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.     

Association between Mycobacterium avium subspecies paratuberculosis infection and milk production in two California dairies

The association between Mycobacterium avium ssp. paratuberculosis (MAP) and milk production was estimated on 2 California dairies using longitudinal data from 5,926 cows. Both study herds had moderate MAP seroprevalence, housed cows in freestalls, and had Johne's disease control programs. Cow MAP status was determined using both serum ELISA and fecal culture results from cows tested at dry-off and from whole-herd tests. Potential confounders were evaluated based on a causal diagram. Mixed models with 2 functions (splines) for days in milk (DIM) representing milk production pre- and postpeak used in similar studies were further modified to use each cow's observed DIM at peak and lactation length. Cows that were seropositive produced 2.5 kg less 4% fat-corrected milk (FCM) per day than their seronegative herdmates. In addition, cows that were fecal-culture positive by liquid culture and confirmed by PCR produced 2.2 kg less 4% FCM per day than their fecal-culture negative herdmates. The decrease in milk production in MAP test-positive compared with test-negative cows started in the second lactation. A switch in MAP status in either ELISA or fecal culture results from positive to negative had no significant association with milk production. Modified DIM functions that used the observed DIM at peak had better model fit than another function that assumed a fixed peak at 60 DIM. Cows that tested positive for MAP on serum ELISA or fecal culture produced less milk than cows that tested negative, and the association between MAP and milk production was not confounded by mastitis, elevated somatic cell counts, or uterine or metabolic cow conditions.     

Short communication: Application of an N-acetyl-l-cysteine-NaOH decontamination method for the recovery of viable Mycobacterium avium subspecies paratuberculosis from milk of naturally infected cows

Mycobacterium avium ssp. paratuberculosis (MAP) is shed into the milk of cattle affected by Johne’s disease and, therefore, is a route of transmission for infection in young stock in dairy herds. The objective of this study was to validate a decontamination and culture protocol for the recovery of MAP from individual bovine milk samples from known infected herds. Decontamination of milk samples (n = 17) with either 0.75% hexadecylpyridinium chloride for 5 h or N-acetyl-l-cysteine-1.5% sodium hydroxide (NALC-1.5% NaOH) for 15 min before culture in BACTEC 12B (Becton Dickinson, Franklin, NJ), para-JEM [Thermo Fisher Scientific (TREK Diagnostic Systems, Inc.), Cleveland, OH], and Herrold’s egg yolk (HEY; Becton Dickinson) media was compared. Treatment with NALC-NaOH resulted in a lower percentage (6%) of contaminated samples than did treatment with hexadecylpyridinium chloride (47%), regardless of culture medium. The decontamination protocol (NALC-1.5% NaOH) was then applied to milk samples (n = 144) collected from cows at 7 US dairies. Recovery of viable MAP from the milk samples was low, regardless of culture medium, with recovery from 2 samples cultured in BACTEC 12B medium, 1 sample cultured in para-JEM medium, and no viable MAP recovered on HEY medium. However, 32 cows were fecal culture positive and 13 milk samples were positive by direct PCR, suggesting that several cows were actively shedding MAP at the time of milk collection. Contamination rates were similar across media, with 39.6, 34.7, and 41.7% of samples contaminated after culture in BACTEC 12B, para-JEM, and HEY media, respectively. Herd-to-herd variation had a major effect on sample contamination, with the percentage of contaminated samples ranging from 4 to 83%. It was concluded that decontamination of milk with NALC-1.5% NaOH before culture in BACTEC 12B medium was the most efficacious method for the recovery of viable MAP from milk, although the ability to suppress the growth of contaminating microorganisms varied greatly between herds.     

Short communication: Effect of homogenization on heat inactivation of Mycobacterium avium subspecies paratuberculosis in milk

Mycobacterium avium ssp. paratuberculosis (MAP) can be present in cow milk and low numbers may survive high-temperature, short-time (HTST) pasteurization. Although HTST treatment leads to inactivation of at least 5 log₁₀ cycles, it might become necessary to enhance the efficacy of HTST by additional treatments such as homogenization if the debate about the role of MAP in Crohn’s disease of humans concludes that MAP is a zoonotic agent. This study aimed to determine whether disrupting the clumps of MAP in milk by homogenization during the heat treatment process would enhance the inactivation of MAP. We used HTST pasteurization in a continuous-flow pilot-plant pasteurizer and evaluated the effect of upstream, downstream, and in-hold homogenization on inactivation of MAP. Reduction of MAP at 72°C with a holding time of 28 s was between 3.7 and 6.9 log₁₀ cycles, with an overall mean of 5.5 log₁₀ cycles. None of the 3 homogenization modes applied showed a statistically significant additional effect on the inactivation of MAP during HTST treatment.     

Clinical disease and stage of lactation influence shedding of Mycobacterium avium subspecies paratuberculosis into milk and colostrum of naturally infected dairy cows

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD). One mode of transmission of MAP is through ingestion of contaminated milk and colostrum by susceptible calves. The objective of this study was to determine if the amount of MAP shed into the milk and colostrum of infected cows was affected by severity of infection as well as the number of days in milk (DIM). Milk was collected over the 305-d lactation period from naturally infected cows in the asymptomatic subclinical (n = 39) and symptomatic clinical (n = 29) stages of disease, as well as 8 noninfected control cows. All milk samples were assayed for MAP by culture on Herrold's egg yolk medium and either BACTEC 12B (Becton Dickinson, Franklin Lakes, NJ) or para-JEM (Thermo Fisher Scientific, Trek Diagnostic Systems Inc., Cleveland, OH) liquid medium, and by direct PCR for the IS900 target gene. Mycobacterium avium ssp. paratuberculosis was detected in 3.8, 4.1, and 12.6% of milk samples collected from cows with subclinical JD after culture in Herrold's egg yolk medium, liquid medium, and direct PCR, respectively. The frequency of MAP positivity increased to 12.9, 18.4, and 49.2% of milk samples collected from cows with clinical JD by these same methods, respectively. None of the milk samples collected from control cows was positive for MAP by any detection method. Viable MAP was primarily isolated from milk and colostrum of subclinically and clinically infected cows collected in early lactation (DIM 0–60), with negligible positive samples observed in mid (DIM 60–240) and late (DIM 240–305) lactation. This study demonstrates that shedding of MAP into milk is affected by infection status of the cow as well as stage of lactation, providing useful information to producers to help break the cycle of infection within a herd.     

Invited review: Modeling within-herd transmission of Mycobacterium avium subspecies paratuberculosis in dairy cattle: A review

Epidemiological models have been developed to test hypotheses on Mycobacterium avium ssp. paratuberculosis (Map) transmission in a herd, and to compare different paratuberculosis control strategies and alternatives for certification-and-surveillance schemes. The models are simplified representations of existing biological processes tailored to the questions they are intended to answer. Such models depend on available knowledge about the underlying processes, notably in relation to pathogen transmission. All decisions relating to integration of specific aspects of the herd structure and transmission mechanisms as well as modeling objective will influence model behavior and simulation results. This paper examines assumptions on pathogen transmission and risk mitigation represented in 8 epidemiological models of within-herd Map transmission in dairy cattle. We describe available models’ structure and examine them in the context of current knowledge about host infection and pathogen transmission pathways. We investigate how population structure and herd management are modeled with regard to their influence on contact structure and pathogen transmission. We show that assumptions about routes of transmission and their contribution within a herd vary greatly among models. Gaps of knowledge that are pivotal to defining transmission equations and parameters, such as variation of susceptibility with age and variability of pattern of shedding, are identified. Quantitative estimates of this incomplete information should be targeted by future research. Existing models could be improved by considering indirect transmission via the environment taking account of Map survival and contact structure between animals in a herd, and by including calf-to-calf transmission, which has recently been proven as being important.     

High herd-level prevalence of Mycobacterium avium subspecies paratuberculosis in Western Canadian dairy farms,based on environmental sampling

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic progressive enteritis in ruminants. The pathogen is present in most countries with modern dairy production, causing substantial economic losses for the industry. The objectives of this study were to estimate dairy herd prevalence of MAP in the Western Canadian provinces of Alberta and Saskatchewan, and to determine whether herd size and housing system (tie-stall versus freestall or loose housing) affected the risk of a herd testing positive for MAP. Six environmental samples were collected on 360 Alberta farms (60% of registered producers) and on 166 Saskatchewan dairy farms (99%). In total, 47% of the sampled farms in Alberta and 53% of the sampled farms in Saskatchewan had at least one environmental sample that was MAP culture positive and were, therefore, defined as infected. Sensitivity of environmental sampling was estimated using 3 subsequent annual tests performed on 82 farms. Because laboratory protocols were continuously improved throughout the project, the sensitivity increased over time. Therefore, a mean of the sensitivity estimates weighted on sampling year was constructed; this resulted in sensitivities of 68 and 69% for Alberta and Saskatchewan, respectively. Implementing those estimates in an approximate Bayesian computation model resulted in a true herd prevalence of 68% (95% probability interval: 60–80%) for Alberta and 76% (95% probability interval: 70–85%) for Saskatchewan. Herds with >200 cows had 3.54 times higher odds of being environmental sample positive and had more positive samples than herds with <50 cows (neither province nor housing system affected those results). In conclusion, the majority of Alberta and Saskatchewan dairy farms were infected with MAP and larger herds were more often MAP positive than smaller herds.     

Mycobacterium avium subsp. paratuberculosis infection in slaughtered domestic pigs for consumption detected by molecular methods

Herein, we report the occurrence of Mycobacterium avium subsp. paratuberculosis (Map), in four domestic pigs slaughtered for consumption that revealed compatible lesions of Mycobacterium spp. Infection in submaxillary and in mesenteric lymph nodes during the post mortem examination was observed. Histopathologic evaluation revealed granulomatous lesions mostly consisted by central necrosis, associated with inflammatory infiltration. Two cases presented dystrophic calcification. Acid-fast bacilli were observed in all cases. Three different PCR assays were carried out to confirm the presence of Map. In swine there are few published cases of Map infection and to the best of our knowledge, this is the first report of molecular detection of Map directly from pig tissues.     

Prevalence of Mycobacterium avium subspecies paratuberculosis in Swiss raw milk cheeses collected at the retail level

A total of 143 raw milk cheese samples (soft cheese, n = 9; semihard cheese, n = 133; hard cheese, n = 1), collected at the retail level throughout Switzerland, were tested for Mycobacterium avium ssp. paratuberculosis (MAP) by immunomagnetic capture plus culture on 7H10-PANTA medium and in supplemented BAC-TEC 12B medium, as well as by an F57-based real-time PCR system. Furthermore, pH and water activity values were determined for each sample. Although no viable MAP cells could be cultured, 4.2% of the raw milk cheese samples tested positive with the F57-based real-time PCR system, providing evidence for the presence of MAP in the raw material. As long as the link between MAP and Crohn's disease in humans remains unclear, measures designed to minimize public exposure should also include a focus on milk products.     

Designing a risk-based surveillance program for Mycobacterium avium ssp. paratuberculosis in Norwegian dairy herds using multivariate statistical process control analysis

Surveillance programs for animal diseases are critical to early disease detection and risk estimation and to documenting a population’s disease status at a given time. The aim of this study was to describe a risk-based surveillance program for detecting Mycobacterium avium ssp. paratuberculosis (MAP) infection in Norwegian dairy cattle. The included risk factors for detecting MAP were purchase of cattle, combined cattle and goat farming, and location of the cattle farm in counties containing goats with MAP. The risk indicators included production data [culling of animals >3 yr of age, carcass conformation of animals >3 yr of age, milk production decrease in older lactating cows (lactations 3, 4, and 5)], and clinical data (diarrhea, enteritis, or both, in animals >3 yr of age). Except for combined cattle and goat farming and cattle farm location, all data were collected at the cow level and summarized at the herd level. Predefined risk factors and risk indicators were extracted from different national databases and combined in a multivariate statistical process control to obtain a risk assessment for each herd. The ordinary Hotelling’s T² statistic was applied as a multivariate, standardized measure of difference between the current observed state and the average state of the risk factors for a given herd. To make the analysis more robust and adapt it to the slowly developing nature of MAP, monthly risk calculations were based on data accumulated during a 24-mo period. Monitoring of these variables was performed to identify outliers that may indicate deviance in one or more of the underlying processes. The highest-ranked herds were scattered all over Norway and clustered in high-density dairy cattle farm areas. The resulting rankings of herds are being used in the national surveillance program for MAP in 2014 to increase the sensitivity of the ongoing surveillance program in which 5 fecal samples for bacteriological examination are collected from 25 dairy herds. The use of multivariate statistical process control for selection of herds will be beneficial when a diagnostic test suitable for mass screening is available and validated on the Norwegian cattle population, thus making it possible to increase the number of sampled herds.     

Reliability of environmental sampling to quantify Mycobacterium avium subspecies paratuberculosis on California free-stall dairies

The reliability of environmental sampling to quantify Mycobacterium avium ssp. paratuberculosis (MAP) based on collector and time was evaluated. Fecal slurry samples were collected using a standardized protocol simultaneously by 2 collectors of different experience levels. Samples were collected from 30 cow pens on 4 dairies every other day on 3 occasions while cow movements between pens were minimal. The 4 study herds had moderate MAP seroprevalence and were housed in free-stall dairies in central California. Results of testing the environmental samples for MAP using PCR and culture were strongly correlated. The reliability of environmental sampling simultaneously by different collectors as estimated by the intraclass correlation coefficient was excellent (81%) for PCR and good (67%) for culture and may justify comparison of quantitative results of samples collected by different investigators. The reliability of environmental sampling over a 5-d period was good (67 and 64% for PCR and culture results, respectively), which justifies the utility of environmental sampling to identify pens with a high MAP bioburden between routine cow pen changes on a dairy. Environmental sampling of free-stall pens using the standardized sampling protocol yielded comparable PCR and culture results across collectors with different experience levels and at different times within a 5-d period.     

Loss of income from cows shedding Mycobacterium avium subspecies paratuberculosis prior to calving compared with cows not shedding the organism on two Minnesota dairy farms

Quantification of the financial effect of Mycobacterium paratuberculosis infection on lactation performance is essential to encourage participation of dairy cattle producers in Johne's disease (JD) control programs. The objective of this study was to evaluate the differences in net income per lactation of cows shedding Mycobacterium paratuberculosis before calving compared with test-negative cows. Two Minnesota dairies were enrolled in the study and fecal samples were collected from 1,048 cows during the close-up period. Milk production, clinical diseases (other than clinical JD), and reproductive performance data were recorded for each cow. Overall, fecal-culture-positive (FCP) cows produced 1,355 kg less than fecal-culture-negative (FCN) cows. Fecal-culture-positive cows that survived their current lactation produced $276 less in milk income than cows that were FCN ($1,956 vs. $1,680; SD $526, $570). Fecal-culture-positive cows were 3.0 (95% confidence interval: 1.6-5.8) times more likely to be culled than FCN cows. The mean days open (number of days from calving to conception) was not statistically significant and the cost differences for clinical disease other than JD were small and neither statistically nor economically significant between FCP and FCN cows. Among all FCP cows, income over feed costs losses were $366 per cow per lactation compared with FCN cows. Among FCP nonculled cows, income over feed costs losses were $276 more compared with FCN cows and this difference was statistically significant. There was a total loss of $155 per lactation for nonculled FCP cows retained in the herd compared with FCN cows retained in the herd. Among culled cows, FCP cow losses were $50 less because of age at culling and $120 for reduced beef value. This totaled a loss of $441 for culled FCP cows compared with culled FCN cows. The losses as a result of lower lactation performance and early culling from the herd should alarm dairy producers and motivate them to implement the appropriate control measures for the disease.     

Short communication: Viable Mycobacterium avium subspecies paratuberculosis in retail artisanal Coalho cheese from Northeastern Brazil

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis and it potentially plays a role in Crohn’s disease. In humans, the main route of transmission of MAP might be the intake of contaminated milk and dairy products. Considering that MAP has already been detected in many types of cheese in different counties, and that Coalho cheese is an important dairy product in northeastern Brazil, the aim of this study was to report the first detection of MAP in retail Coalho cheese in Brazil by PCR and culture. Of 30 retail Coalho cheese samples, 3 (10%) amplified fragments of a similar size to that expected (626 bp) were obtained and viable MAP was recovered by culture from 1 (3.3%) sample. The DNA from the positive culture sample was sequenced and showed 99% identity with the insertion sequence IS900 deposited in GenBank. It was possible to identify the presence of MAP-specific DNA in the analyzed samples for the first time in Brazil, and to recover viable cells from retail Coalho cheese.     

Application of IS900 PCR for detection of Mycobacterium avium subsp. paratuberculosis directly from raw milk

A polymerase chain reaction (PCR)-based assay was developed for detection of insertion sequence 900 (IS900) of Mycobacterium avium subsp. paratuberculosis in raw milk. This IS900 PCR assay included DNA extraction and PCR assay using commercially available kits. The DNA extraction and PCR assay were optimized to detect the IS900 sequence directly from raw milk. The IS900 PCR assay was evaluated by inoculating raw bulk milk and Middlebrook's 7H9 broth with 0 to 10(8) cfu/ml of each of four American Type Culture Collection strains of M. paratuberculosis. Under experimental conditions, both milk culture on Herrold's egg yolk medium slants, and IS900 PCR could detect 10 to 100 cfu/ml of M. paratuberculosis. Detection of M. paratuberculosis by IS900 PCR was consistent (24/24 PCR assays) when about 100 cfu/ml were present, whereas detection was variable (12/24 PCR assays) at concentrations as low as 10 cfu/ml. Based on the findings of the experimental study, IS900 PCR was further evaluated with pooled quarter milk samples from 211 cows from five herds with known history of Johne's disease. Out of 211 animals examined, nine (4%) and 69 (33%) were positive for M. paratuberculosis by milk culture and IS900 PCR from milk, respectively. A total of 20 bulk tank milk sample aliquots (one sample, four aliquots from each herd) were also examined, of which 10 (50%) were positive for M. paratuberculosis by IS900 PCR. By contrast, only one out of 20 (5%) bulk tank milk sample aliquots was positive by culture. The IS900 PCR amplified product of 229-bp obtained on testing of quarter milk and bulk tank milk samples was confirmed to be the IS900 of M. paratuberculosis by DNA sequence analysis. The results of this study suggest that M. paratuberculosis can be detected directly from quarter milk and bulk tank milk by IS900 PCR.     

Localization of Mycobacterium avium subspecies paratuberculosis in artificially inoculated milk and colostrum by fractionation

The purpose of the study was to determine the distribution of Mycobacterium avium ssp. paratuberculosis (MAP) across the main milk and colostrum fractions (cream, curd, and whey). Raw milk and colostrum were inoculated with 1 of 2 MAP strains, ATCC 19698 or S-23, yielding initial concentrations of 10⁶ to 10⁷ cfu/mL. After fractionation, for milk as well as for colostrum, 80 to 90% of the recovered MAP cells were found in the curd fraction and 10 to 20% in the cream fraction. Total MAP colony counts in milk whey were 4 to 5 log₁₀ units lower than colony counts of inoculated milk. In colostrum, colony counts were 2 to 3 log₁₀ units lower in whey than in inoculated colostrum. Because of the slow growth of MAP and to proceed more smoothly with set-up and optimization of the method, luminescent MAP strains were used. The high correlation coefficient (r = 0.960) between colony counts and luminescence measurements showed that the use of luminescent MAP strains during method development was plausible.     

Short communication: Bacterial ecology of high-temperature, short-time pasteurized milk processed in the United States

To determine the microbial ecology of pasteurized milk within the United States, 2% fat pasteurized fluid milk samples were obtained from 18 dairy plants from 5 geographical areas representing the Northeast, Southeast, South, Midwest, and West. Of the 589 bacterial isolates identified using DNA sequence-based subtyping methods, 346 belonged to genera characterized as gram-positive endospore-forming bacteria (i.e., Bacillus and Paenibacillus). Of the 346 gram-positive endospore-forming bacteria isolated in the present study, 240 were classified into 45 allelic types identical to those previously identified from samples obtained in New York State, indicating the widespread presence of these microbes in fluid milk production and processing systems in the United States. More than 84% of the gram-positive spore-forming isolates characterized at d 1, 7, and 10 were of the genus Bacillus, whereas more than 92% of isolates characterized at d 17 of shelf life were of the genus Paenibacillus, indicating that the predominant gram-positive spoilage genera shifts from Bacillus spp. to Paenibacillus spp. during refrigerated storage.     

Osteopontin: a novel cytokine involved in the regulation of Mycobacterium avium subspecies paratuberculosis infection in periparturient dairy cattle

Osteopontin (Opn), an important mediator of the cell-mediated immune response, enhances the host immune response against mycobacterial infections. Infections caused by Mycobacterium avium ssp. paratuberculosis (MAP) have a devastating effect on the dairy industry. We sought to characterize Opn at the level of gene and protein expression in periparturient dairy cows naturally infected with MAP. Peripheral blood mononuclear cells (PBMC) were isolated from control, subclinical, and clinical periparturient dairy cows naturally infected with MAP beginning 3 wk precalving to 5 wk postcalving and incubated with medium alone (non-stimulated: NS), concanavalin A (ConA), or a whole-cell sonicate of MAP (MPS). Real-time PCR was performed to evaluate expression of Opn and classical Th1 and Th2 cytokines. Results demonstrated greater Opn expression in nonstimulated PBMC isolated from subclinical cows compared with control and clinical cows. For clinical cows, there was a strong correlation between Opn expression and expression of the Th1 cytokines IFN-γ and IL-1α for nonstimulated PBMC and IFN-γ and IL-12 for PBMC stimulated with MPS. Expression of tumor necrosis factor-α was greater in clinical cows than the other groups. Nonstimulated, ConA, and MPS-stimulated PBMC from subclinical cows secreted more IFN-γ, and MPS-stimulated PBMC from clinical cows secreted more IL-4 compared with the other groups. Immunoblot analysis of PBMC detected 4 Opn proteins at 60, 52, 34, and 27 kDa. This is the first study to evaluate the role of Opn on the immune response of dairy cows naturally infected with MAP, and results suggest Opn may be a key regulator against MAP infection.     

Short communication: Survey of animal-borne pathogens in the farm environment of 13 dairy operations

A survey was conducted on 13 dairies to determine the occurrence of 5 animal-borne pathogens (Salmonella enterica, Escherichia coli O157:H7, Campylobacter jejuni, Mycobacterium avium ssp. paratuberculosis, and Cryptosporidium parvum) and their distributions across farm elements (feces, bedding, milk filters, stored manure, field soil, and stream water). Presence of C. parvum was measured only in feces and stored manure. All but one farm were positive for at least one pathogen species, and 5 farms were positive for 3 species. Escherichia coli O157:H7 was detected on 6 farms and in all farm elements, including milk filters. Mycobacterium avium ssp. paratuberculosis was detected on 10 of 13 farms and in all farm elements except for milk filters. Salmonella enterica and C. jejuni were detected at lower frequencies and were not identified in soil, stream water, or milk filters on any of the 13 farms. Cryptosporidium parvum was detected in feces but not in stored manure. Stored manure had the highest occurrence of pathogens (73%), followed by feces (50%), milk filters, bedding, soil, and water (range from 23 to 31%). Association of pathogen presence with farm management factors was examined by t-test; however, the small number of study farms and samples may limit the scope of inference of the associations. Pathogens had a higher prevalence in maternity pen bedding than in calf bedding, but total pathogen occurrence did not differ in calf compared with lactating cow feces or in soils with or without manure incorporation. Herd size and animal density did not appear to have a consistent effect on pathogen occurrence. The extent of pathogen prevalence and distribution on the farms indicates considerable public health risks associated with not only milk and meat consumption and direct animal contact, but also potential dissemination of the pathogens into the agroecosystem.     

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.     

Short communication: Attempts to identify Clostridium botulinum toxin in milk from three experimentally intoxicated Holstein cows

Three adult lactating Holstein cows were injected in the subcutaneous abdominal vein with 175 ng/kg of body weight of Clostridium botulinum type C toxin (451 cow median toxic doses) to determine if this botulinum toxin crosses the blood-milk barrier. Whole blood (in sodium heparin) and clotted blood serum samples were taken at 0 min, 10 min, and 3, 6, 9, and 12 h postinoculation. Milk samples were taken at 0 min and at 3, 6, 9 and 12 h postinoculation. All samples were tested for the presence of the toxin using the mouse bioassay and immunostick ELISA test. The immunostick ELISA identified the toxin in whole blood and the mouse bioassay identified the toxin in serum at all times examined in all 3 animals. Toxin was not identified by either detection method in milk samples collected from the 3 animals. From these results, it appears that Clostridium botulinum type C toxin does not cross from the blood to the milk in detectable concentrations.