Comparison of Coxiella burnetii shedding in milk of dairy bovine, caprine, and ovine herds

The shedding of Coxiella burnetii in bovine, caprine, and ovine milk was measured using PCR, in 3 herds for each species, the bulk tank milk samples of which were positive at the time of their selection. Milk samples of 95 cows, 120 goats, and 90 ewes were sampled over 16 wk, as was the bulk tank milk. The shedding of C. burnetii in vaginal mucus and feces was checked at the beginning of the experiment and 2 mo later. The clinical signs in the selected herds as well as the duration and the shedding routes differed among the 3 species. The cows were asymptomatic and shed C. burnetii almost exclusively in milk. In one of the caprine herds, abortions due to C. burnetii were reported. The goats excreted the bacteria mainly in milk. In contrast, the ewes, which came from flocks with abortions due to Q fever (C. burnetii infection), shed the bacteria mostly in feces and in vaginal mucus. This could explain why human outbreaks of Q fever are more often related to ovine flocks than to bovine herds. These excretions did not seem more frequent when the samples were taken close to parturition. The samples were taken from 0 to 421 d after parturition in bovine herds and from 5 to 119 d and 11 to 238 d after parturition in the caprine and ovine herds, respectively. The shedding in milk was sometimes intermittent, and several animals shed the bacteria but were negative by ELISA: 80% of the ewes were seronegative, underscoring the lack of sensitivity of the ELISA tests available for veterinary diagnosis. The detection of antibodies in milk seems more sensitive than it is in serum.     

Nachweis des Q-Fieber-Erregers Coxiella burnetii in Milch

The etiological agent of Q fever, Coxiella burnetii, has its relevance for food especially concerning to raw milk and raw milk products. Today PCR is a quick and sensitive method for the detection of the Q fever agent and may be a useful tool for the protection of consumers.     

Nachweis des Q-Fieber-Erregers Coxiella burnetii in Milch

The etiological agent of Q fever, Coxiella burnetii, has its relevance for food especially concerning to raw milk and raw milk products. Today PCR is a quick and sensitive method for the detection of the Q fever agent and may be a useful tool for the protection of consumers. Eingegangen: 18. Januar 2007     

Short communication: Investigation of Coxiella burnetii occurrence in dairy sheep flocks by bulk-tank milk analysis and antibody level determination

To estimate the prevalence of Coxiella burnetii in the dairy sheep population from the Basque Country (northern Spain), a study was carried out combining molecular and serological techniques. First, bulk-tank milk samples from 154 flocks belonging to the Latxa Breed Farmers Association were analyzed by PCR, with 22% of flocks testing positive for C. burnetii. Then, a selection of 34 flocks (7 PCR positive and 17 negative) was investigated for the presence of serum antibodies by ELISA test on 1,011 ewes (approximately 30 ewes per flock). A total of 8.9% of the animals were seropositive, 67.6% of the flocks had at least one seropositive animal, but only in 14.7% of them was seroprevalence greater than 25%. Older ewes showed a significantly greater prevalence (17.5%) compared with yearlings (7.5%) or replacement lambs (1.5%). A marginally significant association was found between seroprevalence and PCR detection of C. burnetii in bulk-tank milk. The widespread distribution of C. burnetii in the region advocates for the implementation of Q fever control strategies and highlights the potential risk of sheep as a reservoir and infection source for other domestic and wildlife species and the human population.     

Bayesian estimation of sensitivity and specificity of a PCR method to detect Coxiella burnetii in milk and vaginal secretions in sheep and goat samples

Coxiella burnetii is a gram-negative and polymorphic rod bacterium that causes Q fever, a common zoonotic disease distributed worldwide. Widespread occurrences of the disease outbreaks indicate the importance of coordinated animal and human health efforts to control these outbreaks. Different tests are available to determine the C. burnetii infection status of a flock, but false negative responses may occur, as infectious animals can shed bacteria in milk intermittently, especially during an asymptomatic infection. In this study, a Bayesian latent class model was implemented to estimate the sensitivity (Se) and specificity (Sp) of a PCR method for the detection of C. burnetii in milk samples (PCR_M) and vaginal swabs (PCR_V) from Iranian sheep and goats. A nested PCR assay was conducted to detect infected animals among 170 milk samples and 170 vaginal swabs from goat flocks and 170 milk samples and 170 vaginal swabs from sheep flocks. We implemented a Bayesian latent class model to estimate the Se and Sp of a PCR method for the detection of C. burnetii in milk samples and vaginal swabs from sheep and goats. Estimations were based on the cross-classified results of PCR_M and PCR_V from the sheep and goat subpopulations. Positivity was 17.6 and 33.5%, respectively, for PCR_M and PCR_V samples among sheep. In goats, the apparent prevalence was 32.9 and 56.4% in PCR_M and PCR_V samples testing positive, respectively. This indicated the lower sensitivity of PCR_M. The Se of PCR_V was significantly higher than Se of PCR_M, which corresponded to a higher rate of vaginal-positive, milk-negative PCR samples. In contrast, Sp of PCR_V was lower than Sp of PCR_M, representing the higher false-positive rate of vaginal swabs. The PCR_V outperformed PCR_M in terms of identifying latently infected sheep and goats; however, neither method could identify all latently infected sheep and goats, thus the combination is recommended to maximize our ability to identify infected animals. The true prevalence of C. burnetii infection was higher in Iranian goats than sheep.     

Potential public health risk due to consumption of contaminated bovine milk with aflatoxin M1 and Coxiella burnetii in the West of Iran

This study aimed to evaluate the probable public health hazard imposed by bovine milk in relation to aflatoxin M1 (AFM1) and Coxiella burnetii in the West of Iran. The assessment of AFM1, using an ELISA test, indicated 62.22% and 21.11% of 45 samples of raw and pasteurised milk were above the permissible content of Codex and Iranian standard levels, respectively. Further, anti‐C. burnetii antibody was detected in 63.04% of bulk tank milk samples, with six samples harbouring the bacterial genome. High AFM1 contamination levels and an endemic pattern of Q fever warrant continuous surveillance programmes in the studied region.     

Prevalence,antimicrobial resistance,and molecular characterization of Campylobacter spp. in bulk tank milk and milk filters from US dairies

Campylobacter spp. are frequently isolated from dairy cows as commensal organisms. Sporadic Campylobacter infections in humans in the United States are generally attributed to poultry, but outbreaks are also commonly associated with dairy products, particularly unpasteurized or raw milk. Bulk tank milk samples and milk filters from US dairy operations were collected during the National Animal Health Monitoring System Dairy 2014 study and analyzed using real-time PCR and traditional culture techniques for the presence of thermophilic Campylobacter species. The weighted prevalence of operations from which we detected Campylobacter spp. in either bulk tank milk or milk filters was 24.9%. We detected Campylobacter spp. in a higher percentage of operations with 100–499 cows (42.8%) and 500 or more cows (47.5%) than in operations with 30–99 cows (6.5%). Campylobacter spp. were also more frequently detected in operations in the west than the east (45.9 and 22.6%, respectively). We isolated Campylobacter spp. from approximately half of PCR-positive samples, representing 12.5% (weighted prevalence) of operations. The majority (91.8%) of isolates were C. jejuni, but C. lari and C. coli were also isolated. We detected resistance to tetracycline in 68.4% of C. jejuni isolates, and resistance to ciprofloxacin and nalidixic acid in 13.2% of C. jejuni isolates. Based on pulsed-field gel electrophoresis, we found that dairy-associated C. jejuni were genotypically diverse, although clonal strains were isolated from different geographic regions. These results suggest that bulk tank milk can be contaminated with pathogenic Campylobacter spp., and that the consumption of unpasteurized or raw milk presents a potential human health risk.     

Changes in the dynamics of Coxiella burnetii infection in dairy cattle: An approach to match field data with the epidemiological cycle of C. burnetii in endemic herds

This study aimed to evaluate changes in the epidemiological status of Coxiella burnetii in dairy cattle herds to better understand the epidemiology of the infection and to predict its evolution. Bulk-tank milk (BTM) and serum samples were collected from 94 dairy cattle herds and analyzed by ELISA (BTM and sera) and PCR (BTM) in study 1 (S₁). Two years later (study 2; S₂), the same farms were visited with a similar sampling approach. To estimate seroconversion during this period, blood samples were collected from the maximum possible number of animals surveyed in S₁. Environmental samples were collected in S₂ to identify active shedding. Farms were allocated into 3 different categories in each study according to PCR and ELISA results: category A, with BTM ELISA and PCR positive herds and at least 1 seropositive animal; category B, with BTM ELISA or PCR positive herds or individual sera positive; and category C, with all negative results among herds. Changes in herd category between S₁ and S₂ were grouped in 9 classes. Two statistical models, one to search for drives of within-herd changes in C. burnetii infection status and another to look for variables modulating individual changes in C. burnetii antibody level, were built. Several herds in category A in S₁ remained in that category 2 yr later, indicating that C. burnetii can remain within a herd for a long time. Most of the herds with seroconversion and detection of the bacterium in the environment belonged to category A, suggesting active and recent infections. Changes in the epidemiological status of herds were driven by local densities of domestic ruminants, showing the implication of neighbor reservoirs; whereas individual changes in antibody levels were modulated by variation in the epidemiological status of herds. Observed changes in epidemiological status allowed depiction of the hypothesized life cycle of C. burnetii within dairy cattle herds, which should be tested by future long-term series studies on C. burnetii infection to help fitting control measures (e.g., vaccination) to within-herd C. burnetii status.     

Mycobacterium bovis: the importance of milk and dairy products as a cause of human tuberculosis in the UK. A review of taxonomy and culture methods,with particular reference to artisanal cheeses

Mycobacterium bovis is the cause of tuberculosis (TB) in cattle and other ruminants. It can also cause tuberculosis in humans, and consumption of unpasteurized milk or products made from infected and untreated animals is thought to be the primary vehicles of transmission. Although a control programme for cattle exists in the UK, the disease is becoming more prevalent in the cattle population. The increasing interest in artisan cheeses made from unpasteurized milk and recent cases of human TB in which cheese was the known cause has highlighted the need for empirical data on the survival kinetics of M. bovis during the manufacture and ripening of such cheeses.     

Toxoplasma gondii infection and food consumption: A systematic review and meta-analysis of case-controlled studies

ABSTRACT

Background: Toxoplasmosis is a zoonotic disease causing severe symptoms in pregnant women and immunocompromised individuals. On average, worldwide, around 30% of people are seropositive. The oral transmission route is of great significance and food, particularly meat, is an important transmission vehicle for T. gondii. However, the role of different food matrices is debated. Objectives: The aim of this review was to assess the risk of humans developing acute T. gondii infection via the foodborne route. Study eligibility criteria: Case-control studies including acute cases of T. gondii infection were included after literature searches, without time limits, in several databases. All studies estimating the risk of acquiring T. gondii infection after consumption of specific food categories were included. Results: Three risk factors proved to be significantly associated with acute T. gondii infection in humans: consumption of raw/undercooked meat, Odds Ratio (OR) 3.44 (1.29–9.16), consumption of raw/undercooked beef, OR 2.22 (1.57–3.12), and consumption of raw/undercooked sheep meat, OR 3.85 (1.85–8.00). Consumption of raw/undercooked pork, raw eggs, and unpasteurized milk proved to be non-significant risk factors. Limitations: Limitations in the present review and meta-analysis are due to the low number of case-control studies available for analysis and the lack of a search strategy targeting gray literature. Conclusion: Consumption of raw/undercooked beef and sheep meat are important risk factors for T. gondii infection. Their consumption should be avoided in order to prevent toxoplasmosis, particularly by those in at-risk categories, including pregnant women. The review protocol is registered in PROSPERO database (CRD42016043295).     

A review of the microbiological hazards of dairy products made from raw milk

This review concentrates on information concerning microbiological hazards possibly present in raw milk dairy products, in particular cheese, butter, cream and buttermilk. The main microbiological hazards of raw milk cheeses (especially soft and fresh cheeses) are linked to Listeria monocytogenes, verocytotoxin-producing Escherichia coli (VTEC), Staphylococcus aureus, Salmonella and Campylobacter. L. monocytogenes, VTEC and S. aureus have been identified as microbiological hazards in raw milk butter and cream albeit to a lesser extent because of a reduced growth potential compared with cheese. In endemic areas, raw milk dairy products may also be contaminated with Brucella spp., Mycobacterium bovis and the tick-borne encephalitis virus (TBEV). Potential risks due to Coxiella burnetii and Mycobacterium avium subsp. paratuberculosis (MAP) are discussed. Pasteurisation ensures inactivation of vegetative pathogenic microorganisms, which increases the safety of products made thereof compared with dairy products made from raw milk. Several control measures from farm to fork are discussed.     

Survey of raw milk cheeses for microbiological quality and prevalence of foodborne pathogens

Cheese may be manufactured in the United States using raw milk, provided the cheese is aged for at least 60 days at temperatures not less than 35 °F (1.7 °C). There is now increased concern among regulators regarding the safety of raw milk cheese due to the potential ability of foodborne pathogens to survive the manufacturing and aging processes. In this study, 41 raw milk cheeses were obtained from retail specialty shops, farmers’ markets, and on-line sources. The cheeses were then analyzed for the presence of Listeria monocytogenes, Salmonella, Escherichia coli O157:H7, Staphylococcus aureus, and Campylobacter. Aerobic plate counts (APC), coliform and yeast/mold counts were also performed. The results revealed that none of the enteric pathogens were detected in any of the samples tested. Five samples contained coliforms; two of those contained E. coli at less than 10² cfu/g. Three other cheese samples contained S. aureus. The APC and yeast-mold counts were within expected ranges. Based on the results obtained from these 41 raw milk cheeses, the 60-day aging rule for unpasteurized milk cheeses appears adequate for producing microbiologically safe products.     

Occurrence of Burkholderia cepacia in foods and waters: clinical implications for patients with cystic fibrosis.

Two hundred forty-eight retail "ready-to-eat" foodstuffs in eight food categories and 134 waters categorized into nine types were analyzed for the presence of the Burkholderia cepacia complex of organisms. Of these, 14 of 26 (53.8%) samples of raw unpasteurized bovine milk were positive for this organism. Consumption of raw unpasteurized milk may therefore act as a potential source of infection with this organism, which is of particular concern for patients with cystic fibrosis, where colonization and infection with this organism can lead to a fatal necrotizing pneumonia and premature death. In addition to the associated risk of infection from fecal pathogens, patients with cystic fibrosis should therefore avoid the consumption of raw unpasteurized milk to minimize the risk of becoming infected with this organism.     

Real-time PCR-based detection of Coxiella burnetii in cheeses

In this study, the presence of Coxiella burnetii (Cb) in cheese produced in Southern Italy from milk of cow, buffalo, and small ruminants has been evaluated. Two tests based on real-time PCR assay targeting CB IS1111 element were performed. First an assay based on the use of Taq-Man probe was performed to screen the samples. The positive samples were confirmed using both a SyBr Green test and the evaluation of the melting temperature of the amplicons. In addition, all cheese samples were also tested to determine the milk species utilized (Regulation EC 273/2008). The samples of cheese produced with a milk mix from different species were not included into the study. A total of 169 cheese samples were tested, and the obtained results showed an overall prevalence of Cb of 21.3 % with variation between species. A positivity rate of 39 % was observed in cow’s cheese while Cb DNA was detected in 26 % of cheese samples made from small ruminants’ milk. However, the bacterium was found only in 6.9 % of buffalo’s cheese samples. A direct association between prevalence and milk used for the production was highlighted (χ ²  = 19.12). The statistical analysis of the prevalence in the samples from cattle and small ruminants compared with those from buffalo shows an OR of 8.4 and 4.9, respectively.     

Short communication: Detection of Mycobacterium avium subspecies paratuberculosis by polymerase chain reaction in bovine milk in Brazil

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of paratuberculosis, or Johne's disease, a chronic granulomatous enteritis that affects all ruminants worldwide. Since the isolation of MAP from intestinal tissue of human patients bearing Crohn's disease, there has been a debate on the possibility of this agent playing a role in the etiology of Crohn's disease. Milk could be the potential vehicle for transmission to humans. Mycobacterium avium ssp. paratuberculosis has already been detected in milk samples worldwide. In Brazil, detection of MAP is uncommon; however, it has already been detected by bacterial isolation and serological test. The aim of this study was to investigate the presence of MAP, by PCR, in raw milk samples in the region of Viçosa, Minas Gerais State, Brazil. Of 222 milk samples evaluated, 8 (3.6%) quarter milk samples amplified fragments of similar size to that expected of 626 bp. These fragments were cloned and sequenced. The genetic analysis revealed a 99% identity match between the sequences obtained in this study and the insertion sequence IS900 deposited in the GenBank. In the analyzed milk samples, MAP DNA was detected, confirming its presence in dairy cattle in the region of Viçosa. This is the first report of MAP presence in raw milk samples in Brazil.     

Dynamics of relationship between the presence of Coxiella burnetii DNA, antibodies, and intrinsic variables in cow milk and bulk tank milk from Danish dairy cattle

Milk samples of 12 Danish dairy herds were collected 3 times during an 11-mo period and tested for Coxiella burnetii DNA by real-time PCR, detecting the IS1111 element, and for the presence of antibodies against the bacterium by ELISA. On average, 25% of 1,514 samples were seropositive and 32% were positive for C. burnetii DNA. Among the 485 DNA-positive samples, quantification cycle values ranging from 15.8 to 37.8 were found. Test sensitivity did not increase after DNA extraction from the cream fraction compared with full milk. The relationship between antibody levels and bacterial shedding was investigated among 166 cows from 9 herds. The prevalence levels of C. burnetii DNA and antibodies in the herds were found to be rather stable for 6 of the herds. The test results were highly influenced by results obtained 3 to 7 mo earlier. A significant association between the antibody titer and the DNA shedding level at the same and the preceding visit was found. In addition, a significant association between the antibody titer and the antibody titers 3 to 11 mo earlier was found. A multivariable analysis identified a significant increase in C. burnetii DNA shedding with increasing parity and increasing protein concentration in milk. The antibody levels in bulk tank milk and prevalence levels of C. burnetii DNA and antibodies in individual cow milk samples were correlated. A significant correlation was also found between the quantification cycle values of the cow samples (weighted according to milk yield) and the C. burnetii concentration in bulk tank milk.     

Short communication: Recovery of viable Mycobacterium avium subspecies paratuberculosis from retail pasteurized whole milk in Brazil

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiological agent of paratuberculosis, a chronic granulomatous enteritis that affects all ruminants worldwide. Some researchers have indicated a possible role of MAP in Crohn's disease. Despite extensive research and large and important advances in the past few decades, the etiology of Crohn's disease remains indefinite. The most probable transmission route of MAP from animals to humans is milk and dairy products. Mycobacterium avium ssp. paratuberculosis has already been detected in milk samples worldwide, and some studies have reported that MAP is resistant to pasteurization. In Brazil, MAP has been reported in raw milk samples; however, Brazilian retail pasteurized milk has not yet been tested for viable MAP. The aim of this study was to investigate MAP in pasteurized milk in the region of Viçosa (Minas Gerais, Brazil). Thirty-seven samples were collected and processed for culture of MAP. One colony similar to MAP was observed and confirmed by IS900-nested PCR and sequencing. Analysis revealed 97 to 99% identity with the MAP K-10 strain. This study is the first report of the presence of MAP in retail pasteurized whole milk in Brazil.     

Detection of nontuberculous mycobacteria from water buffalo raw milk in Brazil

Milk is an important nutritional source to man and water buffalo raw milk is used to produce mozzarella cheese. Products from unpasteurized milk have been associated with certain infectious diseases and can carry pathogenic mycobacteria. Nontuberculous mycobacteria (NTM) are emerging pathogens causing opportunistic infections in humans and animals. The objectives of this study were to demonstrate the presence of mycobacteria in water buffaloes’ milk and to determine their role as possible sources of NTM infections. In this study, raw milk samples from dairy water buffaloes (Bubalus bubalis) (N = 23) were decontaminated by Petroff method and inoculated on to Löwenstein–Jensen and Stonebrink medium. After confirming positive colonies for acid fast bacilli (AFB) by Ziehl-Neelsen technique, the isolated mycobacteria were identified by PCR-Restriction Enzyme Analysis (PRA) and mycolic acids analysis by thin-layer chromatography (TLC). Mycobacterium simiae (2 isolates), Mycobacterium kansasii (2 isolates), Mycobacterium flavescens (2 isolates), Mycobacterium gordonae (3 isolates) and Mycobacterium lentiflavum (1 isolate) were identified by these techniques. The isolation of opportunistic pathogens such as M. kansasii, M. simiae and M. lentiflavum from raw milk represent a risk for the consumers of mozzarella cheese made by this milk.     

Microbial quality of raw horse milk

Consumption of horse milk has become popular in developed countries, especially among people suffering from bowel problems and skin diseases. Since the positive effect is supposedly not observed after pasteurisation, the product is mostly consumed as raw milk. Since the microbiological quality of this milk has not been systematically surveyed, in this study we examined the presence of spoilage- and pathogenic microorganisms in 123 samples of horse milk collected in The Netherlands and Belgium. Hygiene and faecal indicators were found in a wide range of numbers. Although Salmonella, Campylobacter and Listeria were not found, these pathogens showed no reduction in challenge tests with artificially contaminated horse milk stored for 1 week at 7 °C. Since faecal indicators were present and able to grow at 7 °C, combined with the fact that pathogens may easily end up and survive in the milk, it is not advised to consume raw horse milk.