Identification of Enterococcus spp. by MALDI-TOF mass spectrometry isolated from clinical mastitis and bulk tank milk samples.

BACKGROUND: Throughout a three-year study period, 1,577 bovine clinical mastitis samples and 302 bulk tank samples were analyzed from ten Brazilian dairy herds. Enterococcus spp. was isolated and identified in 93 (5.9%) clinical mastitis samples. In addition, 258 Enterococcus spp. were isolated from the bulk tank samples of the same herds. The identification of Enterococcus spp. isolated from bulk tanks and milk samples of clinical mastitis were accomplished by phenotypic characteristics and confirmed by MALDI-TOF Mass Spectrometry (MS). Fisher test was performed to verify the difference between bulk tanks and mastitis samples. RESULTS: The following species were identified from clinical mastitis: E. saccharolyticus (62.4%), E. faecalis (19.4%), E. faecium (15.1%), E. hirae (1.1%), E. mundtii (1.1%), E. durans (1.1%). Furthermore, from 258 bulk tank milk samples, eight enterococci species were isolated: E. faecalis (67.8%), E. hirae (15.1%), E. faecium (4.6%), E. saccharolyticus (4.6%), E. mundtii (3.1%), E. caseliflavus ( 2.7%), E. durans (1.2%), E. galinarum (0.8%). CONCLUSIONS: The difference in species predominance in bulk tank samples (67.8% of E. faecalis) and clinical mastitis (62.4% of E. saccharolyticus) was unexpected and caught our attention. Although Enterococcus spp. are traditionally classified as an environmental mastitis agent, in the present study, E. saccharolyticus behaved as a contagious agent of mastitis, which consequently changed the control patterns to be implemented.

Influence of pathogens causing clinical mastitis on reproductive variables of dairy cows.

In dairy cattle, mastitis is a disease of the mammary gland caused by pathogens such as bacteria, viruses, fungi, and algae. Mastitis causes economic losses to dairy farms as well as public health concerns. The reproductive efficiency of commercial dairy herds has important implications for the economic success of dairy operations and is strongly associated with the health status of cows. Mastitis has previously been linked with decreased fertility of dairy cows, but the effect of specific pathogens on the severity of fertility reduction is still unclear. In this study, cows diagnosed with mastitis caused by major pathogens (Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli, Klebsiella spp., Mycoplasma spp., and environmental Streptococcus) needed more artificial inseminations (AI) than did cows with mastitis caused by minor pathogens (coagulase-negative Staphylococcus and Corynebacterium spp.) and healthy cows. Cows diagnosed with mastitis, independent of what pathogen was causing mastitis, had more days open compared with nonmastitic cows. The percentage of cows that successfully established pregnancy at first AI was greater for the control group than for the major pathogens group but not significantly different from the minor pathogens group. Pregnancy loss was lower in the control group than in the major pathogens group; however, there was no difference compared with the minor pathogen group. Mastitis caused by gram-negative bacteria decreased the percentage of pregnancy per first AI and increased days open and pregnancy loss compared with the control group. Cows with mastitis caused by gram-positive bacteria also had increased days open compared with control cows. This study shows that different mastitis-causing bacteria can affect the fertility of cows differently. Mastitis events caused by major pathogens and gram-negative bacteria were associated with the greatest decrease in reproductive efficiency.

Putative virulence factors of extra-intestinal Escherichia coli isolated from bovine mastitis with different clinical scores.

We investigated the genes kpsMTII, iucD, sfaDE, afaBC, papA and papC, (proposed to be involved in extra-intestinal pathogenic Escherichia coli-ExPEC), phylogroup classification and the in vitro swimming and swarming motility in 50 E. coli isolated from bovine mastitis with different clinical severity scores (mild, moderate and severe). The aforementioned genes were detected in 12 (n = 12/50; 24·0%) isolates. kpsMTII and iucD were the most frequent genes identified in six (n = 6/50; 12·0%) and four (n = 4/50; 8·0%) of the isolates, respectively. In only one (n = 1/50; 2·0%) isolate, more than one gene was simultaneously identified: iucD and kpsMTll were detected whereas sfaDE and afaBC were not detected. Mild, moderate and severe clinical signs were observed in 40·0% (n = 20/50), 28·0% (n = 14/50) and 32·0% (n = 16/50) of the cases. Commensal phylogroups B1 (n = 19/50; 38·0%) and A (n = 19/50; 38·0%) were prevalent; whereas pathogenic phylogroups B2 and D were observed in only 10·0% (n = 5/50). Swarming and swimming motilities were observed in 90·0% (n = 45/50) and 68·0% (n = 34/50) of the isolates, respectively; and there was a significant association (P = 0·0036) between swarming motility and severe clinical cases (score 3). To the best of our knowledge, this is the first study where clinical severity of bovine mastitis cases and the genes proposed to classify ExPEC were assessed in relation to swarming and swimming motility. SIGNIFICANCE AND IMPACT OF THE STUDY: Escherichia coli is classified as extra-intestinal (ExPEC) when strains contain at least two of the genes kpsMTII, iucD, sfaDE, afaBC and papA and/or papC. We investigated in vitro motility and the presence of these genes in 50 E. coli isolated from bovine mastitis with different clinical scores (mild, moderate and severe). Clinical severity was not associated with the genes studied. Swarming motility was associated with severe cases (score 3) of clinical mastitis. Results of this study contribute to a better understanding of the factors that determine the severity of clinical mastitis.

Expression of fibroblast growth factor 22 (FGF22) and its receptor, FGFR1B, during development and regression of bovine corpus luteum.

The aim of this study was to determine the expression of fibroblast growth factor 22 (FGF22) in the bovine corpus luteum (CL) and to investigate the effects of in vivo total or partial cloprostenol-induced luteolysis on the mRNA abundance of FGF22 and its receptor, FGFR1B. Corpora lutea at different stages of development were then dissected from abattoir ovaries (n = 10/stage); a portion of the tissue samples was fixed in paraformaldehyde and the remaining samples were homogenized and subjected to total RNA extraction. To assess mRNA abundance of target genes during induced luteolysis, nineteen cows were synchronized and then randomly assigned to a Latin square design as follows: Control; 2 administrations of prostaglandin F2α (PGF2α, total luteolysis; 2 × 250 µg of cloprostenol sodium) and 1/6PGF2α (partial luteolysis; 83.33 µg of cloprostenol sodium). FGF22 and FGFR1B expression levels were measured by RT-qPCR, and FGF22 protein expression was detected by immunohistochemistry. In summary, FGF22 mRNA was detected at all stages of CL development, and FGF22 protein was also detected in luteal tissue. FGF22 mRNA expression was lower at stage IV than at stage III (P < 0.05), and the same pattern was observed in luteal immunoreactivity. Furthermore, cloprostenol-induced luteolysis, both total and partial, increased FGFR1B mRNA abundance in luteal tissue (P < 0.05), but did not affect FGF22 mRNA abundance. In conclusion, these data suggest a potential role for the FGF22-FGFR1B system during development and regression of bovine CL.