Presence of Campylobacter and Arcobacter species in in-line milk filters of farms authorized to produce and sell raw milk and of a water buffalo dairy farm in Italy

The objectives of this study were to investigate the presence of Campylobacter spp. and Arcobacter spp. in dairy herds authorized for the production and sale of raw milk and in a water buffalo dairy farm, and to test the antimicrobial susceptibility of the isolates. A total of 196 in-line milk filters were collected from 14 dairy farms (13 bovine and 1 water buffalo) for detection of Campylobacter spp. and Arcobacter spp. by microbiological culture. For each farm investigated, 1 isolate for each Campylobacter and Arcobacter species isolated was tested using the Etest method (AB Biodisk, Solna, Sweden) to evaluate the susceptibility to ciprofloxacin, tetracycline, chloramphenicol, ampicillin, erythromycin, and gentamicin. A total of 52 isolates were detected in 49 milk filters in 12 farms (85.7%) out of 14 and the isolates were identified as Campylobacter jejuni (6), Campylobacter hyointestinalis ssp. hyointestinalis (8), Campylobacter concisus (1), Campylobacter fetus ssp. fetus (1), Arcobacter butzleri (22), and Arcobacter cryaerophilus (14). The small number of isolates tested for antimicrobial susceptibility precludes any epidemiological consideration but highlights that all Campylobacter isolates were susceptible to macrolides, which are the first-choice drugs for the treatment of campylobacteriosis, and that resistance to fluoroquinolones and tetracycline was detected; for Arcobacter isolates, resistance to ampicillin and chloramphenicol was detected. The sale of raw milk for human consumption by self-service automatic vending machines has been allowed in Italy since 2004 and the presence of C. jejuni in in-line milk filters confirms that raw milk consumption is a significant risk factor for human infection. The high occurrence of emerging Campylobacter spp. and Arcobacter spp. discovered in dairy farms authorized for production and sale of raw milk represents an emerging hazard for human health.     

Prevalence of Salmonella enterica,Listeria monocytogenes,and pathogenic Escherichia coli in bulk tank milk and milk filters from US dairy operations in the National Animal Health Monitoring System Dairy 2014 study

The dairy farm environment is a well-documented reservoir for zoonotic pathogens such as Salmonella enterica, Shiga-toxigenic Escherichia coli, and Listeria monocytogenes, and humans may be exposed to these pathogens via consumption of unpasteurized milk and dairy products. As part of the National Animal Health Monitoring System Dairy 2014 study, bulk tank milk (BTM, n = 234) and milk filters (n = 254) were collected from a total of 234 dairy operations in 17 major dairy states and analyzed for the presence of these pathogens. The invA gene was detected in samples from 18.5% of operations and Salmonella enterica was isolated from 18.0% of operations. Salmonella Dublin was detected in 0.7% of operations. Sixteen Salmonella serotypes were isolated, and the most common serotypes were Cerro, Montevideo, and Newport. Representative Salmonella isolates (n = 137) were tested against a panel of 14 antimicrobials. Most (85%) were pansusceptible; the remaining were resistant to 1 to 9 antimicrobials, and within the resistant strains the most common profile was resistance to ampicillin/clavulanic acid, ampicillin, cefoxitin, ceftiofur, ceftriaxone, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline. Listeria spp. were isolated from 19.9% of operations, and L. monocytogenes was isolated from 3.0% of operations. Serogroups 1/2a and 1/2b were the most common, followed by 4b and 4a. One or more E. coli virulence genes were detected in the BTM from 30.5% of operations and in the filters from 75.3% of operations. A combination of stx₂, eaeA, and γ-tir genes was detected in the BTM from 0.5% of operations and in the filters from 6.6% of operations. The results of this study indicate an appreciable prevalence of bacterial pathogens in BTM and filters, including serovars known to infect humans.     

Genotypic analysis of nontuberculous mycobacteria isolated from raw milk and human cases in Wisconsin

Nontuberculous mycobacteria (NTM) compose a group of mycobacteria that do not belong to the Mycobacterium tuberculosis complex group. They are frequently isolated from environmental samples such as water, soil, and, to a lesser extent, food samples. Isolates of NTM represent a major health threat to humans worldwide, especially those who have asthma or are immunocompromised. Human disease is acquired from environmental exposures and through consumption of NTM-contaminated food. The most common clinical manifestation of NTM disease in human is lung disease, but lymphatic, skin and soft tissue, and disseminated disease are also important. The main objective of the current study was to profile the farm-level contamination of cow milk with NTM by examining milk filters and bulk tank milk samples. Five different NTM species were isolated in one dairy herd in Wisconsin, with confirmed 16S rRNA genotypes including Mycobacterium fortuitum, Mycobacterium avium ssp. hominissuis, Mycobacterium abscessus, Mycobacterium simiae, and Mycobacterium avium ssp. paratuberculosis (Mycobacterium paratuberculosis). In tank milk samples, M. fortuitum was the predominant species in 48% of the samples, whereas M. chelonae/abscessus and M. fortuitum were the only 2 species obtained from 77 and 23% of the examined filters, respectively. Surprisingly, M. avium ssp. hominissuis, M. paratuberculosis, and M. simiae were isolated from 16.7, 10.4, and 4% of the examined milk samples, respectively, but not from milk filters. Interestingly, NTM isolates from human clinical cases in Wisconsin clustered very closely with those from milk samples. These findings suggest that the problem of NTM contamination is underestimated in dairy herds and could contribute to human infections with NTM. Overall, the study validates the use of bulk tank samples rather than milk filters to assess contamination of milk with NTM. Nontuberculous mycobacteria represent one type of pathogens that extensively contaminate raw milk at the farm level. The significance of our research is in evaluating the existence of NTM at the farm level and identifying a simple approach to examine the potential milk contamination with NTM members using tank milk or milk filters from dairy operations. In addition, we attempted to examine the potential link between NTM isolates found in the farm to those circulating in humans in Wisconsin.     

A survey of foodborne pathogens in bulk tank milk and raw milk consumption among farm families in pennsylvania

A 2-part study was conducted to determine the risk of exposure to human pathogens from raw milk. The first part of the study focused on determining raw milk consumption habits of dairy producers. A total of 248 dairy producers from 16 counties in Pennsylvania were surveyed. Overall, 105 (42.3%) of the 248 dairy producers consumed raw milk and 170 (68.5%) of the 248 dairy producers were aware of foodborne pathogens in raw milk. Dairy producers who were not aware of foodborne pathogens in raw milk were 2-fold more likely to consume raw milk compared with dairy producers who were aware of foodborne pathogens. The majority of dairy producers who consumed raw milk indicated that taste (72%) and convenience (60%) were the primary factors for consuming raw milk. Dairy producers who resided on the dairy farm were nearly 3-fold more likely to consume raw milk compared with those who lived elsewhere. In the second part of the study, bulk tank milk from the 248 participating dairy herds was examined for foodborne pathogens. Campylobacter jejuni (2%), Shiga toxin-producing Escherichia coli (2.4%), Listeria monocytogenes (2.8%), Salmonella (6%), and Yersinia enterocolitica (1.2%) were detected in the milk samples. Salmonella isolates were identified as S. enterica serotype Typhimurium (n = 10) and S. enterica serotype Newport (n = 5). Of the 248 bulk tank milk samples, 32 (13%) contained ≥1 species of bacterial pathogens. The findings of the study could assist in developing farm community-based educational programs on the risks of consuming raw milk.     

Tracking heat-resistant, cold-thriving fluid milk spoilage bacteria from farm to packaged product

Control of psychrotolerant endospore-forming spoilage bacteria, particularly Bacillus and Paenibacillus spp., is economically important to the dairy industry. These microbes form endospores that can survive high-temperature, short-time pasteurization; hence, their presence in raw milk represents a major potential cause of milk spoilage. A previously developed culture-dependent selection strategy and an rpoB sequence-based subtyping method were applied to bacterial isolates obtained from environmental samples collected on a New York State dairy farm. A total of 54 different rpoB allelic types putatively identified as Bacillus (75% of isolates), Paenibacillus (24%), and Sporosarcina spp. (1%) were identified among 93 isolates. Assembly of a broader data set, including 93 dairy farm isolates, 57 raw milk tank truck isolates, 138 dairy plant storage silo isolates, and 336 pasteurized milk isolates, identified a total of 154 rpoB allelic types, representing an extensive diversity of Bacillus and Paenibacillus spp. Our molecular subtype data clearly showed that certain endospore-forming bacterial subtypes are present in the dairy farm environment as well as in the processing plant. The potential for entry of these ubiquitous heat-resistant spoilage organisms into milk production and processing systems, from the dairy farm to the processing plant, represents a considerable challenge that will require a comprehensive farm-to-table approach to fluid milk quality.     

Characterization of Staphylococcus aureus isolates in milk and the milking environment from small-scale dairy farms of São Paulo,Brazil, using pulsed-field gel electrophoresis

This research aimed to evaluate the occurrence of Staphylococcus aureus isolates in milk and in the milking environment of 10 small-scale farms (<400 L/d) located in the regions of Franca and Ribeirão Preto, state of São Paulo, Brazil. Two-hundred twenty samples of milk were collected from individual cows, along with 120 samples from bulk tank milk, 389 samples from milking equipment and utensils (teat cups, buckets, and sieves), and 120 samples from milkers’ hands. Fifty-six Staph. aureus strains were isolated from 849 analyzed samples (6.6%): 12 (5.5%) from milk samples of individual cows, 26 (21.7%) from samples of bulk tank milk, 14 (3.6%) from samples collected from equipment and utensils, and 4 (3.3%) from samples from milkers’ hands. Pulsed-field gel electrophoresis typing of the 56 Staph. aureus isolates by SmaI restriction enzyme resulted in 31 profiles (pulsotypes) arranged in 12 major clusters. Results of this study indicate a low incidence, but wide distribution of Staph. aureus strains isolated from raw milk collected from individual cows and surfaces of milkers’ hands and milking equipment in the small-scale dairy farms evaluated. However, the high percentage of bulk milk samples found with Staph. aureus is of public health concern because raw, unprocessed milk is regularly consumed by the Brazilian population.     

Antimicrobial resistance and heat sensitivity of oxacillin-resistant, mecA-positive Staphylococcus spp. from unpasteurized milk

Eight Staphylococcus spp. carrying the mecA gene were isolated from oxacillin enrichments of 70 unpasteurized milk samples. The isolates were identified as five Staphylococcus epidermidis, two Staphylococcus lentus, and one Staphylococcus haemolyticus. No mecA-positive Staphylococcus aureus were isolated. All isolates carried genes for other antibiotic resistances in addition to mecA. The results establish that mecA-carrying coagulase-negative Staphylococcus spp. in unpasteurized milk have the potential to be a reservoir of other genes encoding antimicrobial resistance. Two S. epidermidis isolates with qacA/B genes were resistant to benzalkonium chloride. Decimal reduction times (D-values) for the mecA-Staphylococcus spp. at 56 degrees C in whole milk ranged from 1.46 to 2.82 min. D-values at 56 degrees C for nine S. aureus milk isolates ranged from 10.8 to 20.1 min. Heat treatments intended to control S. aureus may be an effective means to protect consumers of milk and dairy products. Contact with or consumption of milk and dairy products that have not been heat treated may lead to the spread of antimicrobial resistance genes in Staphylococcus spp. to animals and humans.     

Prevalence of Salmonella enterica, Listeria monocytogenes, and Escherichia coli virulence factors in bulk tank milk and in-line filters from U.S. dairies

The zoonotic bacteria Salmonella enterica, Listeria monocytogenes, and Escherichia coli are known to infect dairy cows while not always causing clinical signs of disease. These pathogens are sometimes found in raw milk, and human disease outbreaks due to these organisms have been associated with the consumption of raw milk or raw milk products. Bulk tank milk (BTM) samples (536) and in-line milk filters (519) collected from dairy farms across the United States during the National Animal Health Monitoring System's Dairy 2007 study were analyzed by real-time PCR for the presence of S. enterica and pathogenic forms of E. coli and by culture techniques for the presence of L. monocytogenes. S. enterica was detected in samples from 28.1% of the dairy operations, primarily in milk filters. Salmonella was isolated from 36 of 75 PCR-positive BTM samples and 105 of 174 PCR-positive filter samples, and the isolates were serotyped. Cerro, Kentucky, Muenster, Anatum, and Newport were the most common serotypes. L. monocytogenes was isolated from 7.1% of the dairy operations, and the 1/2a complex was the most common serotype, followed by 1/2b and 4b (lineage 1). Shiga toxin genes were detected in enrichments from 15.2% of the BTM samples and from 51.0% of the filters by real-time PCR. In most cases, the cycle threshold values for the PCR indicated that toxigenic strains were not a major part of the enrichment populations. These data confirm those from earlier studies showing significant contamination of BTM by zoonotic bacterial pathogens and that the consumption of raw milk and raw milk products presents a health risk.     

Prevalence and survival of Campylobacter in Egyptian dairy products

Campylobacter is a leading foodborne pathogen that poses an interesting paradox of being frequently transmitted by foodstuffs, yet showing sensitivity to food preservation procedures. In the present study, 227 samples of milk and dairy products were randomly collected and examined for the presence of Campylobacter spp. Samples were collected from the Egyptian area Abou-Homos, where the pathogen had been previously shown to be a major causative agent of intestinal disease. Potential Campylobacter isolates were speciated using a multiplex PCR assay targeting the housekeeping gene lpxA. Only raw milk and fresh Domiati cheese samples were found to contain Campylobacter jejuni at low incidence rates. Using a selected C. jejuni isolate recovered in this study, it was shown that the pathogen maintained its viability in Domiati cheese more than in yoghurt, yet it survived in both products more than expected. This suggests that this foodborne strain of C. jejuni may develop adaptive strategies that aid survival under food preservation conditions, which contradicts with what is known about this pathogen as a stress-sensitive organism.     

The temporal,PFGE and resistance pattern associations suggest that poultry products are only a minor source of human infections in Western Finland

In order to compare human and retail poultry meat thermophilic Campylobacter isolates originating in a regional area in Western Finland, minimum inhibitory concentration (MICs) for six antimicrobials (96 isolates) and pulsed-field gel electrophoresis (PFGE) (102 isolates) were analysed. Campylobacter spp. were detected in 10.5% out of 305 fresh poultry products studied; 29 (90.5%) isolates were identified as Campylobacter jejuni. Among the 70 human isolates, 66 (94.3%) isolates were identified as C. jejuni. Only one C. jejuni domestic poultry isolate showed resistance (ampicillin), whereas domestic human C. jejuni isolates were more commonly resistant to ciprofloxacin, nalidixic acid, ampicillin and tetracycline. The resistance in foreign human isolates was significantly more common than among domestic isolates. PFGE analysis with KpnI restriction enzyme resulted in 59 different PFGE types among the poultry and human isolates. Three types were detected first in poultry meat and thereafter during the following month in domestic human samples, whereas the other conjoint types were detected only after many months. This study suggests that poultry products play only a minor role in human campylobacteriosis in the study area and that the resistance found in domestic human isolates is not likely related to retail poultry meat products.     

Short communication: Methicillin-resistant Staphylococcus aureus detection in US bulk tank milk

Staphylococcus aureus is a major cause of mastitis in dairy cattle. This study estimated the herd prevalence of methicillin-resistant Staph. aureus (MRSA) among US dairy herds by testing bulk tank milk (BTM) samples using genotypic and phenotypic methods. A nationally representative sample of 542 operations had BTM cultured for Staph. aureus, and 218 BTM samples were positive upon initial culture. After 4 wk to 4 mo of frozen storage, 87% of 218 samples (n = 190) were still culture positive for Staph. aureus on blood agar, but none were positive for MRSA on the selective indicator medium CHROMagar MRSA. A duplex PCR was used to detect the Staph. aureus-specific nuc gene and the methicillin resistance gene, mecA, in mixed staphylococcal isolates from the 190 BTM samples that were positive for Staph. aureus after storage. Seven samples tested positive for nuc and mecA, and 2 samples tested positive for mecA only. MecA-positive Staphylococcus spp., but not MRSA, were subsequently isolated from 5 samples, whereas neither mecA-positive Staphylococcus spp. nor MRSA was isolated from the remaining 4 samples. Presence of methicillin-resistant, coagulase-negative Staphylococcus spp. may complicate the detection of MRSA by means of PCR on BTM. Bulk tank milk in the United States is not a common source of MRSA.     

Occurrence and survival of Campylobacter spp. in dairy matrices investigated by viability qPCR

The aim of this study was to investigate the survival of Campylobacter jejuni in dairy matrices by viability qPCR. Survival in UHT milk was examined first and the cheese outbreak strain, C. jejuni 11 218 showed enhanced tendency to become viable but nonculturable (VBNC). It survived for long at different pH values, in raw and fermented milk and cheese. Viability qPCR was also used to examine the occurrence of viable Campylobacter spp. in raw milk, detecting the bacteria in 40.9% of 25 mL samples negative on culture. Results showed that viability qPCR is valuable in estimating Campylobacter risk in dairy products.     

Detection of multiple Mycoplasma species in bulk tank milk samples using real-time PCR and conventional culture and comparison of test sensitivities

The objective of this study was to further validate a SYBR PCR protocol for Mycoplasma spp. by comparing it with standard microbial culture in the detection of Mycoplasma spp. in bulk tank milk samples. Additionally, we identified Mycoplasma spp. present by analysis of PCR-generated amplicons [dissociation (melt) temperature (T_m), length, and DNA sequence]. The research presented herein tests the hypothesis that the SYBR PCR protocol is as sensitive as conventional culture for the detection of Mycoplasma spp. in bulk tank milk samples. Mycoplasmas cause several important disease syndromes in cattle, including mastitis in dairy cows. The standard diagnostic method at the herd level has been microbial isolation of mycoplasmas on 1 of several specialized media and speciation through biochemical or immunological techniques; repeated sampling schemes are recommended. The development of a real-time SYBR PCR protocol offers advantages in decrease of time to detection, cost, and complexity. The T_m of the double-stranded DNA generated from the PCR reaction was used to detect the presence of and tentatively identify the species of mycoplasmas other than Mycoplasma bovis. In the SYBR PCR protocol, the presence of multiple species of mycoplasmas is indicated by an atypical dissociation curve. Gel electrophoresis and sequencing of the amplicons was used to confirm the mycoplasma species present when a non-M. bovis organism was detected (T_m not equal to M. bovis) and used to identify all the mycoplasma species present for the samples with atypical dissociation curves. Mycoplasma bovis was identified in 83% of SYBR PCR mycoplasma-positive bulk tank samples. Another mycoplasma was identified either alone or in addition to M. bovis in 25% of SYBR PCR mycoplasma-positive bulk tank milk samples. Four species of mycoplasma other than M. bovis (Mycoplasma alkalescens, Mycoplasma arginini, Mycoplasma bovigenitalium, and Mycoplasma gateae) were identified in bulk tank milk samples tested with this method. Five farms had 2 mycoplasma species occurring at different times in their bulk tanks. Two mycoplasma species were identified in the same bulk tank sample in 7 instances on 2 farms. The finding of multiple Mycoplasma spp. coexisting on a farm and even in the same bulk tank milk sample indicates that the clinical significance of multiple mycoplasma species in the pathology of intramammary infections should be investigated further. In comparison with conventional culture, the SYBR PCR protocol was slightly (but not statistically significantly) more sensitive in the detection of mycoplasmas in bulk tank milk.     

Canadian National Dairy Study: Herd-level milk quality

The objective of this study was to estimate Canadian national milk quality parameters and estimate the bulk tank milk (BTM) prevalence of 4 mastitis pathogens, Staphylococcus aureus, Streptococcus agalactiae, Mycoplasma bovis, and Prototheca spp., on Canadian dairy farms. A questionnaire was sent to all Canadian dairy producers. Of the 1,062 producers who completed the questionnaire, 374 producers from across the country were visited and milking hygiene was assessed. Farm-level milk quality data for all Canadian dairy producers was collected from the provincial marketing boards and combined with the questionnaire and farm visit data. In addition, a BTM sample was collected either during the farm visit or by the marketing board in November of 2015 and was tested for 4 major mastitis pathogens using the PathoProof Mastitis Major 4 PCR Assay (Thermo Fisher Scientific Inc., Waltham, MA). Apparent herd-level prevalence was 46% for S. aureus, 6% for Prototheca spp., 0% for M. bovis, and 0% for Strep. agalactiae. Due to the low prevalence of M. bovis and Strep. agalactiae and a lack of significant factors associated with farms testing positive for Prototheca spp., an association analysis could only be carried out for Staph. aureus-positive farms. Factors associated with Staph. aureus-positive farms were not fore-stripping cows before milking (odds ratio = 1.87), milking with a pipeline system (odds ratio = 2.21), and stall bases made of a rubberized surface (mats and mattresses), whereas protective factors were using blanket dry cow therapy (odds ratio = 0.49) and applying a tag or visible mark on cows known to have chronic mastitis infections (odds ratio = 0.45). The Canadian national production-weighted geometric mean somatic cell count was determined to be 208,000 cells/mL. This is the first national dairy study conducted in Canada. Participating farms had higher milk yield; were more likely to have a loose housing system, parlor, or automated milking system; and had lower weighted mean BTM somatic cell count than the national level. Sampling larger farms with better milk quality means the apparent prevalence of the 4 mastitis pathogens likely underestimates the true levels.     

Relationships between milk culture results and milk yield in Norwegian dairy cattle

Associations between test-day milk yield and positive milk cultures for Staphylococcus aureus, Streptococcus spp., and other mastitis pathogens or a negative milk culture for mastitis pathogens were assessed in quarter milk samples from randomly sampled cows selected without regard to current or previous udder health status. Staphylococcus aureus was dichotomized according to sparse (≤1,500 cfu/mL of milk) or rich (>1,500 cfu/mL of milk) growth of the bacteria. Quarter milk samples were obtained on 1 to 4 occasions from 2,740 cows in 354 Norwegian dairy herds, resulting in a total of 3,430 samplings. Measures of test-day milk yield were obtained monthly and related to 3,547 microbiological diagnoses at the cow level. Mixed model linear regression models incorporating an autoregressive covariance structure accounting for repeated test-day milk yields within cow and random effects at the herd and sample level were used to quantify the effect of positive milk cultures on test-day milk yields. Identical models were run separately for first-parity, second-parity, and third-parity or older cows. Fixed effects were days in milk, the natural logarithm of days in milk, sparse and rich growth of Staph. aureus (1/0), Streptococcus spp. (1/0), other mastitis pathogens (1/0), calving season, time of test-day milk yields relative to time of microbiological diagnosis (test day relative to time of diagnosis), and the interaction terms between microbiological diagnosis and test day relative to time of diagnosis. The models were run with the logarithmically transformed composite milk somatic cell count excluded and included. Rich growth of Staph. aureus was associated with decreased production levels in first-parity cows. An interaction between rich growth of Staph. aureus and test day relative to time of diagnosis also predicted a decline in milk production in third-parity or older cows. Interaction between sparse growth of Staph. aureus and test day relative to time of diagnosis predicted declining test-day milk yields in first-parity cows. Sparse growth of Staph. aureus was associated with high milk yields in third-parity or older cows after including the logarithmically transformed composite milk somatic cell count in the model, which illustrates that lower production levels are related to elevated somatic cell counts in high-producing cows. The same association with test-day milk yield was found among Streptococcus spp.-positive pluriparous cows.     

Biofilm in milking equipment on a dairy farm as a potential source of bulk tank milk contamination with Listeria monocytogenes

The objective of this study was to assess the presence of a Listeria monocytogenes-containing biofilm in milking equipment as a potential source of bulk tank milk contamination on a dairy farm where milk contamination had been previously documented. Samples were collected from milking equipment and milking parlor premises on 4 occasions and analyzed for the presence of L. monocytogenes. Pulsed-field gel electrophoresis (PFGE) typing was conducted on L. monocytogenes isolates from the milking equipment, parlor and storage room floors, bulk tank milk, and in-line milk filters. Pieces from milk meters and rubber liners were obtained to visually assess the presence of a biofilm using scanning electron microscopy. A total of 6 (15%), 4 (25%), and 1 (6%) samples were culture-positive for L. monocytogenes in the first, second, and third sample collection, respectively. Two samples were L. monocytogenes hly PCR-positive but were culture-negative in the fourth sample collection. Combined AscI and ApaI restriction analysis yielded 6 PFGE types for 15 L. monocytogenes isolates obtained from milking equipment, parlor, bulk tank milk, and milk filters. A predominant and persistent PFGE type (PFGE type T) was observed among these L. monocytogenes isolates (9/15 isolates). Scanning electron microscopy of samples from the bottom cover of 2 milk meters showed the presence of individual and clusters of bacteria, mainly associated with surface scratches. The presence of a bacterial biofilm was observed on the bottom covers of the 2 milk meters. Prevention of the establishment of biofilms in milking equipment is a crucial step in fulfilling the requirement of safe, high-quality milk.     

Short communication: Parapoxvirus and Orthopoxvirus coinfection in milk of naturally infected cows

Several studies have shown the occurrence of poxvirus infections associated with exanthematic lesions in cattle from many Brazilian states. Coinfection between viruses belonging to 2 genera, Orthopoxvirus (OPXV) and Parapoxvirus (PPV), was already identified from the lesions of affected cows and humans. The DNA and infectious viral particles of Vaccinia virus, an OPXV, have been detected in milk of naturally and experimentally infected cows. However, to date no reports have described the detection of Pseudocowpox virus, a PPV, in milk. Thus, we investigated the presence of PPV and OPXV in milk samples obtained from dairy cows from a Brazilian region with exanthematic disease outbreaks. From 2011 to 2014, 6 dairy farms with exanthematic disease outbreaks involving dairy cows, calves, and humans were visited. Twelve crusts of cows' teat lesions and 60 milk samples were collected. The crusts and milk samples were analyzed by PCR to detect OPXV or PPV DNA. According to the analyzed crusts, we detected PPV infection in 4 of the 6 visited farms, from which we investigated the PPV contamination in milk. From the 40 milk samples tested, PPV DNA was detected in 12 samples. Of these milk samples, 8 were positive for both PPV and OPXV. This is the first report of PPV DNA detection in milk samples from affected cows, indicating that the virus may be present in milk and potentially contaminating dairy products associated or not with OPXV. In addition to the lesions caused by direct contact, the presence of 2 or more poxvirus species in milk showed that the effect of zoonotic exanthematic diseases on public health and animal husbandry is relevant and cannot be overlooked.     

Occurrence of Campylobacter spp., Salmonella spp. and shiga toxin-producing Escherichia coli in inline milk filters from Swedish dairy farms

This study investigated the occurrence of shiga toxin-producing Escherichia coli (STEC), thermotolerant Campylobacter spp. and Salmonella spp. in Swedish dairy milk. A total of 302 inline milk filters were analyzed. Salmonella was not isolated from any filters. Polymerase chain reaction screening detected thermotolerant Campylobacter in 30.5% of the milk filters analyzed and it was isolated from 12.6% of filters. The stx genes (stx ₁, stx ₂, or both) were screened from 71% of the filters and STEC was isolated from 14% of these. Of the STEC isolates, 21 contained the stx ₁ gene, 19 the stx ₂ gene, and five a combination of both stx ₁ and stx ₂ genes. Whole genome sequence typing on 34 of the 45 STEC showed that they belonged to 21 different serotypes, of which STEC O145:H28 was the most common (2%). STEC O157:H7 was only found from one (0.3%) of the filters. A combination of stx ₂ and eae genes was found from 0.7% of the total number of inline milk filters analyzed, while stx _2a was found in 24% of the whole genome-sequenced isolates. There was a significant positive correlations between number of animals per farm and presence of pathogens on milk filters.     

The main spoilage-related psychrotrophic bacteria in refrigerated raw milk

Refrigerated raw milk may contain psychrotrophic microorganisms that produce thermoresistant exoproteases and lipases, which may compromise the quality of processed fluid milk and dairy products during storage. The aim of this work was to quantify and identify the deteriorating psychrotrophic microbiota in Brazilian refrigerated raw milk using genetic diversity analysis. The mean psychrotrophic count was 1.1 × 10⁴ cfu/mL. Of the total isolates, 47.8 and 29.8% showed deteriorating activity at 35°C within 48 h and 7°C within 10 d, respectively. Among the proteolytic species, more isolated by this study were Lactococcus lactis (27.3%), Enterobacter kobei (14.8%), Serratia ureilytica (8%), Aerococcus urinaeequi (6.8%), and Bacillus licheniformis (6.8%). Observed among lipolytics were E. kobei (17.7%), L. lactis (15.6%), A. urinaeequi (12.5%), and Acinetobacter lwoffii (9.4%). The isolates S. ureilytica, E. kobei, Pseudomonas spp., and Yersinia enterocolitica potentially produced alkaline metalloprotease (aprX). Despite the low counts, a considerable portion of the psychrotrophic microbiota presented spoilage potential, which reaffirms the need for rigor in the control of contamination and the importance of rapid processing as factors that maintain the quality of milk and dairy products.     

A study on the prevalence of gram-negative bacteria in bulk tank milk

Bulk tank milk from 131 dairy herds in eastern South Dakota and western Minnesota were examined for coliforms and noncoliform bacteria. Coliforms were detected in 62.3% of bulk tank milk samples. Counts ranged from 0 to 4.7 log10 cfu/ml. The mean count was 3.4 log10 cfu/ml. Gram-negative noncoliform bacteria were observed in 76.3% of bulk tank milk. Counts ranged from 0 to 6.2 log10 cfu/ml. The mean count was 4.8 log10 cfu/ml. A total of 234 isolates from bulk tank milk were examined to species level; 205 isolates belonged to 28 species. Coliforms and gram-negative noncoliform bacteria accounted for 32.9 and 67.1% of the total isolates, respectively. Organisms such as Agrobacterium radiobacter, Bordetella spp., Comamonas testosteroni, Listonella damsela, Ochrobactrum anthropi, and Oligella urethralis were isolated from bulk tank milk in this study. These organisms have not been reported previously in bulk tank milk. A total of 116 isolates of Pseudomonas spp. were isolated from raw milk; 98 isolates belonged to nine Pseudomonas spp., and the remaining 18 isolates could not be identified to their species level. Pseudomonas was the most predominant genus. Pseudomonas fluorescens was the most predominant species isolated from bulk tank milk and accounted for 29.9% of all isolates examined. The results of the study suggest that counts of coliforms and noncoliform bacteria in bulk tank milk vary considerably. The isolates represent a wide variety of Gram-negative bacterial species. Examination of bulk tank milk for coliforms and noncoliform bacteria could provide an indication of current and potential problems associated with bacterial counts and milk quality.