A screening sampling plan to detect Mycobacterium avium subspecies paratuberculosis-positive dairy herds

Abstract Mycobacterium avium ssp. paratuberculosis (MAP) is the etiological agent of paratuberculosis, a chronic contagious bacterial disease primarily affecting dairy cattle. Paratuberculosis represents a dual problem for the milk production chain: in addition to economic losses to affected herds, MAP may have zoonotic potential. Infected herds must be identified in order to implement programs designed to reduce the incidence of disease within and between herds and to prevent MAP from entering the food chain. The objective of this study was to evaluate the sensitivity and specificity of a screening sampling plan (SSP) to detect MAP-positive dairy herds by repetitive analysis of bulk tank milk (BTM) samples by ELISA and in-line milk filter (ILMF) samples by PCR. Samples from BTM and ILMF were collected twice from 569 dairy herds in southern Italy. Additionally, 12,016 individual milk samples were collected: 9,509 from 102 SSP-positive herds (SSP MAP-positive) and 2,507 from 21 randomly selected SSP-negative herds (SSP MAP-negative). There was a total of 126 SSP MAP-positive herds (i.e., 21.3% SSP MAP-positive herds; 95% confidence interval=18.0–24.9); the within-herd apparent prevalence (AP) ranged between 0.00 and 22.73% (mean 6.07%). A significant difference in within-herd AP was shown between SSP MAP-positive herds and SSP MAP-negative herds. A highly significant association was shown between the median AP herd status (>5%) and positivity to at least one ILMF or BTM sample. The SSP detected a minimum of 56.25% of low AP herds (AP ≤2.0%) up to a maximum of 100% of herds with a within-herd AP ≥8.0%. Overall, the SSP detected 85.57% of herds in which at least one individual milk sample was positive by ELISA. The proposed SSP was an inexpensive and useful tool to detect MAP-positive herds with a higher risk of infection diffusion and milk contamination. Although the SSP cannot be used for MAP-free certification of herds, it could be useful to prioritize appropriate control measures aimed at reducing the prevalence of infection in dairy herds and milk contamination

Effectiveness of combination of Mini-and Microsatellite loci to sub-type Mycobacterium avium subsp. paratuberculosis Italian type C isolates

<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium avium </it>subsp. <it>paratuberculosis </it>(Map) is the etiological agent of paratuberculosis. The aim of our study was to combine Mini-and Microsatellite loci analysis in order to explore the effectiveness of this sub-typing method in a group of Map isolates. For this purpose, 84 Italian Type C Map isolates, each from a different cattle herd, were submitted to MIRU-Variable-Number Tandem-Repeats (VNTRs) typing and Short Sequence repeats (SSRs) sequencing. Moreover, the method was used to analyse the variability inside 10 herds (from three to 50 isolates per herd).</p> <p>Results</p> <p>The molecular sub-typing, carried out using three SSR and 10 MIRU-VNTR loci, differentiated the 84 isolates into 33 clusters, reaching a Simpson's Discriminatory Index (SID) value of 0.952 (0.933 to 0.972, 95% confidence intervals). Among all considered loci, six (SSR2, MIRU2, SSR1, SSR8, VNTR3527 and VNTR1067) showed relevant allelic variability. Thirty-eight% of the isolates were clustered into four genotypes, differing from each other for the SSR2 locus. The other isolates, characterised by differences in two or more loci, were spread among the rest of the clusters. The intra-herd analysis revealed more than one genotype in most herds with a similar distribution of clusters.</p> <p>Conclusions</p> <p>Our results revealed the advantage of using both Mini-and Microsatellite approaches for successfully discriminating among Map Type C isolates from the same geographic area, host species and herd. These data suggest that the combination of loci here proposed could be a useful molecular tool for regional epidemiological studies.</p

Estimation of Mycobacterium avium subsp. paratuberculosis load in raw bulk tank milk in Emilia-Romagna Region (Italy) by qPCR

Consumption of milk and dairy products is considered one of the main routes of human exposure to Mycobacterium avium subsp. paratuberculosis (MAP). Quantitative data on MAP load in raw cows’ milk are essential starting point for exposure assessment. Our study provides this information on a regional scale, estimating the load of MAP in bulk tank milk (BTM) produced in Emilia‐Romagna region (Italy). The survey was carried out on 2934 BTM samples (88.6% of the farms herein present) using two different target sequences for qPCR (f57 and IS900). Data about the performances of both qPCRs are also reported, highlighting the superior sensitivity of IS900‐qPCR. Seven hundred and eighty‐nine samples tested MAP‐positive (apparent prevalence 26.9%) by IS900 qPCR. However, only 90 of these samples were quantifiable by qPCR. The quantifiable samples contained a median load of 32.4 MAP cells mL (−1) (and maximum load of 1424 MAP cells mL (−1)). This study has shown that a small proportion (3.1%) of BTM samples from Emilia‐Romagna region contained MAP in excess of the limit of detection (1.5 × 10(1) MAP cells mL (−1)), indicating low potential exposure for consumers if the milk subsequently undergoes pasteurization or if it is destined to typical hard cheese production

Estimation of Performance Characteristics of Analytical Methods for Mycobacterium avium subsp. paratuberculosis Detection in Dairy Products

Paratuberculosis is a chronic enteric infection, caused by Mycobacterium avium subsp. paratuberculosis (MAP), affecting virtually all ruminants as well as other animals. MAP is also suspected to be involved in the etiology of some human diseases, like Crohn’s disease and others. In surveillance studies, different analytical methodologies were employed to detect MAP, showing different results and incidence in dairy products. The aim of this study was to evaluate the performance characteristics of three analytical methods [culture, quantitative PCR (qPCR) and peptide-mediated magnetic separation (PMS) phage-based assay] for MAP detection in raw, heat-treated and powdered milk. The methods were evaluated according to performance characteristics defined for qualitative methods in ISO 16140-2:2016. To estimate sensitivity (including trueness) and LOD, 720, and 900 test portions, respectively, were blind tested by two laboratories. Considering all matrices, different sensitivities, expressed as the percentage of positives from the total of true positive test portions, were obtained for IS900 qPCR (94%), f57 qPCR (76%), culture (83%), and PMS-phage (40%). Trueness, expressed as results correctly assigned (including positive and negative) to the reference value, was 93% for the IS900 qPCR method, 89% for culture and 49% for the PMS-phage. The LODs obtained in this study were similar to the LODs previously published for cultural and qPCR methods. However, for the PMS-phage method, the obtained results showed higher LOD values compared to the limited data available in the scientific literature. Our results highlight that while the PMS-phage assay is workable in pure liquid culture for estimation of MAP counts, its usage for surveillance of dairy matrices should be treated with a lot of caution as performance characteristics obtained were lower than for the two other methods tested. qPCR and culture are the most appropriate methods to detect MAP in milk-based matrices according to ISO 16140 methodology. Cultural techniques are considered the gold standard for detection of viable MAP, but qPCR, which is widely used in analytical and surveillance studies, can be considered a suitable and recommendable alternative to cultural methods for screening, if confirmation of MAP’s viability is not requested

Quantitative risk assessment of human salmonellosis and listeriosis related to the consumption of raw milk in Italy.

Two quantitative risk assessment (RA) models were developed to describe the risk of salmonellosis and listeriosis linked to consumption of raw milk sold in vending machines in Italy. Exposure assessment considered the official microbiological records monitoring raw milk samples from vending machines performed by the regional veterinary authorities from 2008 to 2011, microbial growth during storage, destruction experiments, consumption frequency of raw milk, serving size, and consumption preference. Two separate RA models were developed: one for the consumption of boiled milk and the other for the consumption of raw milk. The RA models predicted no human listeriosis cases per year either in the best or worst storage conditions and with or without boiling raw milk, whereas the annual estimated cases of salmonellosis depend on the dose-response relationships used in the model, the milk storage conditions, and consumer behavior in relation to boiling raw milk or not. For example, the estimated salmonellosis cases ranged from no expected cases, assuming that the entire population boiled milk before consumption, to a maximum of 980,128 cases, assuming that the entire population drank raw milk without boiling, in the worst milk storage conditions, and with the lowest dose-response model. The findings of this study clearly show how consumer behavior could affect the probability and number of salmonellosis cases and in general, the risk of illness. Hence, the proposed RA models emphasize yet again that boiling milk before drinking is a simple yet effective tool to protect consumers against the risk of illness inherent in the consumption of raw milk. The models may also offer risk managers a useful tool to identify or implement appropriate measures to control the risk of acquiring foodborne pathogens. Quantification of the risks associated with raw milk consumption is necessary from a public health perspective

Four-Year Monitoring of Foodborne Pathogens in Raw Milk Sold by Vending Machines in Italy

Prevalence data were collected from official microbiological records monitoring four selected foodborne pathogens (Salmonella spp., Listeria monocytogenes, Escherichia coli O157:H7 and Campylobacter jejuni) in raw milk sold by self-service vending machines in seven Italian Regions (n. 60907 samples from 1239 vending machines) during the years 2008 to 2011. Data of samples analyzed both by culture-based and real-time PCR methods were collected in one Region. A total of 100 raw milk consumers in four regions were interviewed while purchasing raw milk from vending machines. One hundred and seventy eight samples out of 60907 were positive for one of the four foodborne pathogens investigated; overall, 18 samples were positive for Salmonella spp., 83 for L. monocytogenes, 24 for E. coli O157:H7 and 53 for C. jejuni in the seven Regions investigated. There were no significant differences in prevalence among Regions, but a significant increase in C. jejuni prevalence was observed over the years. A comparison of the two different analysis methods showed that real-time PCR is from 2.71 to 9.40 times more sensitive than culture-based method. Data on consumer habits showed that some behaviors may enhance the risk of infection due to raw milk consumption: 37% of consumers do not boil milk before consumption, 93% never use an insulated bag to transport raw milk home, and raw milk is consumed by children under five years of age. The study emphasizes that end-product controls alone are not sufficient to guarantee an adequate level of consumer protection. The beta distribution of positive samples in this study and the data on raw milk consumer habits are useful and appropriate for the development of a National Quantitative Risk Assessment of Salmonella spp., L. monocytogenes, E. coli O157 and C. jejuni related to raw milk consumption

Evidence of Common Isolates of Streptococcus agalactiae in Bovines and Humans in Emilia Romagna Region (Northern Italy)

Streptococcus agalactiae (group B Streptococcus, GBS) is one of the most important agents of bovine mastitis and causes remarkable direct and indirect economic losses to the livestock sector. Moreover, this species can cause severe human diseases in susceptible individuals. To investigate the zoonotic potential of S. agalactiae, 203 sympatric isolates from both humans and cattle, isolated in the same time frame (2018) and in the same geographic area (Emilia Romagna region, Northern Italy), were characterized by molecular capsular typing (MCT), pilus island typing (PI), and multi-locus sequence typing (MLST). In addition, antibiotic-resistant phenotypes were investigated. The distribution of the allelic profiles obtained by combining the three genotyping methods (MCT-PI-MLST) resulted in 64 possible genotypes, with greater genetic variability among the human compared to the bovine isolates. Although the combined methods had a high discriminatory power (&gt;96,2%), five genotypes were observed in both species (20,9% of the total isolates). Furthermore, some of these strains shared the same antibiotic resistance profiles. The finding of human and bovine isolates with common genotypes and antibiotic resistance profiles supports the hypothesis of interspecies transmission of S. agalactiae between bovines and humans

Active surveillance of paratuberculosis in Alpine-dwelling red deer (Cervus elaphus)

Paratuberculosis (Johne’s disease) is a globally widespread infectious disease affecting domestic and wild ruminants, caused by Mycobacterium avium subsp. paratuberculosis (MAP). The bacterium is excreted in the feces and is characterized by high environmental resistance. The new Animal Health Law (Regulation EU 2016/429) on transmissible animal diseases, recently in force throughout the European Union, includes paratuberculosis within the diseases requiring surveillance in the EU, listing some domestic and wild Bovidae, Cervidae, and Camelidae as potential reservoirs. Taking advantage of a culling activity conducted in the Stelvio National Park (Italy), this study investigated MAP infection status of red deer (Cervus elaphus) between 2018 and 2022, and evaluated the probability of being MAP-positive with respect to individual and sampling-level variables. A total of 390 subjects were examined macroscopically and tested for MAP, using different diagnostic tools: IS900 qPCR, culture, histopathology, and serology. Twenty-three of them were found positive for MAP by at least one test, with an overall prevalence of 5.9% (95% CI 4.0–8.7), that, respectively, ranged from 12.4% in the first culling season to 2.0 and 2.1% in the 2019–2020 and 2021–2022 culling seasons. Quantitative PCR assay on ileocecal valve and mesenteric lymph nodes detected the highest number of MAP positive animals. The results of the study showed the increased probability of being MAP-positive with increasing age and that red deer with lower body mass values were more likely to be infected with MAP. Overall, the absence of signs of clinical paratuberculosis and gross lesions together with the low level of shedding witness early phases of the disease among the positive red deer and support an improvement of the paratuberculosis status of this population, as shown by the decreased prevalence of the disease over the years

Control of paratuberculosis: who, why and how. A review of 48 countries

Paratuberculosis, a chronic disease affecting ruminant livestock, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). It has direct and indirect economic costs, impacts animal welfare and arouses public health concerns. In a survey of 48 countries we found paratuberculosis to be very common in livestock. In about half the countries more than 20% of herds and flocks were infected with MAP. Most countries had large ruminant populations (millions), several types of farmed ruminants, multiple husbandry systems and tens of thousands of individual farms, creating challenges for disease control. In addition, numerous species of free-living wildlife were infected. Paratuberculosis was notifiable in most countries, but formal control programs were present in only 22 countries. Generally, these were the more highly developed countries with advanced veterinary services. Of the countries without a formal control program for paratuberculosis, 76% were in South and Central America, Asia and Africa while 20% were in Europe. Control programs were justified most commonly on animal health grounds, but protecting market access and public health were other factors. Prevalence reduction was the major objective in most countries, but Norway and Sweden aimed to eradicate the disease, so surveillance and response were their major objectives. Government funding was involved in about two thirds of countries, but operations tended to be funded by farmers and their organizations and not by government alone. The majority of countries (60%) had voluntary control programs. Generally, programs were supported by incentives for joining, financial compensation and/or penalties for non-participation. Performance indicators, structure, leadership, practices and tools used in control programs are also presented. Securing funding for long-term control activities was a widespread problem. Control programs were reported to be successful in 16 (73%) of the 22 countries. Recommendations are made for future control programs, including a primary goal of establishing an international code for paratuberculosis, leading to universal acknowledgment of the principles and methods of control in relation to endemic and transboundary disease. An holistic approach across all ruminant livestock industries and long-term commitment is required for control of paratuberculosis

Sale of Raw Milk in Northern Italy: Food Safety Implications and Comparison of Different Analytical Methodologies for Detection of Foodborne Pathogens

The safety of raw milk sold in Northern Italy was investigated in relation to hygiene quality parameters and presence of Salmonella spp., Listeria monocytogenes, thermotolerant Campylobacter, and Verocytotoxin producing Escherichia coli O157:H7. The performance of different analytical methods used—official culture method (ISO), modified Bacteriological Analytical Manual cultural method (mBAM), and polymerase chain reaction (PCR)— was evaluated. The presence of Mycobacterium avium subsp. paratuberculosis (Map) was investigated only by PCR. All samples met regulations for alkaline phosphatase and inhibitory substance, while 18% and 44.8% of samples collected from vending machines had, respectively, somatic cell count (SCC) > 300,000/mL and total bacterial count (TBC) > 50,000CFU/mL. The correlation between hygienic quality parameters in samples collected from bulk tank and vending machines showed a significant increase of TBC in vending machines meaning that raw milk was mishandled during distribution and sale. All pathogens investigated were detected in raw milk sold at vending machines; a total of five samples (5%) had at least one pathogen, of which two were detected by PCR and three by mBAM. None of the samples was positive by cultural ISO methods. Even if the comparison of analytical methods showed that none performs significantly better than the others, testing a higher volume of milk (25 versus 210 mL) affects significantly the detection rate of pathogens. Three samples (3%) were positive for Map, suggesting that raw milk is a significant source of Map exposure for consumers. The observed TBC increase and the detection of several pathogenic bacteria pose questions on the safety of raw milk; the use of ISO seems inefficient in detecting a low contamination level of pathogens in milk and consequently not appropriate as official method for testing. In order to ensure consumer’s safety, a new approach for the raw milk chain is required