Bovine Bulk Tank Milk Research Articles

Prevalence, Serotypes, Antimicrobial Resistance and Biofilm-Forming Ability of Listeria monocytogenes Isolated from Bulk-Tank Bovine Milk in Northern Greece.

The prevalence of Listeria monocytogenes in bovine bulk-tank milk (BTM) in Greece has not been previously investigated. The aim of the study was to estimate the prevalence of L. monocytogenes in bovine BTM in Greece and to characterize the isolates in terms of carriage of genes encoding for pathogenic determinants, assess the isolates' biofilm-forming ability and determine their susceptibility against 12 antimicrobials. Samples (n = 138) of bovine BTM were obtained from farms located throughout Northern Greece and were analyzed qualitatively and quantitatively for L. monocytogenes. Five samples (3.6%) tested positive for L. monocytogenes. The pathogen's populations in these positive samples were below 5 CFU/mL. Most isolates belonged to the molecular serogroup "1/2a, 3a". All isolates carried the virulence genes inlA, inlC, inlJ, iap, plcA and hlyA, but actA was detected in only three isolates. The isolates displayed weak to moderate biofilm-forming ability and distinct antimicrobial resistance profiles. All isolates were characterized as multidrug resistant, with resistance to penicillin and clindamycin being a common feature. Considering that L. monocytogenes constitutes a serious public health threat, the key findings of the study, related to the carriage of virulence genes and multidrug resistance, highlight the importance of continued monitoring of the pathogen in farm animals.

Detection of Coxiella burnetii and characterisation by multiple-locus variable-number tandem repeat analysis in bovine bulk tank milk samples.

Coxiella burnetii is the aetiological agent of Q fever, which is highly prevalent in Turkiye, but information on the genetic profiles of the bacterium is limited. This study aimed to investigate the presence of C.burnetii in bovine bulk tank milk (BTM) samples by polymerase chain reaction (PCR) and toinvestigate the genotypes bymeans ofmultiple-locus variable-number tandem repeat analysis (MLVA). Atotal of25markets that sold raw cow's milk were analysed byconventional PCR analysis. AnMLVA analysis was performed atsix loci, namely MS23, MS24, MS27, MS28, MS33, and MS34, todetermine the genotypic variations ofC.burnetii found inthepositive DNA samples. The DNA ofC.burnetii was detected in16% ofthe BTM samples. The C.burnetii strains identified inthe bovine milk samples collectedinthis study were found tobelong tothe same genotypic group asthose detected inthe bovine milk samples gathered inGreece. Asaresult, both the presence and genotyping studies ofC.burnetii onthe BTM samples inTurkiye will contribute tothe determination ofthe geographical distribution ofthe agent.

Effect of ‘chlorine‐free’ cleaning of milking equipment on the microbiological quality and chlorine‐related residues in bulk tank milk

The influence of new chlorine‐free milking equipment cleaning protocols was compared with that of traditional chlorine‐based protocols from the perspective of the microbiological quality and chlorine‐related residues in bovine bulk tank milk. Commercial dairy farms using different cleaning protocols were identified, and bulk tank milk from these farms was sampled and tested on three occasions during the milk production season for microbial load and the chlorine‐related residues trichloromethane and chlorate. Total bacterial counts and residue levels were lower with chlorine‐free than with chlorine‐based protocols, demonstrating the new chlorine‐free cleaning protocols had a positive impact on milk quality when implemented on commercial farms.

Prevalence and characterization of mecC MRSA in bovine bulk tank milk in Great Britain, 2017-18.

ObjectivesTo evaluate the current prevalence status of mecC MRSA among dairy farms in England and Wales 5 years after a previous survey conducted in 2011–12.MethodsA convenience sample of 697 dairy farms in England and Wales was used for the study, conducted in 2017–18, testing bulk tank milk samples for the presence of mecC MRSA using high salt broth enrichment and chromogenic MRSA agar selection. All putative MRSA isolates were screened by PCR for the presence of mecA and mecC genes and subjected to antimicrobial susceptibility testing using both the disc diffusion method and VITEK® 2. MRSA isolates were also sequenced for genomic characterization.Results mecC MRSA were detected on 4 out of 697 dairy farms in England and Wales (prevalence 0.57%, 95% CI 0.16%–1.46%). Three of the mecC isolates were ST425 and one was ST4652 (in the CC130 lineage). Two mecA MRSA were also isolated: one ST5 and one ST398.ConclusionsThese results indicate that there has been a substantial reduction in the prevalence of mecC MRSA in England and Wales with a 72% reduction (2.15% to 0.57%) compared with a previous study. While the levels of mecA MRSA remain very low the continued presence of ST398, a livestock-associated MRSA, suggests that this lineage is established in the UK.

Prevalence and characterisation of methicillin-resistant staphylococci from bovine bulk tank milk in England and Wales

ObjectivesTo investigate the prevalence and characteristics of methicillin-resistant staphylococci on dairy farms in England and Wales including zoonotic MRSA. MethodsBulk tank milk was sampled from 363 dairy farms in 2015-2016 and methicillin-resistant staphylococci were isolated by salt broth enrichment and plating on MRSA Brilliance selective agar. Isolates were characterised through antimicrobial susceptibility testing and whole-genome sequencing. ResultsMethicillin-resistant staphylococci were isolated from ∼5% of dairy farms and belonged to six different species, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lentus, Staphylococcus saprophyticus, Staphylococcus fleurettii and Staphylococcus sciuri. Whole-genome sequencing revealed a large variety of antimicrobial resistance genes and SCCmec elements were present, including mecA and mecC alleles. Potentially zoonotic methicillin-resistance S. aureus were found at a low prevalence (0.83% of sampled dairy farms). Whole-genome sequencing also provided evidence for the mobility of a primordial mec gene complex, independently of a SCCmec element, which appears to have been acquired by S. saprophyticus from S. fleurettii. ConclusionsThese data give new insight into the epidemiology of veterinary methicillin-resistant staphylococci to inform future surveillance and zoonotic risk evaluation. Our data indicate that MRSA has likely decreased in prevalence since earlier survey work in England and Wales during 2011-12 and highlights the diversity of methicillin resistance and other resistance determinants among bovine-associated staphylococci with implications for veterinary and human medicine.

Short communication: Characterization of Staphylococcus aureus from bulk tank milk of dairy cattle in Lombardy (northern Italy)

Staphylococcus aureus is one of the most important pathogens causing mastitis in cattle, and it is responsible for economic losses in dairy herds worldwide. The PCR amplification of the 16S-23S rRNA intergenic spacer (ribosomal spacer PCR, RS-PCR) allows a rapid classification of the strains in genotypes and genotypic clusters (CL), which are characterized by different epidemiological and clinical properties. Both RS-PCR and multi-locus sequence typing (MLST) were performed on strains isolated from bovine bulk tank milk (BTM) collected from dairy herds located in the Lombardy region (northern Italy), to outline the distribution of Staph. aureus genotypes in this geographical area. Out of 844 examined samples, 398 were positive for Staph. aureus, with a variable count (cfu/mL) Up to 8 colonies from each sample were genotyped. A total of 1,101 Staph. aureus strains were analyzed with RS-PCR, and only a selection of them (n = 86), in relation to their frequency and geographical origin, underwent MLST. This study revealed 8 major genotypic clusters (CLB, CLC, CLR, CLS, CLI, CLF, CLAO, and CLZ), of which Staph. aureus CLB (29.3%) was the most common. Samples of BTM positive for CLB had a Staph. aureus cfu/mL count significantly higher than the non-CLB positive ones. Our MLST analysis showed genotypes already known as bovine-associated in literature, such as clonal complexes CC8, CC97, and CC151. The same selection of 86 strains was also analyzed for the presence of the adlb gene, which was recently proposed as a possible marker of contagiousness. Most Staph. aureus belonging to CLB or CC8 carried the adlb gene (85%), whereas this gene was detected in only 9% of non-CLB strains (CLAA, CLBI, CLBJ, CLS). In conclusion, the present study confirms that Staph. aureus CLB, which is recognized as a contagious genotype, is a particularly relevant agent of intramammary infection in dairy cows in Lombardy, and indirectly supports the idea that adlb can be a possible marker of contagiousness of isolates.

Arcobacter spp. in bovine milk: An emerging pathogen with potential zoonotic risk.

The aim of the present study was to assess the prevalence and genetic characteristics of Arcobacter spp. in bovine bulk tank milk produced in Apulia Region (Italy). Samples collected from 396 dairy farms, after enrichment in a selective broth, were subjected to an Arcobacter genus - specific Real Time PCR. Positive broths, previously filtered, were seeded on Karmali, MCCD and Columbia Blood Agar plates; presumptive Arcobacter spp. colonies were identified using an amplification and sequencing method and then characterized by Multi-Locus Sequence Typing (MLST). Prevalence of Arcobacter spp. in bovine milk samples was 5% (20/396); A. butzleri was the only isolated species, in agreement with previous studies that reported A. butzleri as the most commonly recovered species in milk and dairy products. MLST analysis of the 20 A. butzleri strains identified 81 alleles and 16 STs. Consistent with previous studies, MLST revealed a high level of heterogeneity between the A. butzleri isolates and confirmed the high discriminatory power of this method and its suitability for epidemiological investigations. This study confirmed the importance of raw milk as a possible source of Arcobacter spp. for humans.

Genome analysis of methicillin resistance in Macrococcus caseolyticus from dairy cattle in England and Wales.

Species of the genus Macrococcus are widespread commensals of animals but are becoming increasingly recognised as veterinary pathogens. They can encode methicillin resistance and are implicated in its spread to the closely-related, but more pathogenic, staphylococci. In this study we have identified 33 isolates of methicillin-resistant Macrococcus caseolyticus from bovine bulk tank milk from England and Wales. These isolates were characterised to provide insight into the molecular epidemiology of M. caseolyticus and to discern the genetic basis for their methicillin resistance. Antimicrobial susceptibility testing was performed by Vitek2 and disc diffusion. Isolates were whole-genome sequenced to evaluate phylogenetic relationships and the presence of methicillin resistance determinants, mecA–D. All 33 isolates were phenotypically methicillin-resistant according to cefoxitin disc diffusion, cefoxitin Etest and oxacillin resistance assessed by Vitek2. In contrast only a single isolate was resistant in the Vitek2 cefoxitin screen. Twenty-seven isolates were positive for mecD and six were positive for mecB. mecA and mecC were not detected. The results of phylogenetic analysis indicated that these methicillin-resistant isolates represented a heterogeneous population with both mecB and mecD found in diverse isolates. Isolates had a widespread distribution across the sampled region. Taken together with the role of M. caseolyticus in veterinary infections, including bovine mastitis, and in the potential spread of methicillin resistance to more pathogenic staphylococci, this work highlights the need to better understand their epidemiology and for increased awareness among veterinary microbiology laboratories.

Detection of genes encoding for enterotoxins, TSST-1, and biofilm production in coagulase-negative staphylococci from bovine bulk tank milk

Coagulase-negative staphylococci (CNS) represent common skin commensals in humans and other mammals. They are opportunistic microorganisms and, in dairy production, are considered as minor pathogens, often associated to subclinical mastitis. It has been recently demonstrated that CNS can produce Staphylococcus aureus superantigens, as well as biofilm. In our study, 74 CNS were isolated from Tuscan (Central Italy) bovine bulk tank milk and phenotypically identified as Staphylococcus xylosus (28.4 %), Staphylococcus chromogenes (20.3 %), Staphylococcus sciuri (12.2 %), Staphylococcus hominis (9.4 %), Staphylococcus haemolyticus (6.8 %), Staphylococcus capitis (5.4 %), Staphylococcus cohnii ssp. cohnii (4.1 %), Staphylococcus saprophyticus (4.1 %), and Staphylococcus simulans (4.1 %). Moreover, one isolate (1.3 %) was detected for each of the following species: Staphylococcus caprae, Staphylococcus epidermidis, Staphylococcus lentus, and Staphylococcus warneri. All isolates were screened by PCR for the presence of genes encoding classical enterotoxins (sea-see), toxic shock syndrome toxin-1 (tst), and biofilm (icaA/D). Forty isolates (54.1 %) presented at least one gene encoding for enterotoxin production, 15 (20.3 %) harbored two or more genes in association. The most frequently detected gene was sea (41.9 %), followed by sec-1 (25.7 %). None of the tested isolates presented genes encoding for enterotoxin B or D. Fifteen isolates (20.3 %) presented genes encoding for biofilm production. Among these, 11 isolates also harbored genes encoding for enterotoxin production. Our results suggest that enterotoxigenic CNS could represent a potential reservoir of pathogenicity factors, which could substantially contribute to enhance S. aureus virulence when present in milk and milk products. Further investigations are required for the evaluation of enterotoxins and biofilm production.

Association between antibody status to bovine herpesvirus 1 and quality of milk in dairy herds in Poland

Bovine herpesvirus 1 (BoHV1) is one of the most important pathogens of cattle; however, its effect on somatic cell count and milk components is not completely understood. The aim of the current study was to examine the effect of BoHV1 infection on quality of bovine bulk tank milk (BTM). A total of 1,790 individual blood samples collected at 28 dairy farms were used to determine the BoHV1 infection status of the herds with ELISA tests. The quality parameters of milk were evaluated by instrumental methods with BTM samples collected at monthly intervals from May 2011 to May 2012. The statistical analysis was performed to study the associations between BoHV1 herd status, quality of BTM, and herd-specific parameters. The risk factors influencing bulk milk somatic cell count (BMSCC) were estimated using the multivariable mixed-effects maximum likelihood regression model. The true prevalences of BoHV1 infection at the animal and herd levels were 49.3 and 64.6%, respectively. The average BMSCC differed significantly between the herds grouped accordingly to their BoHV1 infection status. Interestingly, the highest BMSCC was observed in the vaccinated herds (240.3×103 cells/mL). Additionally, the BoHV1 herd status had a significant effect on the fat content of BTM. The largest herds that were investigated had a BoHV1 seroprevalence over 30%. The herd status was considerably influenced by the numbers of cows in the herds. Besides, no significant differences in total bacterial count or protein content in milk from BoHV1-infected und uninfected herds were observed. An increase in BMSCC was observed during summer compared with the winter months regardless of the BoHV1 status of the herds. In the final multivariable regression model, the main risk factors associated with BMSCC were BoHV1 herd status, the percentage of BoHV1 infected animals in a herd, the number of cows in a herd, and the season. Our study suggests that BoHV1 infection may influence BMSCC levels, which are key parameters of BTM quality and a reference for subclinical mastitis in a herd. In conclusion, BoHV1 infection may cause economic losses by decrease both of quantity and quality of milk.

Prevalence assessment of Coxiella burnetii and verocytotoxin-producing Escherichia coli in bovine raw milk through molecular identification

Raw milk has become increasingly appreciated by consumers and in the EU there is general acceptance of this product, the sale of which is regulated by EU Directives. However, unpasteurised milk can be contaminated by zoonotic agents, thus representing a risk for human health. The aim of our work was the assessment of Coxiella burnetii and verocytotoxin-producing Escherichia coli (VTEC) presence in bovine bulk tank milk of Marche region (Central Italy) destined to direct human consumption. PCR and real-time PCR well-established protocols have been used for pathogen identification, showing high sensitivity and specificity. C. burnetii prevalence was 27%, while VTEC serogroup were found as follows: 3.5% for both O157 and O145; 2.3% for O26; 4.7% for O103. In contrast, the immunomagnetic separation-based protocol (IMS) was able to identify only one sample (1.2%) contaminated by VTEC O157.These results indicate that a combination of real-time PCR and IMS proved to be more appropriate for VTEC serogroup identification, thanks to improved sensitivity. Moreover, the prevalence of these pathogens would suggest that non-O157 VTEC and C. burnetii should be included among microbiological criteria for raw milk, implementing control strategies to limit possible negative impact to consumers' health.

Molecular identification and thermoresistance to boiling of Nocardia farcinica and Nocardia cyriacigeorgica from bovine bulk tank milk

Two strains of Nocardia spp. were isolated from bovine milk of two individual bulk tank. Molecular identification classified the strains as Nocardia farcinica and Nocardia cyriacigeorgica. The thermorresistance to boiling of the isolates was carried out and was observed bacterial growth after boiling. Our findings indicate the potential risk of pathogen transmission to humans through contaminated milk with Nocardia spp.

Application of a Peptide-Mediated Magnetic Separation-Phage Assay for Detection of Viable Mycobacterium avium subsp. paratuberculosis to Bovine Bulk Tank Milk and Feces Samples

Naturally contaminated bovine bulk tank milk (n = 44) and feces (n = 39) were tested for the presence of viable Mycobacterium avium subsp. paratuberculosis by a novel peptide-mediated magnetic separation-phage (PMS-phage) assay. Counts of viable M. avium subsp. paratuberculosis cells ranging from 1 to 110 PFU/50 ml of milk and 6 to 41,111 PFU/g of feces were indicated by the PMS-phage assay.

Relationship between somatic cell count, polymorphonuclear leucocyte count and quality parameters in bovine bulk tank milk

The somatic cell count (SCC) in bovine bulk tank milk is presently used as an indicator of raw milk quality, reflecting the udder health status of the herd. During mastitis, SCC increases, mostly owing to an influx of polymorphonuclear leucocytes (PMN) from blood into milk, with a concomitant change in milk composition. Bulk tank milk samples were categorized according to their SCC, as well as polymorphonuclear leucocyte count (PMNC), to study relationships between SCC, PMNC and various raw milk quality traits, i.e. contents of total protein, whey protein, casein, fat and lactose, casein number, proteolysis and rheological properties. The proportion of PMN, obtained by direct microscopy, was significantly higher in samples with high SCC compared with low SCC samples. SCC and PMNC were strongly correlated, yielding a correlation coefficient of 0.85. High SCC samples had lower lactose and casein contents, lower casein number and more proteolysis than low SCC samples. Samples with high PMNC had a lower casein number than low PMNC samples. Samples with high and low SCC or PMNC did not differ in respect to rheological properties. Our results do not indicate that PMNC is a better biomarker than SCC for raw bulk tank milk quality, as previously proposed.

Milk Lipoprotein Lipase Distribution in the Major Fractions of Bovine Milk

Raw, bovine bulk tank milk and milks from selected cows were separated by ultracentrifugation into four major fractions: casein, sloughed membrane material, serum, and milk fat globule membrane. Milk lipoprotein lipase activity was measured by the pH stat method and protein determinations were made by the Lowry procedure for each of the four fractions in order to calculate specific activity (units per milligram of protein). In six farm-cooled bulk milk samples stored ⩽ 24h, casein had a significantly higher milk lipoprotein lipase total activity, 35.66 units/ml of milk, than all of the fractions. Serum had the next highest activity with 11.69 units/ml of milk. Fluff and milk fat globule membrane had activities of .80 and .41 units/ml of milk, respectively. The specific activity of the fluff was 3.3 milk lipoprotein lipase units/mg of protein, which was significantly higher than the casein and serum fractions in pooled milk. Milks from five cows in midlactation were assayed individually for milk lipoprotein lipase activity and protein content immediately after milking and after 12, 24, 48 and 72h of cold (4°C) storage. Fresh warm milk was characterized by the absence of fluff. Casein had the highest mean activity (29.91 units/ml), followed by serum (10.25 units/ml) and milk fat globule membrane (.26 units/ml) in the warm milk from the individual cows. Upon cooling to 4°C, significant increases in enzyme activity in the fluff and milk fat globule membrane fractions were observed at 12h. In the cooled milks, milk lipoprotein lipase units/mg of protein for casein, fluff, and milk fat globule membrane were not significantly different but were significantly higher than the serum fraction. The specific activity of the fluff following cooling and storage of fresh, raw individual cow's milk suggests an association of the enzyme with sloughed membrane material.