2s-linked exon 3 variation to CD18 counts. Meta-analysis of RNA-Seq data revealed a significant negative correlation (R = -0.51) between expression of CD32 and CD18 in milk. Assembly of DNA-Seq reads uncovered FCGR copy-number variation and a variant, designated V7, was abundant in dairy cattle, probably reflecting adaptation to selection pressure for low SCC in Holstein milk.

A cross sectional study of prevalence and risk factors associated with subclinical mastitis and intramammary infections, in dairy herds linked to milk collection centers in Rwanda.

The objective of this cross-sectional study was to investigate prevalence, causative udder pathogens and their antimicrobial resistance (AMR), as well as cow and herd risk factors associated with subclinical mastitis (SCM = cows with at least one udder quarter with california mastitis test (CMT) score > 2) and intramammary infections (IMI) caused by Staphylococcus(S.) aureus or Non aureus staphylococci (NAS) in dairy cows linked to Milk Collection Centers (MCCs) in Rwanda. Screening for SCM with the CMT was done on 572 cows from 404 herds linked to two MCCs in each of four provinces. Milk from udder quarters with a CMT score ≥3 (scale 1-5) was sampled for bacteriological analysis. Antimicrobial resistance was evaluated in 60 selected S. aureus isolates. Multivariable mixed effect and ordinary logistic regression analyses were performed to identify cow and herd level risk factors associated with SCM, NAS or S. aureus IMI in cows. The prevalence of SCM was 37.3 % at quarter level and 62.0 % at cow level. Bacteria were isolated 73.7 % of the cultured milk samples, whereas 23.3 % were culture-negative and 3.0 % were contaminated. Staphylococcus aureus and NAS were the most prevalent pathogens, representing more than half of all bacteriological findings. Staphylococcus chromogenes and S. epidermidis were the most prevalent NAS species identified. Of the S. aureus strains 83.3 % were resistant for penicillin, 100 % for clindamycin and 20 % tetracycline. The risk factor analysis showed that an increased stage of lactation, dirty udder and legs in single cow herds and lack of calf suckling the dam, dirty udder and legs and lack of feeding cow after milking in multiple cow herds were significantly (P < 0.05) associated with an increased odds of SCM. Similarly, increasing stage of lactation in single cow herds, and housing cows in individual cattle kraal or on earthen floor and hand washing between cows during milking in multiple cow herds were associated with increased odds for NAS IMI. Poor hygiene of milking area in single cow herds and absence of foremilk stripping in multiple cow herds were significantly (P < 0.05) associated with increased odds for S. aureus IMI in cows. In conclusion, SCM prevalence was high across MCCs. The majority of identified pathogens were contagious in nature and they exhibited resistance to penicillin. Control of the identified risks factors and improved biosecurity through adoption of best practices, and farmer training could contribute to lowering SCM prevalence in Rwanda.

In vitro algicidal effect of polypyrrole on Prototheca species isolates from bovine mastitisAlgicidal activity of polypyrrole on Prototheca spp.

Algae of the genus Prototheca are microorganisms involved in the occurrence of diseases in humans and animals. In bovine species, Prototheca spp. cause environmental mastitis, productive losses in dairy herds, mainly leading to the discard of infected cows. Currently, there are no effective anti-Prototheca spp. drugs to combat this infection. Thus, the search for an efficacious therapy for Prototheca spp. infections have become essential. Highly soluble polypyrrole (Ppy) is a molecule with known antimicrobial activity. This study aimed to characterize Prototheca spp. isolates from bovine mastitis as well as to evaluate the susceptibility profile and to verify the morphological alterations on Prototheca spp. isolates treated with Ppy. In this research, 36 Brazilian isolates of Prototheca spp. were characterized by restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) assay for the mitochondrial cytB gene. Additionally, Ppy algicidal activity against these isolates of Prototheca spp. was assessed by minimal microbicidal concentration method in microplates. Further, scanning electron microscopy (SEM) was performed in order to verify the morphological alterations on Prototheca spp. isolates in response to Ppy. The isolates were characterized as belonging to Prototheca zopfii genotype 2 (35/36) and Prototheca blaschkeae (1/36). Ppy had an algicidal effect on all isolates tested at concentrations ranging from 15.625 µg ml-1 to 62.5 µg ml-1. SEM showed changes on planktonic and sessile P. zopfii, including a decrease of the number of cells with the presence of an amorphous substance involving the cells. The algicidal activity of Ppy suggests the therapeutic potential of this molecule in the prevention and treatment of Prototheca spp. in bovine mastitis.

Prevalence of sub-clinical mastitis and its association with milking practices in an intensive dairy production region of Uganda.

A cross-sectional study was conducted to investigate the risk factors for sub-clinical mastitis (SCM) in Mbarara District, an intensive dairy production region of Uganda where hand-milking is dominant. In 30 farms, herd-level milking practices and SCM prevalence were studied. The SCM prevalences were 68.6% (417/608, 95% confidence interval (CI): 64.9-72.2%) and 39.2% (946/2,411, 37.3-41.2%) at the cow- and quarter-levels, respectively. A preventive factor for SCM was cow calmness at the end of milking (OR: 0.20, 95%CI: 0.05-0.79, P=0.021); a risk factor was rough teat-end (OR: 1.75, 95%CI: 1.14-2.68, P=0.011). Good cow hygiene was negatively associated with environmental mastitis (P=0.002). Appropriate hand-milking practices that avoid teat damage are expected to reduce SCM in Uganda.

Risk factors of S. aureus intramammary infection in pre partum dairy heifers under grazing conditions and molecular characterization of isolates from heifers and cows.

The aims of the research reported here were to identify potential risk factors associated with the presence of Staphylococcus aureus intramammary infection (IMI) in pre partum dairy heifers on 17 dairy farms from three provinces of Argentina and to characterize, at molecular level, isolates from those heifers and lactating cows from two selected herds. A total of 1474 heifers and 4878 lactating cows were studied. The prevalence of Staphylococcus aureus IMI in the heifers, heifers at quarter level and lactating cow mammary quarters was 14.41, 4.82, and 14.65%, respectively. Univariate analysis showed the key variables associated with S. aureus IMI presence in the heifers were: S. aureus IMI prevalence in cows of the lactating herd, the time calves stayed with their dam after birth, the calf rearing system, the place of rearing (own farm or other dairy farm) and fly control on the farm. None of the variables included in the multivariable analysis was associated with the presence of S. aureus IMI in the pre partum heifers, probably due to low variability among management practices used by the farms for rearing the heifer calves. At the molecular level, S. aureus isolates were grouped into three main PFGE clusters and several genotypes within the clusters. Isolates from mammary secretion of pre partum heifers and milk of lactating cows comprised different PFGE clusters in both herds, although two exceptions occurred. The absence of gene fnbpB, which codifies for a virulence factor protein involved in cell invasion by S. aureus, was significantly more frequent in pre partum heifer secretion isolates than in isolates from lactating cow milk. These results suggest that, under these management conditions, isolates from mammary secretions of pre partum heifers do not originate from the milk of lactating cows, but rather other sources to which the heifer is exposed.

Microbiological and molecular detection of Mycoplasma bovis in milk samples from bovine clinical mastitis / Detecção microbiológica e molecular de Mycoplasma bovis em amostras de leite oriundas de mastite clínica bovina

The genus Mycoplasma includes more than 200 bacterial species that cause disease in animals. It is responsible for causing mastitis in bovines and may be related to other manifestations, such as arthritis and pneumonia in calves and heifers. The present study aimed to detect Mycoplasma bovis isolated from milk samples of bovine clinical mastitis, and to compare the isolation rates in two culture media: Hayflick and SP4. An initial screening was performed in order to detect the presence of the class Mollicutes in 1166 milk samples from clinical mastitis by the conventional Polymerase Chain Reaction (PCR) technique. According to the 1166 milk samples evaluated, 8.6% (100/1166) were positive to class Mollicutes. Regarding molecular analyses, 1.1% (13/1166) of conventional PCR for positive M. bovis was obtained and 0.9% (11/1166) in real-time PCR. The results of the microbiological culture of the 100 samples previously screened demonstrated that 6% (6/100) of colony growth have been developed when using the Hayflick medium, and 11% (11/100) when using the SP4 medium (including the positive on Hayflick medium). Concerning the 11 isolates obtained in the microbiological culture, conventional PCR confirmed M. bovis in nine of them, and two cultures were negative. In the phylogenetic analysis of the isolates, all of them were grouped in M. bovis and M. agalactiae clusters. The results confirmed the importance of the presence of M. bovis in the etiology of bovine clinical mastitis and reinforced the need for further studies to elucidate other Mycoplasma species that may be involved in bovine clinical mastitis in Brazil.(AU)

O gênero Mycoplasma inclui mais de 200 espécies que causam doenças nos animais. É responsável por quadros de mastite em bovinos, podendo também estar relacionado à outras manifestações como artrite e pneumonia em bezerros e novilhas. O presente estudo objetivou a detecção de Mycoplasma bovis isolados a partir de amostras de leite de mastite clínica bovina, bem como, a comparação da taxa de isolamento em dois meios de cultura: Hayflick e SP4. Para efeito de triagem amostral, foram avaliadas quanto à presença da classe Mollicutes 1166 amostras de leite de casos de mastite clínica pela técnica de PCR convencional. Das 1166 amostras de leite avaliadas, 8,6% (100/1166) foram positivas à classe. Nas análises moleculares, obteve-se 1,1% (13/1166) de positividade para Mycoplasma bovis na PCR convencional e 0,9% (11/1166) na PCR em tempo real. Os resultados do cultivo microbiológico das 100 amostras triadas previamente demonstraram 6% (6/100) de crescimento de colônias ao se utilizar o meio Hayflick e 11% (11/100) ao se utilizar o meio SP4 (incluindo as positivas ao primeiro). A partir dos 11 isolados obtidos no cultivo microbiológico, a PCR convencional confirmou Mycoplasma bovis em nove deles, e dois foram negativos para o agente. Na análise filogenética dos isolados, todos agruparam no cluster Mycoplasma bovis e Mycoplasma agalactiae. Frente aos resultados, ressalta-se a importância da presença de Mycoplasma bovis na etiologia da mastite clínica bovina e reforça a necessidade de estudos mais aprofundados para a elucidação de outras espécies de micoplasmas que possam estar envolvidas na mastite bovina.(AU)

Inhibition of the growth of major mastitis-causing pathogens by non-aureus Staphylococcus isolates using the cross-streaking method / Comunicação: Inibição do crescimento dos principais patógenos causadores de mastite por isolados de estafilococos não aureus pelo método cross-streaking

O objetivo do presente estudo foi avaliar a capacidade de estafilococos não aureus (NAS) isolados de diferentes nichos ecológicos (leite, ambiente e ápice do teto), associados a vacas leiteiras, de inibir os principais agentes etiológicos da mastite bovina (Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis e Escherichia coli). Neste estudo, 38 isolados NAS de diferentes nichos ecológicos foram avaliados quanto à capacidade de inibir o crescimento in vitro de importantes patógenos causadores de mastite pelo método cross-streaking. No total, 19 (50%) isolados de NAS (oito isolados de S. chromogenes, 10 de S. fleurettii e um de S. haemolyticus) apresentaram inibição contra os principais patógenos causadores de mastite. No entanto, a inibição dos patógenos causadores da mastite bovina por isolados de NAS foi maior contra bactérias Gram-positivas. Além disso, o presente estudo não sugeriu que os nichos ecológicos influenciam a capacidade do NAS de inibir os principais patógenos causadores da mastite bovina. Com base nesses resultados, concluiu-se que certos isolados de NAS apresentam potencial efeito protetor contra os principais patógenos da mastite, pelo menos in vitro.(AU)

Three Novel Players: PTK2B, SYK, and TNFRSF21 Were Identified to Be Involved in the Regulation of Bovine Mastitis Susceptibility via GWAS and Post-transcriptional Analysis.

Bovine mastitis is a common inflammatory disease caused by multiple factors in early lactation or dry period. Genome wide association studies (GWAS) can provide a convenient and effective strategy for understanding the biological basis of mastitis and better prevention. 2b-RADseq is a high-throughput sequencing technique that offers a powerful method for genome-wide genetic marker development and genotyping. In this study, single nucleotide polymorphisms (SNPs) of the immune-regulated gene correlative with mastitis were screened and identified by two stage association analysis via GWAS-2b-RADseq in Chinese Holstein cows. We have screened 10,058 high quality SNPs from 7,957,920 tags and calculated their allele frequencies. Twenty-seven significant SNPs were co-labeled in two GWAS analysis models [Bayesian (P < 0.001) and Logistic regression (P < 0.01)], and only three SNPs (rs75762330, C > T, PIC = 0.2999; rs88640083, A > G, PIC = 0.1676; rs20438858, G > A, PIC = 0.3366) were annotated to immune-regulated genes (PTK2B, SYK, and TNFRSF21). Identified three SNPs are located in non-coding regions with low or moderate genetic polymorphisms. However, independent sample population validation (Case-control study) data showed that three important SNPs (rs75762330, P < 0.025, OR > 1; rs88640083, P < 0.005, OR > 1; rs20438858, P < 0.001, OR < 1) were significantly associated with clinical mastitis trait. Importantly, PTK2B and SYK expression was down-regulated in both peripheral blood leukocytes (PBLs) of clinical mastitis cows and in vitro LPS (E. coli)-stimulated bovine mammary epithelial cells, while TNFRSF21 was up-regulated. Under the same conditions, expression of Toll-like receptor 4 (TLR4), AKT1, and pro-inflammatory factors (IL-1ß and IL-8) were also up-regulated. Interestingly, network analysis indicated that PTK2B and SYK are co-expressed in innate immune signaling pathway of Chinese Holstein. Taken together, these results provided strong evidence for the study of SNPs in bovine mastitis, and revealed the role of SYK, PTK2B, and TNFRSF21 in bovine mastitis susceptibility/tolerance.

Association of dry matter intake and energy balance prepartum and postpartum with health disorders postpartum: Part II. Ketosis and clinical mastitis.

The main objective of this study was to determine the association of dry matter intake as percentage of body weight (DMI%BW) and energy balance (EB) prepartum (-21 d relative to parturition) and postpartum (28 d) with ketosis (n = 189) and clinical mastitis (n = 79). For this, DMI%BW and EB were the independent variables and ketosis and clinical mastitis were the dependent variables. A secondary objective was to evaluate prepartum DMI%BW and EB as predictors of ketosis and clinical mastitis. For this, ketosis and clinical mastitis were the independent variables and DMI%BW and EB were the dependent variables. Data from 476 cows from 9 experiments were compiled. Clinical mastitis was diagnosed if milk from 1 or more quarters was abnormal in color, viscosity, or consistency, with or without accompanying heat, pain, redness, or swelling of the quarter or generalized illness, during the first 28 d postpartum. Ketosis was defined as the presence of acetoacetate in urine that resulted in any color change [5 mg/dL (trace) or higher] in the urine test strip (Ketostix, Bayer, Leverkusen, Germany). Cows that developed ketosis had lesser DMI%BW and lesser EB on d -5, -3, -2, and -1 than cows without ketosis. Each 0.1-percentage point decrease in the average DMI%BW and each 1-Mcal decrease in the average of EB in the last 3 d prepartum increased the odds of having ketosis by 8 and 5%, respectively. Cut-offs for DMI%BW and EB during the last 3 d prepartum to predict ketosis were established and were ≤1.5%/d and ≤1.1 Mcal/d, respectively. Cows that developed ketosis had lesser postpartum DMI%BW and EB and greater energy-corrected milk (ECM) than cows without ketosis. Cows that developed clinical mastitis had lesser DMI%BW but similar prepartum EB compared with cows without clinical mastitis. Each 0.1-percentage point decrease in the average DMI%BW and each 1-Mcal decrease in the average EB in the last 3 d prepartum increased the odds of having clinical mastitis by 10 and 8%, respectively. The average DMI%BW and EB during the last 3 d prepartum produced significant cut-offs to predict clinical mastitis postpartum, which were ≤1.2%/d and ≤1.0 Mcal/d, respectively. Cows that developed clinical mastitis had lesser postpartum DMI%BW from d 3 to 15 and on d 17; greater EB on d 18, from d 21 to 23, and on d 26; and lesser ECM. The main limitation in this study is that the time-order of disease relative to DMI%BW and ECM is inconsistent such that postpartum outcomes were measured before and after disease, which was diagnosed at variable intervals after calving. In summary, measures of prepartum DMI were associated with and were predictors of ketosis and clinical mastitis postpartum, although the effect sizes were small.

Production of lipid mediators in mastitic milk of cow.

Bovine mastitis is one of the most prevalent and costly diseases in the dairy industry. Lipid mediators are signaling molecules which coordinately and intricately modulate inflammation. They are produced from polyunsaturated fatty acids (PUFAs) in the cellular membrane via several enzymes including cyclooxygenase (COX) and lipoxygenase (LOX). In the present study, we performed comprehensive analysis of lipid production in milk obtained from clinical or subclinical mastitic cows using liquid chromatography/mass spectrometry. We detected 26, 24, and 40 kinds of lipid constantly in healthy, subclinical, and clinical mastitic milk, respectively. In clinical mastitic milk, the amount of a major n-6 PUFA, arachidonic acid (AA), tended to increase, whereas amounts of major n-3 PUFAs, eicosapentaenoic acid and docosahexaenoic acid, tended to decrease. The amounts of several AA-derived lipids including COX-catalyzed prostaglandin (PG) D2 and PGE2 , and LOX-catalyzed leukotriene (LT) B4 were increased in clinical mastitic milk. Although subclinical mastitic milk represented similar trend of lipid production to healthy milk, amounts of several lipids such as LTD4 , 14,15-dihydroxyeicosatrienoic acid, and 14-epoxyeicosatrienoic acid changed. These findings would be helpful for better understanding of mastitis pathology and give us some insights to develop a new diagnostic and therapeutic strategy.

Caracterização fenotípica pela técnica de MALDI-TOF de Staphylococcus coagulase negativa isolados de mastite bovina em rebanhos leiteiros do Sul de Minas Gerais / Phenotypic characterization by the MALDI-TOF technique of Staphylococcus coagulase negative isolated from bovine mastitis in dairy herds of the South of Minas Gerais

O objetivo deste trabalho foi identificar espécies de Staphylococcus coagulase negativa em leite de vacas leiteiras com mastite por testes fenotípicos e pela técnica de MALDI-TOF MS. Foram utilizadas 85 isolados de estafilococos provenientes de leite de vacas com mastite. Todos os isolados foram caracterizados fenotipicamente por avaliação da morfologia das colônias, coloração de Gram, testes de oxidase, catalase e coagulase. Posteriormente, foram analisados os espectros de proteínas gerados no MALDI-TOF MS, seguida pela separação e detecção de íons pelo tempo de voo (TOF).Foram identificadas sete espécies, sendo a de maior ocorrência S. chromogenes 65 (76%), seguida por, S. hyicus 5 (6%), S. epidermidis 4 (5%).A técnica de MALDI-TOF demonstrou-se efetiva na caracterização de espécies de Staphylococcus causadores de mastite bovina.

Levantamento de agentes causadores de mastite e sua resistência a antibióticos / Survey agents of mastitis causes and their resistance to antibiotics

Dentre as enfermidades que acometem o rebanho leiteiro a mastite é a que apresenta o maior impacto econômico, assim com o objetivo de identificar os agentes causadores de mastite e de avaliar a sua resistência a antibióticos, analisou-se 119 amostras de leite, coletadas de 47 unidades produtoras distribuídas em 10 municípios da microrregião de Pelotas no RS. Das amostras analisadas, 76,7% agentes foram classificados como Gram positivos (G+) e 23,3% como Gram negativos (G-) . Das amostras G+ analisados, os que obtiveram maior resistência por parte dos microrganismos isolados, classificados como Gram positivos, foram trimetropin (70% ), seguido da penicilina (65,2%). Já os antimicrobianos com maior resistência por parte dos isolados de G-, foram cefalotina (85,7%), seguido de trimetropin (80,9%). Por fim, as 16 amostras de Staphylococcus coagulase positiva, foram 100% resistentes a estreptomicina. Verifica-se assim a importância de um adequado manejo para o correto uso dessas drogas.