Comparison of the immune responses associated with experimental bovine mastitis caused by different strains of Escherichia coli.

We studied the mammary immune response to different mammary pathogenic Escherichia coli (MPEC) strains in cows, hypothesising that the dynamics of response would differ. E. coli is a major aetiologic agent of acute clinical bovine mastitis of various degrees of severity with specific strains being associated with persistent infections. We compared challenge with three distinct pathogenic MPEC strains (VL2874, VL2732 and P4), isolated from different forms of mastitis (per-acute, persistent and acute, respectively). A secondary objective was to verify the lack of mammary pathogenicity of an environmental isolate (K71) that is used for comparison against MPEC in genomic and phenotypic studies. Twelve cows were challenged by intra-mammary infusion with one of the strains. Cellular and chemokine responses and bacterial culture follow-up were performed for 35 d. All cows challenged by any of the MPEC strains developed clinical mastitis. Differences were found in the intensity and duration of response, in somatic cell count, secreted cytokines (TNF-α, IL-6 and IL-17) and levels of milk leucocyte membrane Toll-like receptor 4 (TLR4). A sharp decrease of TLR4 on leucocytes was observed concomitantly to peak bacterial counts in milk. Intra-mammary infusion of strain K71 did not elicit inflammation and bacteria were not recovered from milk. Results suggest some differences in the mammary immune response to distinct MPEC strains that could be correlated to their previously observed pathogenic traits. This is also the first report of an E. coli strain that is non-pathogenic to the bovine mammary gland.

Alternative Traits for Genetic Evaluation of Mastitis Based on Lifetime Merit.

Genetic selection has achieved little progress in reducing mastitis incidence. Mastitis traits are problematic due to the lack of sensitivity of the data and reliance on clinical diagnosis, often missing subclinical cases, and/or on monthly somatic cell count (SCC) measurements. The current measure for mastitis is the lactation average of the somatic cells score (LSCS). We studied two datasets: (1) 148 heifers divided into non-intramammary infected, sub-clinically infected and clinical mastitis groups; (2) data from 89,601 heifers from Israeli Holsteins through the same period divided into "udder healthy" (UH) and "non-healthy" (UNH) by a threshold of SCC 120,000 cells/mL in all nine monthly milk recordings. In study 1, non-infected heifers had significantly (p < 0.05) more partum, production days and overall lifetime milk production compared to clinical and sub-clinically infected. In study 2, UH heifers (20.3%) had significantly higher (p < 0.01) lifetime milk, production days, and lactations. Subdividing datasets by sires, the same analyses detected differences in percentages of UH daughters between the sire groups. Lifetime milk production correlated (r = +0.83, p < 0.001) with udder health status. SCC threshold of less than 120,000 cells/mL during all first lactation measurements indicated healthy udder, providing a valuable insight that this dichotomous trait is advantageous for calculating lifetime net-merit index (NM$) over LSCS.

Nonbactericidal secreted phospholipase A2s are potential anti-inflammatory factors in the mammary gland.

The recent burst of duplication and divergence of the bovine PLA2G2D genes is considered typical of immune response genes, and it was recently shown that PLA2G2D is abundantly expressed in mouse leukocytes and acts as an immunosuppressive phospholipase. Analysis of 1,143 Holstein bulls indicated that the four common haplotypes spanning PLA2G2D display copy number variation ranging from 1 to 4 per haploid genome. Association of the fourth haplotype with negative total merit remained significant (P < 0.002) when corrected for population relatedness. We compared the lipase and bactericidal activities of bovine pancreatic PLA2G1B with human PLA2G2A and G2D and bovine PLA2G2D1 and G2D4 proteins, which had been subcloned, expressed, and refolded by us, and the impact of point mutations in the calcium binding site was investigated. All tested phospholipases were ineffective bactericides of Escherichia coli isolated from bovine mastitis. However, in lactating mice treated with E. coli or lipopolysaccharide (LPS), intramammary injection of bovine PLA2G1B relieved visual and histological inflammation and reduced blood levels of infiltrating lactose. Further studies are warranted to determine whether the observed anti-inflammatory effect involves competitive binding of the receptor Pla2r1 which may mimic the LPS resistance effect in Pla2r1-deficient mice.

Longitudinal study on the effects of growth-promoting and therapeutic antibiotics on the dynamics of chicken cloacal and litter microbiomes and resistomes.

BACKGROUND: Therapeutic and growth-promoting antibiotics are frequently used in broiler production. Indirect evidence indicates that these practices are linked to the proliferation of antimicrobial resistance (AMR), the spread of antibiotic-resistant bacteria from food animals to humans, and the environment, but there is a lack of comprehensive experimental data supporting this. We investigated the effects of growth promotor (bacitracin) and therapeutic (enrofloxacin) antibiotic administration on AMR in broilers for the duration of a production cycle, using a holistic approach that integrated both culture-dependent and culture-independent methods. We specifically focused on pathogen-harboring families (Enterobacteriaceae, Enterococcaceae, and Staphylococcaceae). RESULTS: Antibiotic-resistant bacteria and antibiotic resistance genes were ubiquitous in chicken cloaca and litter regardless of antibiotic administration. Environment (cloaca vs. litter) and growth stage were the primary drivers of variation in the microbiomes and resistomes, with increased bacterial diversity and a general decrease in abundance of the pathogen-harboring families with age. Bacitracin-fed groups had higher levels of bacitracin resistance genes and of vancomycin-resistant Enterococcaceae (total Enterococcaceae counts were not higher). Although metagenomic analyses classified 28-76% of the Enterococcaceae as the commensal human pathogens E. faecalis and E. faecium, culture-based analysis suggested that approximately 98% of the vancomycin-resistant Enterococcaceae were avian and not human-associated, suggesting differences in the taxonomic profiles of the resistant and non-resistant strains. Enrofloxacin treatments had varying effects, but generally facilitated increased relative abundance of multidrug-resistant Enterobacteriaceae strains, which were primarily E. coli. Metagenomic approaches revealed a diverse array of Staphylococcus spp., but the opportunistic pathogen S. aureus and methicillin resistance genes were not detected in culture-based or metagenomic analyses. Camphylobacteriaceae were significantly more abundant in the cloacal samples, especially in enrofloxacin-treated chickens, where a metagenome-assembled C. jejuni genome harboring fluoroquinolone and ß-lactam resistance genes was identified. CONCLUSIONS: Within a "farm-to-fork, one health" perspective, considering the evidence that bacitracin and enrofloxacin used in poultry production can select for resistance, we recommend their use be regulated. Furthermore, we suggest routine surveillance of ESBL E. coli, vancomycin-resistant E. faecalis and E. faecium, and fluoroquinolone-resistant C. jejuni strains considering their pathogenic nature and capacity to disseminate AMR to the environment. Video Abstract.

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.

Milk quality and milk transformation parameters from infected mammary glands depends on the infecting bacteria species.

The current study measured the influence of milk of subclinically infected glands by different bacteria species on the cow's milk. The effects of bacterial infection or inflammation on gland milk yield were related to the bacteria species that caused the infection. The volume of milk of the inflamed gland from the cow's milk yield was significantly lower (P<0.001) for the glands previously infected by Escherichia coli (PIEc) and those infected with Streptococcus dysgalactiae. Coagulation properties, rennet clotting time (RCT) and curd firmness (CF) also depended on the bacteria causing the infection. RCT values of all the inflamed glands were significantly longer (P<0.001) and CF values were significantly lower than that of the healthy ones. Moreover, in the whole milk, CF was also significantly lower and not proportional to the volume of the milk from the inflamed gland of the cow's milk. Calculation of the predicted 40% dry matter curd weight (PCW) on the cow level, including the healthy and inflamed glands or the healthy glands alone, showed that for 10 of 13 PIEc cows, the presence of the affected gland's milk in the whole cow milk resulted in a lower PCW value. Likewise, 7 of 20 cows infected by S. dysgalactiae had negative delta values. Unlike the latter bacteria, PCW from milk of glands infected with CNS increased, although in a lower magnitude than in the healthy glands. No correlation was found between logSCC in the whole cow milk (healthy and inflamed glands) and PCW.

Application of pancreatic phospholipase A2 for treatment of bovine mastitis.

Recent findings have indicated that secreted phospholipases A2 (sPLA2s) have anti-inflammatory functions, including relief of symptoms in a mouse model of mastitis. This prompted us to investigate the therapeutic application of sPLA2, PLA2G1B, for bovine mastitis. Initial testing of PLA2G1B's effect on bovine mammary epithelial cell (bMEC) line PS revealed no changes in cell viability or cytokine-secretion pattern. However, when cells were first treated with lipopolysaccharide endotoxin (LPS) or live bacteria (Escherichia coli or Staphylococcus aureus), incubation with PLA2G1B significantly improved cell viability, suggesting involvement of sPLA2s in protecting membranes from lipid-peroxidation damage, rather than a bactericidal action. When PLA2G1B was applied simultaneously with LPS, a significant short-term reduction in interleukin-8 secretion was observed compared with bMECs treated only with LPS, supporting previous reports that PLA2G1B affects interleukin-8 signaling in similar cells. Following the favorable outcome of the in vitro experiments, we tested PLA2G1B in vivo by mammary infusion into infected glands. In one of a small sample (n = 4) of lactating cows chronically infected with Streptococcus dysgalactiae, a single PLA2G1B treatment completely cleared inflammation and bacteria, demonstrating its potential to cure subclinical mastitis. PLA2G1B treatment did not affect coagulase-negative staphylococci infection. These types of mastitis may involve formation of a resistant biofilm, and its elimination may relate to sPLA2s' characteristic ability to aggregate with cellular debris, facilitating their internalization by macrophages. In a bovine model of clinical mastitis based on introduction of E. coli via the streak canal, a single mammary infusion of PLA2G1B led to faster recovery to pre-infection milk-yield levels and decrease of somatic cell counts. In this case, all of sPLA2s' modes of resolving inflammation may apply, including competitive binding of the sPLA2s' receptor, the inactivation of which confers resistance to endotoxic shock. Hence, this study strongly supports further research into PLA2G1B as a cure for bovine mastitis.

Postgenomics Characterization of an Essential Genetic Determinant of Mammary Pathogenic Escherichia coli.

Escherichia coli are major bacterial pathogens causing bovine mastitis, a disease of great economic impact on dairy production worldwide. This work aimed to study the virulence determinants of mammary pathogenic E. coli (MPEC). By whole-genome sequencing analysis of 40 MPEC and 22 environmental ("dairy-farm" E. coli [DFEC]) strains, we found that only the fec locus (fecIRABCDE) for ferric dicitrate uptake was present in the core genome of MPEC and that it was absent in DFEC genomes (P < 0.05). Expression of the FecA receptor in the outer membrane was shown to be citrate dependent by mass spectrometry. FecA was overexpressed when bacteria were grown in milk. Transcription of the fecA gene and of the inner membrane transport component fecB gene was upregulated in bacteria recovered from experimental intramammary infection. The presence of the fec system was shown to affect the ability of E. coli to grow in milk. While the rate of growth in milk of fec-positive (fec+) DFEC was similar to that of MPEC, it was significantly lower in DFEC lacking fec Furthermore, deletion of fec reduced the rate of growth in milk of MPEC strain P4, whereas fec-transformed non-mammary gland-pathogenic DFEC strain K71 gained the phenotype of the level of growth in milk observed in MPEC. The role of fec in E. coli intramammary pathogenicity was investigated in vivo in cows, with results showing that an MPEC P4 mutant lacking fec lost its ability to induce mastitis, whereas the fec+ DFEC K71 mutant was able to trigger intramammary inflammation. For the first time, a single molecular locus was shown to be crucial in MPEC pathogenicity.IMPORTANCE Bovine mastitis is the major infectious disease in dairy cows and the leading cause of economic loss to the global dairy industry, directly contributing to the price of dairy products on supermarket shelves and the financial hardships suffered by dairy farmers. Mastitis is also the leading reason for the use of antibiotics in dairy farms. Good farm management practices in many countries have dramatically reduced the incidence of contagious mastitis; however, the problems associated with the incidence of environmental mastitis caused by bacteria such as Escherichia coli have proven intractable. E. coli bacteria cause acute mastitis, which affects the health and welfare of cows and in extreme cases may be fatal. Here we show for the first time that the pathogenicity of E. coli causing mastitis in cows is highly dependent on the fecIRABCDE ferric citrate uptake system that allows the bacterium to capture iron from citrate. The Fec system is highly expressed during infection in the bovine udder and is ubiquitous in and necessary for the E. coli bacteria that cause mammary infections in cattle. These results have far-reaching implications, raising the possibility that mastitis may be controllable by targeting this system.

Effects of intra-mammary bacterial infection with coagulase negative staphylococci and stage of lactation on shedding of epithelial cells and infiltration of leukocytes into milk: comparison among cows, goats and sheep.

The effects of mammary gland bacterial infection and stage of lactation on leukocyte infiltration into the mammary gland were compared among cows, goats and sheep. Animals were at two stages of lactation: mid or late. In mid-lactation animals, bacterial-free glands and coagulase negative Staphylococcus (CNS)-infected glands were compared. In late lactation only uninfected glands were studied. Of mid-lactation bacteria-free animals, goats had the highest number of leukocytes and % polymorphonuclears (PMNs), whereas sheep had the lowest and leukocytes number in cows were intermediate between sheep and goats. Based on %PMN, two cell clusters were found in sheep, which overlapped with the parallel cell clusters of cows and goats, but with a slightly higher number of leukocytes in each cell cluster. At late lactation, goats had higher values for %PMN and leukocyte numbers in comparison to cows, which had a similar cellular profile to sheep. The cellular immune response to CNS infection was similar for the three animal species, although the number of cells was different, while the basal cell level at mid-lactation and especially at the end of lactation was species specific.

Vaccine development for the prevention of staphylococcal mastitis in dairy cows.

Staphylococci are the most common and costly mammary disease of dairy cattle worldwide. Target of RNAIII Activating Protein (TRAP), a membrane associated 167AA protein, is highly conserved among staphylococci and was shown in Staphylococcus aureus to be involved in bacterial stress response. The aims of this study were to test the safety and efficacy of recombinant TRAP (rTRAP) vaccine in dairy animals. The vaccine was safe as 2-3 subcutaneous injections of rTRAP (54-100 µg) with adjuvant ISA 206 to cows and goats did not lead to any abnormal symptoms of sensitivity to the vaccine. The rTRAP vaccine was immunogenic and caused the induction of a humoral immune response that remained high for at least 160 d post second immunization. The rTRAP vaccine was efficacious; at parturition only 13.5% (5/37) heifers in the immunized group were infected with Staphylococcus chromogenes as compared to 42.9% (18/42) in the non-immunized group. Additionally, when cows were immunized in mid-lactation, the difference between somatic cell count (SCC) in immunized and control animals was profound (45 ± 7 vs. 470 ± 194, respectively). At the same time, the difference in milk yield was also evident (48.3 ± 1.4 L d(-1)vs. 44.3 ± 0.9 L d(-1), respectively). Put together, these studies indicate the value of the rTRAP vaccine in preventing new udder infections by staphylococci, which significantly lead to lowered SCC and some increase in milk yield. TRAP is conserved among all strains and species and is constitutively expressed in any strain of S. aureus or CNS tested so far, including those isolated from cows. TRAP may thus serve as a universal anti-staphylococcus vaccine.

Phenotypic and genotypic characterization of Pseudomonas aeruginosa strains isolated from mastitis outbreaks in dairy herds.

During 1998-2002 outbreaks of Pseudomonas sp. mastitis among more than 15 Israeli sheep and goat dairy herds were observed. The animals presented a wide spectrum of clinical signs ranging from subclinical to gangrenous udder. Ninety-five isolates of Pseudomonas sp. were isolated from clinical and subclinical mastitis of 47 sheep, 17 goats and 31 cows from 34 different farms. Biochemical and genetic analyses revealed that the all-causative organism was Ps. aeruginosa. Selections of isolates were further analysed on the bases of colony morphology, biochemical traits and capacity to form biofilm. All the strains displayed a wide heterogeneity in all the tested traits. No association between bacterial isolates, farm of origin and type of animal was found. Pulsed-field gel electrophoresis and cluster analysis showed no clonality among the tested strains. The present study revealed that a large variety of Ps. aeruginosa strains may cause mastitis outbreaks in sheep, goat and cattle in Israel.

Staphylococcus aureus leucocidin, a virulence factor in bovine mastitis.

The involvement of Staphylococcus aureus exosecretions in bovine udder infection (Younis et al. 2003) suggests that four different monomer protein bands appearing between 36 and 31 kDa, are associated with the severity of the cow's infection response. Three out of these four bands have been identified by means of protein sequencing. Band B, with a MW of 35 kDa was identified as Panton-Valentaine leucocidin LukF'-PV chain- Staph. aureus; band C, with a MW of 32 kDa was identified as leucocidin chain LukM precursor- Staph. aureus; and band D was found to be similar, but not identical, to phosphatidylinositol-specific phospholipase-C-X. Bands B and C were purified by gel filtration using FPLC. The ability of these proteins to induce udder inflammation in vivo, and proliferation response in vitro and cytokine secretion were tested for both the crude exosecretions and purified bands. Three cows were inoculated intracisternally, with three quarters receiving either 0.007-0.008 mg (as total proteins) of Staph. aureus FR2449/1 bacterial exosecretion, pooled fraction 39-41 (bands B and C), or culture broth medium. The fourth quarter was left free as a control. Quarters that received fraction 39-41 of Staph. aureus FR2449/1, exhibited induced inflammation, which was indicated by increased somatic cell count and enhanced NAGase activity that was significantly higher than that of the original Staph. aureus FR2449/1 bacterial exosecretion. Proliferation tests of bovine blood lymphocytes in vitro showed that the pooled fraction 39-41 stimulated bovine proliferation of mononuclear cells much more than the original Staph. aureus FR2449/1 bacterial exosecretion. Secretion of TNF-alpha, IL-1beta, IL-6 and IL-8 was in accordance with the contents of LukF'-PV and LukM precursor in the exosecretions. The results suggest that LukM/ LukF' induce inflammation into the udder by a mechanism similar to that of LPS or by a unique mechanism(s) which requires further investigation.