Mastitic Samples Research Articles

The status of Mycoplasma bovis infection in clinical mastitis cases in China

In this study, ELISA and PCR were used to examine paired serum and milk samples (n = 850) from China and milk samples (n = 860) from the Inner Mongolia Autonomous Region from 2008 to 2015. The annual proportion of Mycoplasma bovis‐positive mastitis samples in China and Inner Mongolia Autonomous Region fluctuated inexplicably, with total average positivity value of 53.0% and 34.2%, respectively. We propose that the monthly variation in the proportion of M. bovis‐positive mastitis milk in Inner Mongolia Autonomous Region is related to the pathogen's survival in the environment, which requires further confirmation. The results highlight the severity of M. bovis infection in clinical mastitis in China.

Phenotypic and genotypic study on antimicrobial resistance patterns of E. coli isolates from bovine mastitis

It is important to evaluate the antimicrobial resistance pattern by genotypic and phenotypic methods in epidemiological studies in order to control antimicrobial resistance and to improve the outcome of the treatments. Four-hundred and thirty clinical mastitis samples were collected from 14 dairy herds in five different cities. The antimicrobial susceptibility test was performed using agar disk diffusion for 70 identified Escherichia coli isolates. The antimicrobial resistance genes including strA, strB, aadA, sulI, sulII, sulIII, ampC were detected by PCR method. Phylogenic groups were determined by Clermont's multiplex PCR method, and RAPD typing was performed on all isolates. Most isolates were resistant to lincomicin and streptomycin, whereas sulfa-trimethoprim has the lowest resistance rate. Moreover, ampC, aadA and sul2 genes had the highest frequency (92.85%, 38.57%, and 32.85% respectively). 20% of all the isolates carried strA and strB genes, and 11.42% of the isolates had sul1 gene and 10% of the isolates had the less frequent sul3 gene. Of the total of 70 E. coli isolates, 26 (37.14%), 20 (28.5%), 17 (24.2%), 8 (11.4%) isolates belonged to B1, A, B2 and D phylogenic groups respectively. strA, strB, sul2and aadA resistance genes had the highest percentage in A phylogenic groups. Based on RAPD-PCR method, E. coli isolates were classified in four clusters. The result showed a high phenotypic and genotypic E. coli resistance to the current antimicrobials with a similar pattern in different cities; also the majority of E. coli isolates belonged to B1 group which mainly contains the commensal E. coli isolates.

Comparison of an on-farm point-of-care diagnostic with conventional culture in analysing bovine mastitis samples.

The performance of a new point-of-care diagnostic (Mastatest), an on-farm test designed to identify bacteria and provide antibiotic sensitivity testing information from milk samples, was compared with standard microbiological culture methods. A total of 292 milk samples from clinical mastitis cases in dairy cows on New Zealand dairy farms were examined, and latent class analysis was used to estimate the performance characteristics of both tests. Two hundred and fifty-six samples (87.7%) demonstrated bacterial infection in standard culture, and 269 (92.1%) using the point-of-care diagnostic. The most common bacterial species detected was Streptococcus uberis, found in 195 samples (66.8%) using standard culture and 190 samples (65.1%) using the point-of-care diagnostic. Latent class analysis found no significant differences in test characteristics between the point-of-care diagnostic and standard culture. The estimated sensitivity and specificity of the point-of-care diagnostic against all targets combined were 94.6 and 72.1% respectively; the corresponding estimates for standard culture were 90.5 and 73.9%. Comparison of antibiotic susceptibility testing using the point-of-care diagnostic and the reference method showed similar trends and, in some cases, identical MIC50 and MIC90 values, with at most one antibiotic dilution difference.

Bovine fetal mesenchymal stem cells exert antiproliferative effect against mastitis causing pathogen Staphylococcus aureus

Staphylococcus aureus is the most commonly isolated pathogen from clinical bovine mastitis samples and a difficult pathogen to combat. Mesenchymal stem cells (MSC) are multipotent progenitor cells equipped with a variety of factors that inhibit bacterial growth. The aim of the present study was to evaluate the in vitro antibacterial potential against S. aureus of conditioned medium (CM) from MSC derived from fetal bovine bone marrow (BM-MSC) and adipose tissue (AT-MSC). BM-MSC, AT-MSC and fetal fibroblasts (FB) cultures were activated by infection with S. aureus. Bacterial growth was evaluated in presence of CM, concentrated CM (CCM), activated CM (ACM) and concentrated ACM (CACM) from BM-MSC, AT-MSC and FB. Gene expression of β-defensin 4A (bBD-4A), NK-lysine 1 (NK1), cathelicidin 2 (CATHL2), hepcidin (HEP) and indoleamine 2,3 dioxygenase (IDO) and protein expression of bBD-4A were determined in activated and non-activated cells. The majority of BM-MSC and AT-MSC expressed CD73, Oct4 and Nanog, and were negative for CD34. Growth of S. aureus decreased when it was exposed to CM from BM-MSC, AT-MSC and FB. Moreover, growth of S. aureus in CCM, ACM and CACM was lower compared to controls of CM from BM-MSC and AT-MSC. Activated AT-MSC increased mRNA levels of bBD4A and NK1, and protein levels of bBD4A in CM. Thus, CM from fetal bovine BM-MSC and AT-MSC has the capacity to reduce in average ~30% of S. aureus relative growth under in vitro conditions. The in vitro antibacterial effect of fetal bovine MSC may be mediated by bBD4A and NK1 activity.

Isolasi dan identifikasi bakteri dari susu kambing peranakan Ettawa (PE) terjangkit mastitis subklinis di Kemiri Kebo, Sleman, Yogyakarta

Subclinical mastitis in Ettawa crosbreed goat (PE) is economic disease which lost milk yield that bacteria caused. The aim of these research was to isolatian and identification bacteria that caused subclinical mastitis in PE goat from Kemiri Kebo, Turi, Sleman, Yogyakarta. A total of 24 goat milk PE subclinical mastitis samples, were used in these research. Determination of subclinical mastitis in PE goat used CMT test which (++) and (+++) results. Isolation and identification conventional bacteria based on biochemical. Subclinical mastitis PE goat in Kemiri Kebo, Turi, Sleman, Yogyakarta was caused Staphylococcus intermedius (38%), Staphylococcus aureus (32%), Staphylococcus epidermidis (25%) , Staphylococcus hycus (12%) , Streptococcus sp (22%), Corynebacterium sp (12%) , Pseudomonas aeruginosa (18%), and E. coli (2%). The research showed that Gram positive bacteria is dominant that caused subclinical mastitis in PE goat compared than Gram negative bacteria.

Immunohistochemical detection of alpha-smooth muscle actin and S-100 in bovine mammary gland with mastitis

Fibrosis is a common finding in chronic mastitis. Epithelial-mesenchymal transition (EMT) plays a prominent role in fibrogenesis in adult tissues. The aim of this study was to investigate whether EMT occurs in bovine mastitis by assessing the expression or localization of alpha-smooth muscle actin (alpha-SMA), S-100 protein and caspase-3. Two normal bovine mammary gland tissue samples and five mastitic samples were used in the study. Antibodies to alpha-SMA, S-100 and caspase-3 were used for immunohistochemical studies. In mastitis there was increased expression of S-100 in the alveolar epithelial cells. Alpha-SMA was highly expressed in the areas where there is fibrosis in chronic mastitis. Further caspase-3 was observed in alveolar cells in mastitic tissue. Overexpression of S-100 and alpha-SMA directly correlates to the severity of mastitis.S-100 and alpha-SMA may serve as biomarkers to know the extent of tissue structural changes in mastitis. EMT could be one of the mechanistic pathways underlying the pathogenesis of fibrosis in mastitis.

Gene expression of multidrug-resistant ATP-binding cassette transporter (MDR1/ABCB1) in bovine mastitis

Purpose: To investigate the expression of multidrug-resistant ATP binding cassette transporter Pglycoprotein (MDR1/ABCB1/P-gp) in mastitis and its correlation with the expression of apoptosis regulation.Methods: Mastitic and normal mammary gland samples were collected after clinical examination and confirmed using field California mastitis test. Total RNA was extracted followed by cDNA synthesis and real-time PCR to detect the expression pattern of P-gp. Tissue sections were examined for pathological changes and immune reactivity to the apoptotic regulator, Bcl-2-associated X protein (BAX).Results: Stained sections from mastitic samples showed congestion, infiltration of inflammatory cells and hemorrhage. Using immunohistochemistry, it was shown that mastitis is associated with overexpression of the apoptosis regulator, Bcl-2-associated X protein (BAX). There was increased expression of the pro-apoptotic factor, Bax, in mastitic alveolar lining and lactic tubules compared with normal mammary gland tissues. Using real-time PCR, the expression level of MDR1/ABCB1 was determined to be 2.6-fold higher in mastitic tissues than in normal mammary tissues.Conclusion: Higher MDR1/P-gp level can modulate drug interventions by preventing intracellular accumulation of chemotherapeutics or anti-inflammatories via continuous efflux or export to extracellular fluids.Keywords: ABC transporter, P-glycoprotein, Drug exporters, Mastitis, Mammary gland

Isolation of multi-drug resistant Klebsiella sp. from bovine mastitis samples in Rangpur, Bangladesh.

Objective:The objective of this study was to identify the multi-drug resistance (MDR) Klebsiella sp. from mastitis milk samples.Materials and Methods:In the current research, 48 clinical mastitis milk samples were collected from Rangpur division, Bangladesh. Confirmation of bovine mastitis (BM) was done by the California Mastitis Test (CMT). All the CMT positive isolates were subjected for the identification of Klebsiella sp. using through a series of cultural and biochemical tests. MDR Klebsiella sp. isolates were determined using the disk diffusion method, and minimum inhibitory zones were measured by following Clinical and Laboratory Standards Institute. MDR patterns of the isolates were also subjected to study by using housefly (Musca domestica).Results:Among the isolates, 62.5% (n = 30/48) revealed the presence of Klebsiella sp. Eight antimicrobial agents including Amoxicillin, Novobiocin, Erythromycin, Vancomycin, Cephradine, Tetracycline, Bacitracin, Methicillin, and housefly (M. domestica) showed complete resistance to Klebsiella sp. On the other hand, Chloramphenicol, Gentamicin, Ciprofloxacin, Azithromycin, Norfloxacin, Levofloxacin, and Nalidixic acid showed sensitivity.Conclusion:This study helps to treat BM with effective antibiotics and helps in an epidemiological study in Rangpur division as well as helps to create public health awareness.

Evaluation of leukocyte esterase test strips for rapid diagnosis of subclinical mastitis in dairy cows

Subclinical mastitis is difficult to detect because it has no visible indications and requires special tests to be detected. Therefore, an untimely and precise diagnosis of subclinical mastitis has been suggested. The purpose of this paper was to estimate the performance of the Analyticon reagent strip test application for the diagnosis of subclinical mastitis as a cow-side test. Milk samples from 111 dairy cows were collected and the leukocyte esterase (LE) test was performed with the reagent strip test. Leukocyte esterase activity was estimated following 10 (LE10) and 15 (LE15) min. Also, milk somatic cell count (SCC) obtained by an automated Fossomatic machine was considered as the gold standard for diagnosing subclinical mastitis. The specificity (Sp), sensitivity (Se), positive likelihood ratio (+LR), negative likelihood ratio (−LR), area under the receiver operating curve (AUC), and kappa coefficient (κ) were determined. The 75.7% (84/111) milk samples with SCC ≥ 200,000/mL were considered as the subclinical mastitis milk. A strong statistically significant correlation was found between SCC, LE test marker, and SCC scoring (0–3) in all milk samples. Based on the cutoff point, the LE15 test had Se = 80%, Sp = 100%, +LR = ∞, and −LR = 0.20. There was a very close relation between LE15 and SCC test with a kappa coefficient of 0.70 (P < 0.0001). Also, according to the cutoff point of 0.5, the LE10 test had Se = 64%, Sp = 100%, +LR = ∞, and −LR = 0.36. In addition, there was a very close relation between LE10 and SCC tests with a kappa coefficient of 0.50 (P < 0.0001). The LE activity was significantly higher (P < 0.05) in subclinical mastitis samples compared to that of healthy milk samples at the time of 10 and 15 min. In summary, these results showed a strong correlation among the LE test markers with the reagent strip test of Analyticon and SCC using the Fossomatic machine. Based on such findings, it is suggested that the LE activity could be used as a clinically useful cow-side test and a valid method for screening the subclinical mastitis.

Identification of Host Defense-Related Proteins Using Label-Free Quantitative Proteomic Analysis of Milk Whey from Cows with Staphylococcus aureus Subclinical Mastitis.

Staphylococcus aureus is the most common contagious pathogen associated with bovine subclinical mastitis. Current diagnosis of S. aureus mastitis is based on bacteriological culture of milk samples and somatic cell counts, which lack either sensitivity or specificity. Identification of milk proteins that contribute to host defense and their variable responses to pathogenic stimuli would enable the characterization of putative biomarkers of subclinical mastitis. To accomplish this, milk whey samples from healthy and mastitic dairy cows were analyzed using a label-free quantitative proteomics approach. In total, 90 proteins were identified, of which 25 showed significant differential abundance between healthy and mastitic samples. In silico functional analyses indicated the involvement of the differentially abundant proteins in biological mechanisms and signaling pathways related to host defense including pathogen-recognition, direct antimicrobial function, and the acute-phase response. This proteomics and bioinformatics analysis not only facilitates the identification of putative biomarkers of S. aureus subclinical mastitis but also recapitulates previous findings demonstrating the abundance of host defense proteins in intramammary infection. All mass spectrometry data are available via ProteomeXchange with identifier PXD007516.

First report of the Staphylococcus aureus isolate from subclinical bovine mastitis in the South of Brazil harboring resistance gene dfrG and transposon family Tn916-1545

The aim of this work was to identify at the molecular level the species of coagulase-positive staphylococci isolates from clinical and subclinical bovine mastitis samples in Southern Brazil, and to evaluate the antimicrobial resistance profile, as well as the presence of resistance genes. According to the PCR assay, all 31 isolates were classified as Staphylococcus aureus. The isolates were tested for resistance to penicillin, ampicillin, oxacillin, cefoxitin, cephalothin, ceftiofur, streptomycin, tobramycin, teicoplanin, erythromycin, clindamycin, enrofloxacin, sulfonamide, trimethoprim-sulfamethoxazole, trimethoprim, and tetracycline by the disk diffusion method. Most of the isolates were resistant to sulfonamide (20), followed by ampicillin and clindamycin (16). Twenty isolates were multidrug-resistant. PCR was used for the detection of several antimicrobial resistance genes (ereB, ermB, ermC, tetA, tetB, tetK, tetL, tetM, tetO, Tn916-1545, strA, strB, sul1, sul2, dfrA, dfrG, dfrK, blaZ, mecA, and mecC). The most prevalent antimicrobial resistance genes were tetK and tetL, ereB, followed by tetM, Tn916-1545 and blaZ, detected in 11, nine and four isolates, respectively. For all the tetM gene positive isolates, the presence of conjugative transposons of the Tn916-1545 family was detected. The presence of multidrug-resistant isolates, antimicrobial resistance genes and transposons suggests a potential risk of spreading multi-resistance genes to other bacteria.

Identification of Emerging Human Mastitis Pathogens by MALDI-TOF and Assessment of Their Antibiotic Resistance Patterns.

Lactational mastitis constitutes one of the main causes of undesired weaning, depriving the mother–infant pair from the benefits of breastfeeding; therefore, this condition should be considered a relevant public health issue. The role of specific microorganisms remains unclear since human milk cultures and antibiotic susceptibility testing (AST) are not routinely performed, despite the fact that this would be key to ensure an early and effective diagnosis and treatment. The objective of this study was to describe the culturable microbial diversity in 647 milk samples from breastfeeding women with clinical symptoms of mastitis by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF) VITEK MS technology and to analyze the antimicrobial susceptibility profiles of a collection of isolates from these samples by the VITEK 2 AST system. Staphylococcus epidermidis was the most common species isolated from mastitis samples (87.6%), while Staphylococcus aureus was detected in 22.1%. Streptococci constituted the second (68.6%) most prevalent bacterial group, with Streptococcus mitis/oralis, Streptococcus salivarius, and Streptococcus parasanguinis detected with frequencies of 40.8, 36.8, and 14.4%, respectively. The antibiotic susceptibility profiles of 642 staphylococcal isolates indicated a remarkable resistance to benzylpenicillin (88.3%) and erythromycin (67.3%) with differences between species. A high percentage of Staphylococcus isolates were resistant to at least one antibiotic (Staphylococcus hominis, 100%; S. epidermidis, 98.2%; S. aureus, 92.9%; Staphylococcus lugdunensis, 90.5%) and the percentage of multidrug-resistance (MDR) isolates was noticeable (S. hominis, 81%; S. epidermidis, 64.4%; S. aureus, 11.5%; S. lugdunensis, 10.5%). In relation to streptococcal isolates (n = 524), AST revealed high or moderate percentages of resistance to erythromycin (68.7%), benzylpenicillin (63.7%), ampicillin (51.5%), and tetracycline (30.8%). Antibiotic resistance to at least one antibiotic was detected in 97.6% of S. parasanguinis, 92.6% of S. salivarius, 83.3% of S. mitis/oralis, and 72.4% of Streptococcus vestibularis isolates. A significant number of MDR streptococcal isolates was also found (S. parasanguinis, 51.2%; S. salivarius, 39.3%; S. mitis/oralis, 34.6%; and S. vestibularis, 19%). The results highlight the important role of coagulase-negative staphylococci and streptococci as human mastitis-causing agents. Moreover, the high rates of antimicrobial resistance among these microorganisms must be contemplated as an issue of clinical relevance in relation to treatment options.

Antimicrobial Cytotoxic Immune Proteins in Human Milk

Abstract Human milk (HM) contains a large variety of immune components providing protection to the infant. The presence of the cytotoxic immune proteins perforin (Per), granulysin (Grly)and granzymes (Grzm)has not yet been shown in HM cells. Per, Grnly and Grzm (A, B, H and M) were examined in cells isolated from samples of prepartum HM (PS) collected during pregnancy and HM collected longitudinally (2–5 sampling occasions) from mothers (n=31) during the first year postpartum, including 3 samples from breast infections. Gene expression was analyzed in RNA sequencing data and using qRT-PCR. Linear modeling and principle component analysis between the genes was conducted. Flow cytometry (n=5) was performed to investigate the presence of these immune proteins in HM cells. Gene expression of all immune proteins was confirmed in PS and in HM during month 1 of lactation, where variation of expression was found between women and time lactation. Strong correlations were found between GrzmA – Per (r2=0.86), GrzmA – CD45 (r2=0.76), Per – CD45 (r2=0.68) and Per – GrzmB (r2=0.67). Comparison between healthy and mastitis samples showed a higher expression of CD45 and all immune peptides in mastitis. Moreover, flow cytometry analysis showed an increase in CD45 positive cells in mastitis in comparison with healthy participants (15.1% and 4.7% respectively of total cell amount). The presence of Per, Grly and Grzm has been confirmed in HM cells. An increase in gene expression of these immune proteins has been confirmed in 3 participants suffering from breast infection. Further investigations are required to elucidate the roles of these immune peptides for the infant and/or mother

Relationship between milk cathelicidin abundance and microbiologic culture in clinical mastitis

The availability of reliable tools to enable the sensitive and specific detection of mastitis in dairy cows can assist in developing control strategies and promote the more rational use of antibiotics. We have developed a milk cathelicidin ELISA that shows high sensitivity and specificity for dairy cow mastitis, based on latent class analysis. In this study, we investigated the effect of microbial agents on cathelicidin abundance in the milk of cows with clinical mastitis. We subjected 535 quarter milk samples (435 from quarters showing signs of clinical mastitis and 100 from healthy quarters as a control) to milk cathelicidin ELISA, somatic cell count (SCC), and microbiologic culture. Of the 435 clinical mastitis samples, 431 (99.08%) were positive for cathelicidin, 424 (97.47%) had SCC >200,000 cells/mL, and 376 (86.44%) were culture-positive. Of the 59 culture-negative samples, 58 (98.30%) were positive for cathelicidin and 55 (93.22%) had SCC >200,000 cells/mL. The abundance of cathelicidin and the extent of SCC increase depended on the causative agent: Streptococcus agalactiae and coagulase-negative staphylococci showed the highest and lowest changes, respectively. We also observed differences in behavior between the 2 markers depending on the pathogen: Streptococcus agalactiae induced the highest cathelicidin abundance, and Serratia spp. induced the highest SCC. Nevertheless, the different ability of microorganisms to induce cathelicidin release in milk did not compromise its value as a mastitis marker, given its higher sensitivity compared to SCC or microbiologic culture. All 100 negative control samples (collected from healthy quarters with SCC <100,000 cells/mL and culture-negative) were also negative for cathelicidin, corresponding to 100% specificity in the evaluated sample cohort. This study confirmed the value of the milk cathelicidin ELISA for detecting bovine mastitis, and highlighted the influence of mastitis-causing microorganisms on cathelicidin abundance. This influence did not compromise diagnostic performance; instead, it may have better reflected disease severity and evolution than SCC.

Longitudinal metagenomic profiling of bovine milk to assess the impact of intramammary treatment using a third-generation cephalosporin.

Antimicrobial usage in food animals has a direct impact on human health, and approximately 80% of the antibiotics prescribed in the dairy industry are used to treat bovine mastitis. Here we provide a longitudinal description of the changes in the microbiome of milk that are associated with mastitis and antimicrobial therapy. Next-generation sequencing, 16 S rRNA gene quantitative real-time PCR, and aerobic culturing were applied to assess the effect of disease and antibiotic therapy on the milk microbiome. Cows diagnosed with clinical mastitis associated with Gram-negative pathogens or negative aerobic culture were randomly allocated into 5 days of Ceftiofur intramammary treatment or remained as untreated controls. Serial milk samples were collected from the affected quarter and the ipsilateral healthy quarter of the same animal. Milk from the mastitic quarter had a higher bacterial load and reduced microbial diversity compared to healthy milk. Resolution of the disease was accompanied by increases in diversity indexes and a decrease in pathogen relative abundance. Escherichia coli-associated mastitic milk samples had a remarkably distinct bacterial profile, dominated by Enterobacteriaceae, when compared to healthy milk. However, no differences were observed in culture-negative mastitis samples when compared to healthy milk. Antimicrobial treatment had no significant effect on clinical cure, bacteriological cure, pathogen clearance rate or bacterial load.

Genotypic and phenotypic detection of capsular polysaccharide and biofilm formation in Staphylococcus aureus isolated from bovine milk collected from Brazilian dairy farms.

Staphylococcus aureus is a pathogen that frequently causes mastitis in bovine herds worldwide. This pathogen produces several virulence factors, including cell-associated adhesins, toxic and cytolytic exoproteins, and capsular polysaccharides. The aim of the present study was to test for the presence of genes involved in capsular polysaccharide production and biofilm formation in S. aureus isolated from bovine mastitis samples collected from 119 dairy herds located in three different Brazilian regions, as well as to assay the production of capsular polysaccharides and biofilm, in vitro. The detection of the cap, icaAD, and bap genes was performed using PCR. The detection and quantification of capsular polysaccharide production was performed using ELISA assays. The ability of the isolates to form a biofilm was examined using the polystyrene surface of microtiter plates. All 159S. aureus isolates investigated harboured the cap gene: 80% carried the cap5 gene and 20% carried the cap8 gene. Sixty-nine percent of the isolates expressed capsular polysaccharide (CP) in vitro, 58% expressed CP5 and 11% expressed CP8. All of the isolates harboured the icaA and icaD genes, and 95.6% of the isolates carried the bap gene. Of the 159 isolates analysed, 97.5% were biofilm producers. A significant association between the capsular genotype and phenotype and the amount of biofilm formation was detected: cap5/CP5 isolates tended to form more biofilm and to produce a thinner CP layer than cap8/CP8 isolates. The results indicate a high potential for pathogenicity among S. aureus isolated from bovine milk collected from three different regions in Brazil.

Culture-dependent assessment of bacterial diversity from human milk with lactational mastitis

Mastitis, an inflammation of mammary gland, is a deliberately painful condition experienced by lactating women. Etiology of mastitis is still underexplored due to the paucity of research conducted on it. The objective of this study was to explore culturable bacterial diversity present in the breast milk of mastitis-suffering women by 16S rDNA sequencing and evaluate its antibiotic susceptibility. The culturable bacteria present in the breast milk of 32 women with lactational mastitis were studied. Thirty-seven bacterial isolates comprised of distinct colony characteristics were checked for antibiotic susceptibility and identified by 16S rDNA sequencing. Sequencing of 16S rDNA and BLAST analysis revealed the presence of 17 genera and 30 different species indicating highly diverse community in mastitis milk. The Proteobacteria (51.35 % of total clones) and Firmicutes (37.83 %) were the most abundant phyla and included representatives from the class Gammaproteobacteria, Alphaproteobacteria, and Bacilli. Pseudomonas (6 species) and Staphylococcus (4 species) were the most prevalent genera while Brevundimonas, Bacillus, Enterococcus, Micrococcus, and Macrococcus were infrequently isolated. Staphylococcus sp. (87.5 % of mastitis samples), Staphylococcus aureus (75 %), Pseudomonas aeruginosa (53.12 %), Klebsiella pneumoniae (43.75 %), and Brevundimonas diminuta (37.5 %) were frequently isolated from mastitis milk. Antibiotics like chloramphenicol, gentamicin, ofloxacin, and ciprofloxacin proved to be most effective against mastitis pathogens. Overall analysis suggested that high bacterial diversity in diseased condition could be the result of colonization of mammary gland by opportunistic pathogens but the actual role of bacteria in mastitis is still a debatable question.

Sensitivity of two methods to detect Mycoplasma agalactiae in goat milk.

BackgroundLaboratory diagnostic techniques able to detect Mycoplasma agalactiae are essential in contagious agalactia in dairy goats. This study was designed: 1) to determine the detection limits of PCR and culture in goat milk samples, 2) to examine the effects of experimental conditions including the DNA extraction method, PCR technique and storage conditions (fresh versus frozen stored milk samples) on these methods and 3), to establish agreement between PCR and culture techniques using milk samples from goats with mastitis in commercial dairy herds. The study was conducted both on artificially inoculated and field samples.ResultsOur findings indicate that culture is able to detect M. agalactiae in goat milk at lower concentrations than PCR. Qualitative detection of M.agalactiae by culture and PCR was not affected by sample freezing, though the DNA extraction method used significantly affected the results of the different PCR protocols. When clinical samples were used, both techniques showed good agreement.ConclusionsThe results from this study indicate that both culture and PCR are able to detect M. agalactiae in clinical goat mastitis samples. However, in bulk tank milk samples with presumably lower M. agalactiae concentrations, culture is recommended within the first 24 h of sample collection due to its lower limit of detection. To improve the diagnostic sensitivity of PCR in milk samples, there is a need to increase the efficiency of extracting DNA from milk samples using protocols including a previous step of enzymatic digestion.

Short communication: Protease activity measurement in milk as a diagnostic test for clinical mastitis in dairy cows

Due to the increasing use of automated milking systems, automated detection of clinical mastitis is becoming more important. Various in- or on-line diagnostic tests are in use, but generally suffer from false mastitis alerts. In this study, we explored a new diagnostic approach based on measurement of protease activity using fluorogenic protease substrates, which can be performed on site, at high speed, and at low costs. Samples from cows with clinical mastitis submitted for bacteriological culture at the University Farm Animal Practice were collected during several months and kept at −20°C until protease activity measurement. A reference set of milk samples from clinically healthy cows were collected on 9 different farms and were tested for protease activity directly and after freezing at −20°C to allow for comparison with the samples from clinical cases. The protease activity in mastitic milk samples was significantly higher than in samples from healthy animals. Based on 71 clinical mastitis samples and 180 milk samples from clinically healthy quarters, the area under the receiver-operating characteristic curve was estimated to be between 0.88 and 0.90, and at a threshold of 38 fluorescence per minute the test had a specificity of 0.99 at a sensitivity of 0.58. Protease activity measured in fresh milk from clinically healthy cows was significantly associated with somatic cell count and parity, but not with electrical conductivity, whereas protease activity in milk that had been frozen was statistically significantly associated with all 3 parameters. This study indicates that protease activity measurement as a stand-alone test can be used for detecting mastitis samples, using milk samples that have been frozen. Because protease activity acts in part on a different biological mechanism than somatic cell count or electrical conductivity, this test may increase the accuracy of mastitis diagnosis in combination with currently available in- or on-line tests in automated milking systems.

Reduction of Lactobacillus in the milks of cows with subclinical mastitis.

Clinical and subclinical bovine mastitis are the most frequent diseases encountered on dairy farms worldwide, which cause significant economic loss and veterinary cost. The mastitic disease status is associated with increases in both milk bacterial pathogens and somatic cell count (SCC). Although it is well established that the mastitic pathogens generally correlate with the milk SCC, to our knowledge, the correlation between the probiotic genus, Lactobacillus, and the mastitic causative bacteria and SCC have not been determined previously. Thus, in this study, milk samples from 12 mild and 28 severe subclinical mastitic dairy cows were collected from the same farm. The overall milk bacterial load was quantified with the total plate count method. The Lactobacillus genus and 4 common clinical and subclinical mastitic pathogens (Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, and Trueperella pyogenes) in the sampled milk were enumerated by quantitative PCR. Mild and severe subclinical mastitic samples were distinctly separated on the principal component analysis score plot generated based on the quantities of these 5 target bacteria, suggesting that clear differences existed in the microbiological composition between the two sample groups. Based on comparison with the pairwise Mann-Whitney test, the mild subclinical mastitic dairy cows had a significantly higher amounts of lactobacilli (P=0.0175), but lower E. coli (P=0.0002), S. aureus (P<0.0001), S. agalactiae (P=0.0001) and T. pyogenes (P=0.0044) quantities, while an opposite trend occurred in the severe subclinical mastitic group. The negative correlation between Lactobacillus and the pathogenic bacteria, as well as the SCC, was confirmed with Spearman correlation analysis. Data generated from the current study may hint to a close relationship between Lactobacillus and the health of bovine udder.