Molecular regulatory mechanism of key LncRNAs in subclinical mastitic cows with folic acid supplementation.

BACKGROUND: Folic acid is a water-soluble B vitamin (B9), which is closely related to the body's immune and other metabolic pathways. The folic acid synthesized by rumen microbes has been unable to meet the needs of high-yielding dairy cows. The incidence rate of subclinical mastitis in dairy herds worldwide ranged between 25%~65% with no obvious symptoms, but it significantly causes a decrease in lactation and milk quality. Therefore, this study aims at exploring the effects of folic acid supplementation on the expression profile of lncRNAs, exploring the molecular mechanism by which lncRNAs regulate immunity in subclinical mastitic dairy cows. RESULTS: The analysis identified a total of 4384 lncRNA transcripts. Subsequently, differentially expressed lncRNAs in the comparison of two groups (SF vs. SC, HF vs. HC) were identified to be 84 and 55 respectively. Furthermore, the weighted gene co-expression network analysis (WGCNA) and the KEGG enrichment analysis result showed that folic acid supplementation affects inflammation and immune response-related pathways. The two groups have few pathways in common. One important lncRNA MSTRG.11108.1 and its target genes (ICAM1, CCL3, CCL4, etc.) were involved in immune-related pathways. Finally, through integrated analysis of lncRNAs with GWAS data and animal QTL database, we found that differential lncRNA and its target genes could be significantly enriched in SNPs and QTLs related to somatic cell count (SCC) and mastitis, such as MSTRG.11108.1 and its target gene ICAM1, CXCL3, GRO1. CONCLUSIONS: For subclinical mastitic cows, folic acid supplementation can significantly affect the expression of immune-related pathway genes such as ICAM1 by regulating lncRNAs MSTRG.11108.1, thereby affecting related immune phenotypes. Our findings laid a ground foundation for theoretical and practical application for feeding folic acid supplementation in subclinical mastitic cows.

The effect of pre-calving injection of trace mineral supplements on periparturient disease incidence in pasture based dairy cows.

Trace minerals, have a role in immune function and a trace mineral supplement (TMS) can improve animal health in dairy herds. This prospective randomised clinical study assessed whether subcutaneous injection of 5.5 mL of TMS (40 mg zinc, 10 mg manganese, 5 mg selenium, 15 mg copper per mL), 14-28 days before planned start of calving (PSC) reduced clinical mastitis (CM), subclinical mastitis (SCM) and purulent vaginal discharge (PVD). From four farms, half of 1700 cows stratified on somatic cell count, age and breed were randomly allocated to treatment or no treatment. Occurrence of CM from - 7 to PSC + 100 days, SCM at PSC + 60 days and PVD at PSC + 24 days was analysed using survival analysis and Bayesian generalised mixed multivariable models. From -7 to PSC +30 days, TMS reduced the adjusted hazard ratio (HR) for CM at quarter and cow level (P < 0.001), with no evidence for an effect beyond 30 days. The adjusted OR (and 95% highest density interval, HDI) for the effect of TMS on CM from -7 to PSC +30 days was 0.40 (95% HDI, 0.26-0.63) at quarter level, 0.51 (95% HDI, 0.38-0.69) at cow level and for SCM, 0.72 (95% HDI, 0.54-0.95). The difference in CM incidence from TMS at the cow level was -2.0% (95% HDI, -3.4 to -1.1%) and -1.2% (95% HDI, -3.2 to - 0.6%) at quarter level. No clear effect was identified of TMS on cumulative incidence of PVD.

Subclinical mastitis in dairy cows in south-Asian countries: a review of risk factors and etiology to prioritize control measures.

Mastitis is a major production disease, causing significant economic losses for dairy farmers in South-Asian countries, as well as other parts of the world. Udder health control programs (UHCP) have been established in developed countries as an effective strategy for mastitis control but have not yet been introduced in South-Asian low-income countries like Bangladesh, India, Pakistan, and Sri Lanka. To launch UHCP successfully in dairy herds in South-Asia, it is important to know the current prevalence and risk factors for subclinical mastitis (SCM). Therefore, a narrative literature review was conducted with the aim to describe the dairy sector, the prevalence of SCM and its causal agents, risk factors for mastitis occurrence and the control measures suggested by different studies conducted in the selected countries. The literature revealed that India had the highest cattle population. Milking was mainly done by hand in all of the studied countries. Stall feeding was done in Bangladesh and Sri Lanka and limited access to grazing was also reported in some farms in India and Pakistan. There was substantial variation in the prevalence of SCM between studies in all 4 countries, ranging from about 20% to about 80%, but the average prevalence across all studies was high (50%). The most common causal agents for SCM were non-aureus staphylococci (NAS), Staphylococcus (S.) aureus, Streptococcus spp. and Escherichia (E.) coli. The management related risk factors reported for SCM were stall feeding of cows, a higher stock density, cracked floors, open drains, the presence of flies, poor drainage, peri-parturient diseases, infrequent dung removal and earth floors. The control measures suggested in these studies were to improve the hygiene and sanitation of cows, to improve the cleanliness of farms and milker's hands, to apply dry cow therapy, supplementing micronutrients and routine screening for SCM combined with taking intervention measures like isolation of cows or milking infected cows last, and proper treatment. Also, full hand milking, complete milking, machine milking, and providing feed and water immediately after milking have been recommended. Finally, we show that current literature often studies the same set of (non-manageable) risk factors, so more research is needed to obtain a comprehensive picture of the determinants of SCM. Randomized controlled trials are needed to truly quantify the effect of intervention under field conditions. Altogether, our work gives an overview of the udder health situation in South-Asia and provides the basis for the design of UHCP in this region.

Lactococcus lactis subsp lactis CRL1655 and Schleiferilactobacillus perolens CRL1724 inhibit the adherence of common bovine mastitis pathogens to mammary gland cells, without causing histological changes in the mammary gland.

AIMS: The present work assessed the ability of two selected lactic acid bacteria (LAB) strains (Schleiferilactobacillus perolens CRL1724 and Lactococcus lactis subsp. lactis CRL1655) to inhibit the adherence of bovine mastitis pathogens to mammary epithelial cells (MAC-T) and their effects (if any) on the structure of the gland after intramammary inoculation at dry-off. METHODS AND RESULTS: Established bovine mammary epithelial cells (MAC-T) were used to assess the LAB strains' ability to inhibit the adherence of bovine mastitis pathogens. Monolayers of MAC-T cells were co-cultured with the LABs and then individual pathogen was added. Both strains prevented the adherence of S. aureus RC108, S. chromogenes, S. uberis UT102 and E. coli ATCC 35218. Adherence of the latter two pathogens was inhibited most strongly in vitro. To evaluate the effect of the LAB on the structure of the bovine udders, quarters were intramammary inoculated with the LAB mixture at dry-off. After slaughtering, the teats were dissected and histopathologically analysed. No modifications were identified post-inoculation in the structure of the epithelial, subepithelial and connective tissues of the mammary gland. CONCLUSIONS: Probiotic strains L. lactis subsp lactis CRL1655 and S. perolens CRL1724 were both able to inhibit the adherence of a number of bovine mastitis pathogens in vitro, and that the intramammary inoculation of these strains at the established dose and concentration did not cause significant alterations in the mammary epithelium nor had undesirable effects on tissues, and may therefore be considered harmless. SIGNIFICANCE AND IMPACT OF STUDY: The promising findings demonstrated in this work support the potential of probiotic micro-organisms as a natural and effective alternative to prevent bovine mastitis during the dry-off period.

Evaluation of fully oxidised ß-carotene as a feed ingredient to reduce bacterial infection and somatic cell counts in pasture-fed cows with subclinical mastitis.

AIMS: To assess the effect of oral supplementation with fully oxidised ß-carotene (OxBC) on bacteriological cure, the incidence of clinical mastitis, and somatic cell counts (SCC) in pasture-fed cows with subclinical intramammary infection. METHODS: Cows from four dairy herds were enrolled in early lactation if they had quarter-level SCC >200,000 cells/mL and they had a recognised bacterial intramammary pathogen in one or more quarters. They were randomly assigned to be individually fed from Day 0, for a mean of 40 days, with 0.5 kg of a cereal-based supplementary feed that either contained 300 mg of OxBC (treatment; n = 129 quarters) or did not (control; n = 135 quarters). Quarter-milk samples were collected on Days 21 and 42 for microbiology and SCC assessment. Bacteriological cure was defined as having occurred when the bacteria present on Day 0 were not isolated from samples collected on Days 21 or 42. A new intramammary infection was defined as a bacterial species isolated either on Day 21 or 42 differing from that isolated on Day 0. Clinical mastitis was diagnosed and recorded by herdowners up to Day 42. RESULTS: The bacteriological cure rate was greater for quarters from cows in the treatment group (13.9 (95% CI = 4.1-23.7)%) than for cows in the control group (6.9 (95% CI = 4.8-9.1)%; p = 0.02). The percentage of quarters that developed a new intramammary infection at Day 21 or 42 was greater for cows in the treatment group (17.9 (95% CI = 6.7-29.1)%) than for cows in the control group (13.0 (95% CI = 4.3-21.8)%; p < 0.01). The prevalence of quarters that were infected on Day 42 was less in cows in the treatment group (79.9 (95% CI = 62.3-97.6)%) than the control group (88.2 (95% CI = 78.4-97.9)%; p = 0.009). The incidence of quarters diagnosed with clinical mastitis by Day 42 was lower in cows from the treatment group (1/129 (0.78 (95% CI = 0.02-4.24)%)) than in cows from the control group (6/135 (4.44 (95% CI = 1.65-9.42)%; p = 0.03)). Mean quarter-level SCC was not statistically different between treatment groups (p = 0.34). CONCLUSIONS AND CLINICAL RELEVANCE: Feeding 300 mg/cow/day of OxBC resulted in a higher bacteriological cure rate, a lower prevalence of intramammary infection, and a lower incidence of clinical mastitis compared to untreated controls. However new intramammary infections increased in treated cows, and the magnitude of the increased bacteriological cure was low, resulting in 80% of cows remaining infected at Day 42. Therefore treatment with OxBC should be considered as an adjunct to other mastitis control measures.

Efficacy of Panax ginseng extract combined with cephalexin as a dry cow therapy.

Our objective was to evaluate the efficacy of intramammary administration, at drying-off, of a Panax ginseng extract (PGe) combined with cephalexin (Ceph) on the post-calving bacteriological cure rate of pre-existing intramammary infections (IMI) and on the occurrence of new IMI during the dry period. In addition, milk yield and somatic cell count (SCC) in the post-treatment lactation were evaluated. One hundred and eight late-lactation cows were randomly divided into two experimental groups and were treated at drying-off with Ceph alone or PGe combined with Ceph.Cure rates for IMI present at drying-off were similar for both treatments (OR = 0.95, 95% CI = 0.33-2.74). Cure rates for Staphylococcus aureus were lower (OR = 15.4, 95% CI = 1.66-142.52) in quarters treated with PGe + Ceph than in those treated with Ceph alone. Intramammary infusion of PGe + Ceph at drying-off had no effect on preventing new dry period IMI (OR = 0.75, 95% CI = 0.38-1.51), compared with infusion of Ceph alone. Milk production and SCC in the ensuing lactation were not affected by PGe + Ceph treatment. In conclusion, addition of PGe to dry cow therapy did not show any advantage over the use of dry cow therapy alone.

Eficácia da suplementação oral com 1, 3-1, 6 betaglucano proveniente de Saccharomyces cerevisiae no controle da mastite bovina / [Efficiency of oral supplementation of 1. 3-1. 6 beta-glucan from saccharomyces cerevisiae on the control of bovine mastitis]

A mastite bovina, uma das principais doenças do rebanho leiteiro, caracteriza-se por um processo inflamatório no úbere. A inviabilidade econômica, o impacto ambiental negativo e os resíduos antimicrobianos têm estimulado a pesquisa de outros tratamentos alternativos para a prevenção e o tratamento de doenças na bovinocultura leiteira. O betaglucano é um agente imunomodulador com potencial ação preventiva para doenças infecciosas, inclusive a mastite. Este estudo teve como objetivo avaliar a eficácia do uso do betaglucano, por meio de administração oral, em animais em lactação. Foram utilizadas 20 vacas lactantes, distribuídas em dois grupos, um controle e um tratamento, com 10 animais em cada grupo. O grupo tratamento recebeu 5g/dia, durante 60 dias, de 1,3-1,6 betaglucano isolado da parede celular de Saccharomyces cerevisiae diluído em ração após a ordenha, enquanto o grupo controle recebia somente a ração. Foram realizados os testes de California Mastitis Test (CMT), contagem de células somáticas (CCS), produção de leite e percentual de gordura e proteína no leite. Não houve diferença estatisticamente significativa entre os grupos quanto à CCS, ao CMT, à composição do leite ou produção. Não se observou, portanto, eficácia do uso do betaglucano purificado, administrado por via oral, no controle e na prevenção da mastite em vacas leiteiras, quando comparadas com o grupo controle. Atribuem-se esses resultados, principalmente, à degradação ruminal do produto testado. Sugerem-se, portanto, mais pesquisas utilizando o 1,3-1,6 betaglucano purificado de parede de S. cerevisiae por outras vias de administração, tais como intramamária e subcutânea.(AU)

Bovine mastitis, one of the main diseases of dairy herds, is characterized by an inflammatory process in the udder. The economic and environmental impacts, as well as the residues of antimicrobial drugs have stimulated the research of novel alternative treatments for the prevention and treatment of diseases in dairy production cows. The beta-glucan is an immunomodulator agent, with potential preventive action for infectious diseases, including mastitis. This study aimed to assess the effectiveness of orally administered beta-glucan in lactating cows. 20 lactating cows were used, distributed into two groups, one control and one treatment, with 10 cows in each group. The treatment group received 5g of 1.3-1.6 betaglucan daily for 60 days, isolated from the cell-wall of Saccharomyces cerevisiae diluted into a grain meal, whereas the animals in the control group received only the ration. The California Mastitis Test (CMT), Somatic Cells Counting (SCC), daily production and assessments of fat and protein content in milk were done. There was no statistically significant difference between the groups concerning subclinical mastitis detected by CMT, SCC, milk production and composition regarding protein and fat content. It was not observed, therefore, the effectiveness of the use of purified beta-glucan orally administered on the control or prevention of mastitis in dairy cows. The results are attributed to the ruminal degradation of the product tested. It is, therefore, suggested that more research should be conducted using the 1.3-1.6 beta-glucan purified from the cell wall of S. cerevisiae by other administration means and ruminal protection technologies for the isolated beta-glucan.(AU)

A meta-analysis and meta-regression of the effects of vitamin E supplementation on serum enrichment, udder health, milk yield, and reproductive performance of transition cows.

Vitamin E is an essential nutrient for cows, but the effect of vitamin E supplementation is often controversially discussed in the published literature. The main goal of this meta-analysis was to evaluate the effects of vitamin E supplementation on its serum and colostrum enrichment, milk yield (MY), and somatic cell counts (SCC), as well as on various reproductive variables of transition cows, by considering a large set of variables that might influence the responses to vitamin E supplementation. After a broad search in journals and databases with keywords related to transition cows supplemented with vitamin E and appropriate filtering of the results, 36 papers including 53 trials were selected, and their data were extracted into a database. A meta-analysis was conducted on the extracted data. The analysis showed enrichment of serum vitamin E both at parturition (effect size: 2.423) and postpartum (effect size: 0.473), but no effects of vitamin E supplementation on IgG concentration in colostrum (effect size: -0.05) were found. There was a tendency for supplemented cows to produce more milk (effect size: 1.29) during the first month of lactation. Because of large heterogeneity, a meta-regression was performed but none of the presumed influencing factors was identified as a potential variable affecting MY. Milk SCC, as an indicator of udder health, was unaffected by vitamin E supplementation. Vitamin E supplementation tended to decrease the calving to first estrus period (CFP), whereby supplementing Se and taking parity into account in the analysis significantly lowered the CFP. Cows receiving additional vitamin E had, on average, 6.1% fewer cases of retained placenta, whereby Se supplementation and breed were key factors improving the effect of vitamin E to reduce retained placenta. In this regard, breeds other than Holstein responded better and these cows showed a lower incidence of retained placenta. The supplemented cows showed fewer days open (effect size: -0.31), and this improvement was affected linearly by increasing the dosage administered. Also, cows showed fewer services per conception with increasing dosage of vitamin E. In conclusion, this analysis showed that supplementing vitamin E did not affect SCC or colostrum quality but improved reproductive performance of transition cows, an effect consistent with increased levels of serum vitamin E and, for some variables, being modulated by Se supplementation.

Randomized controlled trial investigating the effect of 2 selective dry-cow therapy protocols on udder health and performance in the subsequent lactation.

The objective of this study was to compare culture- and algorithm-guided selective dry-cow therapy (SDCT) programs with blanket dry-cow therapy (BDCT) in a multi-site, randomized, natural exposure clinical trial for the following cow-level outcomes: clinical mastitis, removal from the herd, and Dairy Herd Improvement Association (DHIA) test-day milk yield and SCC measures during the first 120 d in milk (DIM). Two days before planned dry-off, cows in each of 7 herds were randomly allocated to BDCT, culture-guided SDCT (cult-SDCT), or algorithm-guided SDCT (alg-SDCT). At dry-off, BDCT cows received an intramammary antibiotic (500 mg of ceftiofur hydrochloride) in all 4 quarters. Antibiotic treatments were selectively allocated to quarters of cult-SDCT cows by only treating quarters from which aseptically collected milk samples tested positive on a rapid culture system after 30 to 40 h of incubation. For alg-SDCT cows, antibiotic treatments were selectively allocated at the cow level, with all quarters receiving antibiotic treatment if the cow met at least one of the following criteria: (1) any DHIA test with a somatic cell count >200,000 cells/mL during the current lactation, and (2) ≥2 clinical mastitis cases during the current lactation. All quarters of all cows were treated with an internal teat sealant. Clinical mastitis and removal from the herd events (i.e., culling or death) and DHIA test-day data from dry-off to 120 DIM were extracted from herd records. Hazard ratios (HR) for the effect of treatment group on clinical mastitis and removal from the herd during 1 to 120 DIM were determined using Cox proportional hazards regression. The effects of treatment group on test-day loge-transformed SCC and milk yield were determined using linear mixed models. Final models indicated that either SDCT program was unlikely to increase clinical mastitis risk (HRcult-SDCT/BDCT = 0.82, 95% CI: 0.58, 1.15; HRalg-SDCT/BDCT = 0.83, 95% CI: 0.63, 1.09) or test-day logeSCC (cult-SDCT minus BDCT = 0.05, 95% CI: -0.09, 0.18; alg-SDCT minus BDCT = 0.07, 95% CI: -0.07, 0.21). Risk of removal from the herd and test-day milk yield were similar between treatment groups. Findings from this study indicate that culture- or algorithm-guided SDCT can be used at dry-off without negatively affecting cow health and performance in early lactation.

Methionine and arginine supplementation alter inflammatory and oxidative stress responses during lipopolysaccharide challenge in bovine mammary epithelial cells in vitro.

Mastitis, inflammation of the udder, is one of the most common diseases hampering milk yield of dairy cows. Methionine (Met) and arginine (Arg) are key nutrients with potential to regulate inflammation and oxidative stress. The aim of this study was to evaluate the effect of increased supply of Met and Arg on mRNA and protein abundance associated with innate immune response and redox balance during lipopolysaccharide (LPS) stimulation in primary bovine mammary epithelial cells (BMEC). Primary BMEC (n = 4 replicates per treatment) were pre-incubated for 12 h in media with the following amino acid combinations: ideal profile of amino acids (control; Con), increased Met supply (incMet), increased Arg supply (incArg), and increased supply of Met and Arg (incMetArg). Subsequently, cells were challenged with or without LPS (1 µg/mL) and incubated for 6 h. Data were analyzed as a 2 × 2 × 2 factorial using the MIXED procedure of SAS 9.4 (SAS Institute Inc., Cary, NC). The downregulation of SLC36A1 and SLC7A1 mRNA abundance induced by LPS was attenuated in the incArg cultures. Although challenge with LPS led to lower abundance of proteins related to the antioxidant response (NFE2L2, NQO1, GPX1), lower levels of ATG7, and lower mRNA abundance of GPX3, we found little effect in cultures with incMet or incArg. Cultures with incMet, incArg, or incMetArg led to attenuation of the upregulation of SOD2 and NOS2 induced by LPS. Abundance of phosphorylated p65 (RELA) was greater after LPS stimulation, but the response was attenuated in cultures with incMet. The greater ratio of pRELA to total RELA in responses to LPS was also attenuated in cultures with incMetArg. The greater mRNA abundance of the proinflammatory cytokine IL1B induced by LPS was attenuated in cultures with incMet, and the same trend induced by LPS on CXCL2 was also alleviated in cultures with incArg. Overall, the data suggest that greater supply of Met and Arg alleviated the proinflammatory responses triggered by LPS through controlling the abundance of proinflammatory cytokines and chemokines and activity of NF-κB. Little benefit on oxidative stress induced by LPS challenge in BMEC was detected with greater supply of Met and Arg.

Atividade antimicrobiana e citotoxicidade in vitro do óleo essencial de Tagetes minuta L. visando à aplicação no controle da mastite bovina / In vitro antibacterial and cytotoxicity activities of Tagetes minuta L. essential oil towards bovine mastitis treatment

O objetivo deste estudo foi avaliar a atividade antimicrobiana in vitro do óleo essencial de Tagetes minuta L. contra Staphylococcus aureus e Escherichia coli e a citotoxicidade sobre células epiteliais da glândula mamária bovina (MAC-T), visando a seu uso no tratamento da mastite bovina. A análise qualitativa do óleo revelou cis-tagetona (24,24%), di-hidrotagetona (16,65%), 1,3,6-octatrieno-3,7-dimetil-E (13,61%), trans-ocimenona (13,52%) e cis-ocimenona (10,06%) como compostos majoritários. Nos ensaios da atividade antimicrobiana, a concentração inibitória mínima (CIM) verificada foi de 1 mg/mL para a cepa padrão (ATCC 25923), cinco isolados de S. aureus provenientes de leite de vacas com mastite e a cepa padrão resistente à meticilina (MRSA) (ATCC 33592). Para a cepa padrão de E. coli (ATCC 8739) e dois isolados de leite de vacas com mastite, a CIM foi de 3 mg/mL. Elevado efeito citóxico do óleo sobre as células da linhagem MAC-T foi constatado. Concentrações superiores a 10 (g/mL do óleo resultaram em mais de 90% de morte celular. Tais resultados sugerem que, apesar da atividade antimicrobiana contra agentes causadores da mastite bovina, a utilização intramamária do óleo de T. minuta não seria recomendada. É importante destacar a sensibilidade da cepa MRSA ao óleo essencial, o que evidencia seu potencial como antisséptico e sanitizante.(AU)

The aim of this study was to evaluate the in vitro antimicrobial activity of Tagetes minuta L. essential oil against Staphylococcus aureus and Escherichia coli, and its cytotoxicity to bovine mammary epithelial cells (MAC-T line), aiming at its use for bovine mastitis treatment. The qualitative analysis of the oil by GC-MS identified cis-tagetone (24.24%), dihydrotagetone (16.65%), 1,3,6-Octatriene 3,7-Dimethyl-E (13.61%); trans-ocimenone (13.52%) and cis-ocimenone (10.06%) as major compounds. Antimicrobial activity was determined by broth microdilution technique and revealed the minimum inhibitory concentration of 1mg/mL for the standard strain of S. aureus (ATCC 25923) and five bacterias isolated from mastitic milk, including a multiresistant strain (ATCC 33592); and 3mg/ml for the standard strain of E. coli (ATCC 8739) and two bacterias isolated from mastitic milk. However, a strong citotoxic effect on MAC-T cells was found. Oil concentrations from 10(g/mL resulted in over 90% of cell death. The results suggest that although the antimicrobial activity was identified against the main agents of bovine mastitis, the intramammary use of T. minuta oil may not be recommended. On the other hand, it is important to highlight the sensibility of the MSRA strain to the essential oil, which evidences its potential as an antiseptic or sanitizer.(AU)

Immunomodulatory molecules regulate adhesin gene expression in Staphylococcus aureus: Effect on bacterial internalization into bovine mammary epithelial cells.

Staphylococcus aureus is a major pathogen of subclinical bovine mastitis that usually is chronic and recurrent, which has been related to its ability to internalize into bovine mammary epithelial cells (bMECs). Previously, we reported that short and medium fatty acids and cholecalciferol reduce S. aureus internalization into pretreated-bMECs with these molecules suggesting a role as immunomodulatory agents. Hence, we assessed the role of sodium butyrate (NaB), sodium octanoate (NaO) and cholecalciferol on S. aureus adhesin expression and its internalization into bMECs. S. aureus pre-treated 2 h with 0.5 mM or 2 mM NaB showed a reduction in internalization into bMECs (∼35% and ∼55%; respectively), which coincided with a down-regulated expression of clumping factor B (ClfB). Also, the S. aureus internalization reduction by 2 mM NaB (2 h) agreed with a down-regulated expression of sdrC. Moreover, the 2 mM NaB (24 h) pre-treatment induced bacterial internalization (∼3-fold), which was related with an up-regulation of spa, clfB and sdrC genes. Also, NaO (0.25 mM and 1 mM) only reduced S. aureus internalization when bacteria were grown 2 h with this molecule but there was no relationship with adhesin expression. In addition, cholecalciferol (50 nM) reduced bacteria internalization at similar levels (∼50%) when bacteria were grown 2 and 24 h in broth supplemented with this compound, which correlated with spa and sdrC mRNA expression down-regulated at 2 h, and fnba and clfB mRNA expression decreased at 24 h. In conclusion, our data support the fact that fatty acids and cholecalciferol regulate adhesin gene expression as well as bacteria internalization in nonprofessional phagocytic cells, which may lead to development of anti-virulence agents for control of pathogens.

Effects of an immunomodulatory feed additive on intramammary infection prevalence and somatic cell counts in a dairy herd experiencing major health issues.

A trial was conducted to determine if feeding OmniGen-AF® (OG) to 22 late lactation cows 60 days prior to and during the early dry period, a time of increased susceptibility to mastitis, could reduce disease incidence in a dairy herd experiencing major health issues. Treated cows (n = 11) consumed a ration containing OG [9 g/100 kg of body weight/day] beginning 60 days before dry-off, during the dry period, and through 30 days in milk (DIM). Control cows received the same ration during the dry period through 30 DIM only. Body weights, body condition scores (BCS), intramammary infection (IMI) prevalence, new IMI rates, somatic cell counts (SCC), milk yield, and adverse health events were measured. No differences were found between treatments for body weight or BCS. Adverse health event data at calving showed no differences between treatments except for percentage of cows with hyperketonemia, which was lower among treated cows (63.6% vs 100%). Prevalence of IMI from calving through 30 DIM for treated cows (6.1%) was lower than controls (11.05%); likewise, new IMI rate during this time for treated cows (0.61%) was lower than controls (5.81%). The SCC from calving through 30 DIM for treated cows (215,000/ml) was lower than controls (493,000/ml). Average production/day at the first DHIA test (~33 DIM) showed that treated cows produced more milk (39.9 kg) than controls (35.34 kg). In conclusion, feeding OG 60 days prior to dry-off reduced hyperketonemia and mastitis, lowered SCC, and numerically increased milk yield in a dairy herd experiencing major health issues.

Mammary Gland Immunobiology and Resistance to Mastitis.

The ability of dairy cattle to prevent infectious pathogens from causing mastitis is related to the efficiency of the mammary immune system. The primary roles of the bovine immune system are to prevent bacterial invasion of the mammary gland, eliminate existing infections, and restore mammary tissues to normal function. Mammary gland immunity uses a multifaceted network of physical, cellular, and soluble factors to protect the cow from the diverse array of mastitis-causing pathogens. Strategies to optimize mammary gland defenses can be an effective way to prevent the establishment of new intramammary infections and limit the use of antimicrobials to treat mastitis.

Effects of pegbovigrastim administration on periparturient diseases, milk production, and reproductive performance of Holstein cows.

The aim of this study was to evaluate the effects of treating Holstein cows with pegbovigrastim on periparturient diseases, milk production, and reproductive performance while exploring the mode of action of an immunomodulatory protein. Cows were randomly allocated to 1 of 2 treatments, untreated control (CTR, n = 423) and pegbovigrastim (PEG, n = 417). At 7 d from the anticipated calving date (d -7), cows allocated to PEG received a subcutaneous injection of 15 mg of pegylated recombinant bovine granulocyte colony stimulating factor (pegbovigrastim injection, Imrestor, Elanco Animal Health, Greenfield, IN). A second injection was administered within 24 h after calving (d 0). Blood samples were obtained from a subset of cows (CTR, n = 103; PEG, n = 102) at -7 and 0, 3, 7, and 14 d relative to parturition. Samples were used for hemogram and quantification of haptoglobin, nonesterified fatty acids, ß-hydroxybutyrate, and trace and macro minerals. Vaginal cytobrush was performed on the same subset cows at d 0, 7, and 14 to assess the relative neutrophil count. Additionally, colostrum samples were collected to measure IgG, IgM, IgA, and lactoferrin concentrations. Postpartum disease occurrence was recorded from calving until 30 d in milk (DIM). Weekly milk yield was recorded for the first 12 wk after calving. Cows treated with PEG had a 3- to 4-fold increase in circulating polymorphonuclear leukocyte, lymphocyte, and monocyte numbers, with a peak at 3 d after treatment followed by a gradual decline, but the counts remained significantly greater compared with CTR at 14 DIM. The administration of PEG did not affect the incidence of clinical and subclinical mastitis, retained fetal membranes, metritis, puerperal metritis, and endometritis. Primiparous cows treated with PEG tended to have lower odds of developing hyperketonemia than CTR [odds ratio (OR) = 0.57, 95% confidence interval (CI) = 0.23 to 1.42]. Cows treated with PEG had higher odds of being diagnosed with lameness within 30 DIM compared with CTR (OR = 1.79, 95% CI = 1.16 to 2.76); however, we found no significant differences by 60 DIM. Treatment with PEG increased the odds of displaced abomasum (OR = 8.27, 95% CI = 1.02 to 66.6). Cows treated with PEG had higher odds of being diagnosed with 1 or more clinical diseases compared with CTR cows (OR = 1.39, 95% CI = 1.02 to 1.90). We observed no differences in linear scores or milk composition between treatments. Furthermore, primiparous cows treated with PEG produced more milk than CTR primiparous cows during the first 12 wk postpartum (PEG = 37.51 ± 0.66; CTR = 35.91 ± 0.65 kg), but no differences were observed on energy-corrected milk. Treatment did not alter reproductive performance; additionally, cows diagnosed with metritis or puerperal metritis and treated with PEG tended to have higher proportion of neutrophils in the vaginal mucosa when compared with CTR metritic cows. Although PEG treatment increased circulating polymorphonuclear leukocyte, monocyte, and lymphocyte numbers, as expected, it was detrimental to cow health because it increased morbidity.

Alternative approach to mastitis management ­ How to prevent and control mastitis without antibiotics? / Abordagem alternativa para o manejo da mastite ­ Como prevenir e controlar a mastite sem antibióticos?

Mastitis affects a high proportion of dairy cows throughout the world and is one of the greater problems faced by the dairy industry today. The disease is still a major cause of economic loss on a dairy farm. Mastitis poses not only negative consequences for the dairy farmer but also for the dairy industry as a number of issues threaten the reputation of milk as a healthy product from healthy animals. The use of antimicrobials is one of those concerns and threats. Antimicrobial usage on dairy farms is most often related to udder health as most medicines are used in prevention and control of mastitis. Antimicrobials remain vital for treatment of bacterial infections in dairy cattle, but in light of the upcoming debate instigated by the potential link between the use of antimicrobial products in animal husbandry and the development of antimicrobial resistance in both animal and human pathogens, there is an urgent need for innovation and alternatives to antibiotic therapy for mastitis treatment and control. Alternative approaches include vaccination, probiotics or beneficial microorganisms and inhibitory substances, immunomodulation, bacteriophages, homeopathy, and plant-derived inhibitory substances, yet only when scientifically-proven evidence is available indicating these alternatives are effective.(AU)

A mastite ocorre em uma alta proporção de vacas leiteiras em todo o mundo e é um dos grandes problemas que atinge a indústria leiteira. A doença ainda é uma das maiores causas de perdas econômicas das granjas leiteiras. A mastite não tem apenas consequências negativas para os fazendeiros, mas atinge também a indústria leiteira, particularmente pela expectativa de que o leite deve ser um produto saudável produzido por animais saudáveis. O uso de antimicrobianos é uma grande preocupação. O emprego de antimicrobianos nas granjas leiteiras está frequentemente relacionado com a saúde do úbere uma vez que a maioria dos medicamentos são usados na prevenção e controle da mastite. Os antimicrobianos ainda são vitais para o tratamento das infecções bacterianas do gado leiteiro, mas na atualidade há um grande questionamento para a ligação potencial entre o uso de produtos antimicrobianos no manejo animal e o desenvolvimento de resistência antimicrobiana tanto de patógenos de animais como de humanos, há portanto a necessidade urgente da introdução de alternativas para o tratamento e controle da mastite com antibióticos. As possibilidades incluem vacinação, probióticos ou microorganismos benéficos e substâncias inibidoras, imunomodulação, bacteriófagos, homeopatia, fitoterápicos inibidores. Contudo todos esses procedimentos necessitam ser cientificamente provadas para demonstrar que são alternativas realmente efetivos. (AU)

Composition of the teat canal and intramammary microbiota of dairy cows subjected to antimicrobial dry cow therapy and internal teat sealant.

Antimicrobial dry cow therapy (DCT) is an important component of mastitis control programs aimed to eliminate existing intramammary infections and prevent the development of new ones during the dry period. However, to what extent the microbiota profiles of different niches of the udder change during the dry period and following administration of DCT remains poorly understood. Therefore, the main objective of the present study was to qualitatively evaluate dynamics of the microbiota of teat canal (TC) and mammary secretions (i.e., milk and colostrum) of healthy udder quarters subjected to DCT using a long-acting antimicrobial product, containing penicillin G and novobiocin, in combination with internal teat sealant. To this end, TC swabs (n = 58) and their corresponding milk (n = 29) and colostrum samples (n = 29) were collected at the time of drying off and immediately after calving from clinically healthy udder quarters of Holstein dairy cows from a commercial dairy farm. All samples were subjected to DNA extraction and high-throughput sequencing of the V1-V2 hypervariable regions of bacterial 16S rRNA genes. Overall, shifts were more pronounced within the microbiota of mammary secretions than the TC. In particular, microbiota of colostrum samples collected immediately after calving were less species-rich compared with the pre-DCT milk samples. Proportions of several bacterial genera belonging to the phylum Proteobacteria, including Pseudomonas, Stenotrophomonas, and unclassified Alcaligenaceae, were enriched within the microbiota of colostrum samples, whereas Firmicutes genera, including Butyrivibrio, unclassified Clostridiaceae, and unclassified Bacillales, were overrepresented in pre-DCT milk microbiota. Apart from shifts in the proportion of main bacterial genera and phyla, qualitative analysis revealed a high degree of commonality between pre-DCT and postpartum microbiota of both niches of the udder. Most importantly, a considerable number of bacterial genera and species commonly regarded as mastitis pathogens or opportunists (or both), including Staphylococcus spp., unclassified Enterobacteriaceae, and Corynebacterium spp., were shared between pre-DCT and postpartum microbiota of mammary secretions. Percentage of shared bacterial genera and species was even higher between pre-DCT and postpartum microbiota of TC samples, suggesting that the DCT approach of the present study had limited success in eliminating a considerable proportion of bacteria during the dry period.

Clinical responses in cows vaccinated with a developed prototype killed Staphylococcus aureus mastitis vaccine.

Mastitis is an inflammatory condition of the udder that occurs as a result of the release of leucocytes into the udder in a response to bacterial invasion. The major causes of mastitis are an array of gram positive and negative bacteria, however, algae, virus, fungi, mechanical or thermal injury to the gland have also been identified as possible causes. Mastitis vaccines are yet to be developed using Malaysian local isolate of bacteria. The objective of the present experimental trial was to develop a monovalent vaccine against mastitis using S. aureus of Malaysian isolate and to evaluate the clinical responses such as temperature, respiratory rates and heart rates in vaccinated cows. S. aureus is a major causative bacteria in clinical and subclinical types of mastitis in cows. Four concentrations of the bacterin (106, 107, 108 and 109 cfu/ml of the local isolate of S. aureus) were prepared using Aluminium potassium sulfate adjuvant. Thirty cows were grouped into four treatment groups (B, C, D and E) with a fifth group as control (A). These groups were vaccinated intramuscularly(IM) with the prepared monovalent vaccine and its influence on the vital signs were intermittently measured. The mean of rectal temperature was significantly different (p˂ 0.05) at 0hr Post Vaccination [1]" in groups D and E (39.5 ±â€¯0.15 °C and 39.4 ±â€¯0.15 °C respectively) and at 3 h PV in groups C, D and E (39.8 ±â€¯0.14 °C, 39.9 ±â€¯0.14 °C and 40.3 ±â€¯0.14 °C respectively) compared to the control group. This indicated a sharp increased rectal temperatures between 0hr and 3 h PV in groups C, D and E which later declined at 24 h PV. The mean of rectal temperature of group E was significantly different (p˂ 0.05) at weeks 1 and 2 PV (39.87 ±â€¯0.19 °C and 39.80 ±â€¯0.18 °C respectively) compared to the control group. The mean of heart rate was significantly different (p˂ 0.05) at week 1 PV in groups D and E (83.0 ±â€¯3.8 beats/minute and 80.0 ±â€¯3.8 °C respectively) compared to control. A trending decrease was however observed in heart rates of group E from weeks through 4 PV and in group D from weeks 1 through 3 PV. The mean of respiratory rates was significantly different (p˂ 0.05) at week 3 PV in group B and D (31.0 ±â€¯1.2 breaths/minute and 28.0 ±â€¯1.2 breaths/minute) compared to control. In conclusion, this study highlights responses of these vital signs due to vaccination against S. aureus causing mastitis in cows. To the best of our knowledge the findings of this study adds value to the shallow literature on vital signs alterations in cows vaccinated against mastitis as elevated levels of temperature and heart rates of group D and E indicated obvious response.

Panax ginseng extract reduces Staphylococcus aureus internalization into bovine mammary epithelial cells but does not affect macrophages phagocytic activity.

Panax ginseng extract (PGe) has been shown to possess immunomodulatory effects in healthy dairy cows at drying off and to trigger an adequate immune response to protect from an experimental intramammary infection (IMI) with Staphylococcus aureus in a murine model. S. aureus is one of the major pathogens isolated from bovine IMI; being capable to invade and survive within mammary epithelial cells. However, the precise mechanism by which PGe interacts with bovine mammary epithelial cells (MAC-T) and bovine macrophages in the course of a S. aureus infection remains unclear. We evaluated the effect of PGe on MAC-T cytokine response and on the internalization of S. aureus into MAC-T. In addition, we evaluated the effect of PGe on the phagocytic activity of macrophages isolated from bovine mammary secretions. Results shown that MAC-T cells TLR4 and NF-κB mRNA expression was not affected by PGe at all evaluated times. IL-6 mRNA expression and protein level and IL-4 protein level were significantly induced in MAC-T treated with 3 mg/ml of PGe. PGe at 3 mg/ml reduced significantly the internalization of two S. aureus strains in MAC-T. In addition, PGe did not affect the percentage of phagocytosis and the NO and ROS production of macrophages co-cultured with two strains of S. aureus. These results, obtained in in vitro models together with those obtained in in vivo previous studies carried out in bovines and mice can contribute to improve the understanding of the effects of PGe following inoculation in bovine mammary glands.

Udder health of dairy cows fed different dietary energy levels after a short or no dry period without use of dry cow antibiotics.

Reports on the effects of length of dry period (DP) on udder health of cows that were not treated with dry cow antibiotics are scarce. Additionally, the effects of a reduced dietary energy level for cows with a 0-d DP on udder health have not yet been studied. The aims of this study were (1) to compare effects of a 0-d or 30-d DP without use of dry cow antibiotics on udder health across the DP and subsequent lactation in dairy cows fed different dietary energy levels and (2) to evaluate associations between udder health and metabolic status of dairy cows. Five weeks before the expected calving date, Holstein-Friesian dairy cows (n = 115) were blocked for parity, expected calving date, and milk yield and somatic cell count (SCC) at their 2 last test days and were randomly assigned to 2 DP lengths: 0-d DP (n = 77) or 30-d DP (n = 38). Quarter milk samples were taken in wk 5 prepartum and in wk 1 and 5 postpartum. Proportion of quarters with elevated SCC (SCC ≥200,000 cells/mL) and proportion of udder pathogens in quarter milk samples did not differ between DP lengths among weeks. After calving, 102 of these cows were randomly assigned to 3 treatments: a 30-d DP with a standard energy level required for expected milk yield (30-d DP SEL; n = 36), a 0-d DP with the same energy level as cows with a 30-d DP (0-d DP SEL; n = 33), and a 0-d DP with a low energy level (0-d DP LEL, n = 33). From wk 8 of lactation onward, cows received either a glucogenic ration consisting of corn silage and grass silage or a lipogenic ration consisting of grass silage and sugar beet pulp at a standard or low energy level. During wk 1 to 7 postpartum, treatment did not affect SCC or SCC corrected for milk yield. During wk 8 to 44 of lactation, 0-d DP SEL cows had a greater SCC than 0-d DP LEL or 30-d DP SEL cows and had a greater SCC corrected for milk yield than 0-d DP LEL cows. During wk 1 to 44 of lactation, occurrence of at least 1 elevation of SCC (SCC ≥200,000 cells/mL after 2 wk of SCC <200,000 cells/mL) was not different among treatments. The 0-d DP SEL cows but not the 0-d DP LEL cows tended to have a 2.17 times greater hazard of having a case of clinical mastitis at any time in lactation than 30-d DP SEL cows. In wk 1 to 44 of lactation, lower fat- and protein- corrected milk yield and energy intake, greater energy balance, and greater plasma insulin concentration were associated with greater SCC. In conclusion, DP length did not affect udder health in the DP and in early lactation but seemed to decrease udder health for 0-d DP SEL cows in later lactation compared with 30-d DP SEL or 0-d DP LEL cows.