Emergence of Group B Streptococcus Disease in Pigs and Porcupines, Italy.

We describe group B Streptococcus linked to disease in farmed pigs and wild porcupines in Italy. Occurrence in pigs was attributed to transmission from nonpasteurized bovine milk whey. Antimicrobial-resistance profiles in isolates from porcupines suggest no common source of infection. Our findings expand the known host range for group B Streptococcus disease.

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.

High clonality of Mycobacterium avium subsp. paratuberculosis field isolates from red deer revealed by two different methodological approaches of comparative genomic analysis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the aetiological agent of paratuberculosis (Johne's disease) in both domestic and wild ruminants. In the present study, using a whole-genome sequence (WGS) approach, we investigated the genetic diversity of 15 Mycobacterium avium field strains isolated in the last 10 years from red deer inhabiting the Stelvio National Park and affected by paratuberculosis. Combining de novo assembly and a reference-based method, followed by a pangenome analysis, we highlight a very close relationship among 13 MAP field isolates, suggesting that a single infecting event occurred in this population. Moreover, two isolates have been classified as Mycobacterium avium subsp. hominissuis, distinct from the other MAPs under comparison but close to each other. This is the first time that this subspecies has been found in Italy in samples without evident epidemiological correlations, having been isolated in two different locations of the Stelvio National Park and in different years. Our study highlights the importance of a multidisciplinary approach incorporating molecular epidemiology and ecology into traditional infectious disease knowledge in order to investigate the nature of infectious disease in wildlife populations.

Development of a Real-Time PCR Assay for the Detection of the Phlebovirus Fermo Virus.

Fermo virus is a Phlebovirus that is increasingly reported in sand flies from northern Italy. The natural cycle is not fully understood, but the virus has been detected by direct methods only in sand flies. Although there is serological evidence that it can infect vertebrates, the virus has not been directly detected in animals or humans. Here, we have developed and reported a specific real-time PCR for Fermo virus. The availability of the described method will be useful to characterize the epidemiology of the FERV, ensuring, compared to previously available protocols, a more sensitive detection in insects and the possible detection in vertebrates to evaluate the presence of reservoirs and the pathogenic potential of the virus in humans or animals.

Validation of digital PCR assay for the quantification of Mycobacterium avium subsp. paratuberculosis in bovine faeces according to the ISO 20395:2019.

Paratuberculosis is an enteric disease caused by Mycobacterium avium subs. Paratuberculosis (MAP). Quantifying the load of MAP in faeces samples offers the advantage of determining the stage of infection and planning control measures. Currently, detection of MAP in faecal specimens relies on cultural assays and quantitative PCR (qPCR), but both methods have limitations such as prolonged isolation times for cultural assay and the absence of nucleic acid standards for qPCR. Digital PCR (dPCR) represents an advancement over qPCR as it allows direct quantification of nucleic acid in a sample without the need for a standard curve. The present paper reports about the validation process, following ISO 20395:2019 guidelines, of a F57 digital PCR assay for quantifying MAP cells in faecal samples. Based on our validation, the Limit Of Detection (LOD) corresponds to 7.85 104 MAP cells/g, and the Limit Of Quantification (LOQ) to 7.85 105 MAP cells, with an efficiency of recovery at LOQ estimated about 4.5%. To assess precision, we evaluated the same faecal sample extracted by two different operators at different times. The standard deviation under repeatability conditions (S Repeatability) and intersession variability conditions (S Intermediate) were calculated, resulting in values of 0.43 and 0.26, respectively. Trueness was determined at LOQ and a value ten times higher, yielding percentages of 3.35% and 5.16%, respectively. Linearity showed a R2 value of 0.998, indicating strong linear correlation. Measurement uncertainty was 26% in absolute value and 3% on a logarithmic base 10 scale. Overall, the assay exhibits good specificity and robustness. Our validation underlines the good performance of the quantification method and allow the laboratory to provide quantitative results of MAP/cells on faecal samples.

Comparison between broth microdilution and agar disk diffusion methods for antimicrobial susceptibility testing of bovine mastitis pathogens.

In order to counter the antibiotic resistance phenomenon, a prudent and rational use of antimicrobials should be driven by an accurate clinical diagnosis and, when possible, by the isolation of the etiological agent followed by susceptibility testing, with the aim to select the most suitable molecule for therapy. Cow mastitis is considered the main cause of antibiotic use in the cattle breeding sector. The purpose of this study was to compare the broth microdilution (BMD) method performed with Sensititre Custom Plates and the agar disk diffusion (ADD) method in determining antimicrobial susceptibility of 215 isolates from bovine mastitis, including contagious pathogens (Staphylococcus aureus, Streptococcus agalactiae) and environmental (Streptococcus uberis, Streptococcus dysgalactiae, Enterococcus spp., Escherichia coli, Serratia marcescens, Klebsiella pneumoniae). We compared results of the following antimicrobials: amoxicillin/clavulanic acid, ampicillin, cefazolin, ceftiofur, enrofloxacin, erythromycin, kanamycin, oxacillin, penicillin, pirlimycin, rifampicin and trimethoprim/sulphonamides. We applied MIC breakpoints and zone diameter breakpoints as recommended by CLSI and EUCAST. MIC and disk diffusion diameters were compared for 1839 microorganism/antimicrobial combination and discrepancies between the two methods were classified as very major discrepancy (VMD), major discrepancy (MD) and minor discrepancy (MiD). The overall agreement between the two methods was found to be 80.7% with a Cohen's kappa coefficient of 0.397, thus indicating a fair concordance. BMD method and ADD method demonstrated a satisfactory agreement (89 to 100%) for S. aureus and S. marcescens and all antimicrobial agents tested. Low agreement was observed for S. uberis and rifampicin (20%), enrofloxacin (49%), penicillin (51%) and pirlimycin (52%), E. coli and ampicillin (20%), S. dysgalactiae and enrofloxacin (44%), S. agalactiae and rifampicin (25%). A possible explanation for the discrepancies detected could be found in the breakpoints used which, sometimes, are not specific for the tissue-matrix of isolation/animal species/pathogen agent. The majority of the discrepancies found were MiD and MD, revealing a higher restrictiveness of the BMD method, while VMD represented only 0.2% of the total observations, a comforting fact since this type of error may result in treatment failure.

Paratuberculosis, Animal Welfare and Biosecurity: A Survey in 33 Northern Italy Dairy Goat Farms.

Paratuberculosis is a notable infectious disease of ruminants. Goats appear to be particularly susceptible. The survey aimed to investigate the spread of paratuberculosis in Italian goat farming and evaluate whether the presence of the disease could be influenced by welfare and biosecurity deficiencies. A serological survey for paratuberculosis in 33 dairy farms in northern Italy was conducted. Contextually, animal welfare and biosecurity were assessed, using a standardized protocol of 36 welfare indicators and 15 biosecurity indicators which assigns to each farm a welfare and biosecurity score from 0 (any application) to 100% (full application). An overall result of less than 60% was considered insufficient. Nineteen farms (58%) tested positive for paratuberculosis, with a mean intra-herd seroprevalence of 7.4%. Total welfare ranged from 39.56 to 90.7% (mean 68.64%). Biosecurity scores ranged from 10.04 to 90.01% (mean 57.57%). Eight farms (24%) showed poor welfare conditions (welfare score < 60%) and 19 (58%) an unsatisfactory biosecurity condition (biosecurity score < 60%). With respect to the explorative character of the study, an indicative association between seven welfare and biosecurity indicators and paratuberculosis seropositivity was identified. The presence of paratuberculosis in northern Italy dairy goat farms was confirmed. The welfare and biosecurity assessment protocol proved to be an accurate tool, capable of identifying critical points for managing health, welfare and productivity.

Microbiological and Chemical Analysis of Food Collected Under Official Control in the Emilia-Romagna Region of Northern Italy, 2014-2019.

This study analyzed data from 6 years (2014-2019) of official controls in the Emilia-Romagna region (northern Italy) to investigate the frequencies of human pathogens and chemical hazards in foods during production and distribution. Campylobacter spp. was the most prevalent pathogen, isolated in 4.4% of the 1,078 food samples examined, followed by Salmonella spp. (2.8%), Shiga toxin-producing Escherichia coli (STEC) (1.9%), and Listeria monocytogenes (0.9%). Salmonella serotyping showed that the isolates belonged to the serotypes most commonly isolated from humans in Emilia-Romagna. These serotypes were as follows: S. Infantis (34.8%), mostly isolated from chicken, monophasic S. Typhimurium (1,4, [5],12:i:-) (12.6%), S. Bredeney (8.9%), and S. Derby (8.6%). No Clostridium botulinum, Yersinia spp., and Shigella spp. were isolated. No positivity was detected for hepatitis A virus, while 5.1% of samples taken in the production phase of the food chain were found to be contaminated with norovirus. The chemical analyses identified environmental contaminants within legal limits (heavy metals, 0.6% positive overall; mycotoxins, 0.4% positive overall), analytes subjected to monitoring (perfluoro-alkyl substances (PFASs), 6.2% positive overall; inorganic arsenic, no positives overall) and process contaminants and additives within legal limits (acrylamide, 9.6% positive overall; permitted or nonpermitted additives, 0.9% positive overall). Only one sample showed dioxins and polychlorinated biphenyls (PCBs) at levels higher than the legal limits. The monitoring by competent authorities (CA) of food contamination can generate useful data that can be used as a basis for estimating the exposure to different food contaminants over time and for evaluating the effects of control measures on the contamination of food.

Isothermal inactivation of Mycobacterium avium subsp. paratuberculosis in curd simulating the stretching phase in pasta-filata cheese process.

Raw milk and dairy products are usually considered the major sources of Mycobacterium avium subsp. paratuberculosis (MAP) exposure for humans. During the production process of mozzarella cheese, as well as of other pasta-filata cheeses made with pasteurized or raw milk, curd is heated and stretched by addition of hot or boiling water. This step is the critical point for the inactivation of MAP during the production process, but, to our knowledge, no studies have been published about the thermal death time values of MAP in curd. The aim of this study was to determine the inactivation kinetics of MAP in curd used to produce pasta-filata cheese in six independent experiments. The milk was inoculated with a mix of MAP strains (field and registered strains) and, with the aim to simulate the thermal treatment of the curd during the stretching step, samples of 10 g of contaminated curd were vacuum packed and treated separately at six different temperatures from 60°C to 75°C in a water bath. MAP survival was then evaluated by plate count method and inactivation parameters were estimated for determining the thermal resistance of the pathogen directly in the curd. D-values increased from 0.15 min (D75-value) to 4.22 min (D60-value) and the calculated z-value was 10.2°C. These data aid: (i) to design food thermal process treatments defining acceptance limits of critical control points to ensure safety against MAP; (ii) to predict the time/temperature combinations needed to obtain a certain MAP log reduction during the curd stretching step; (iii) to optimize or validate pasta-filata cheese process.

Validation of IS900- qPCR assay to assess the presence of Mycobacterium avium subs. paratuberculosis in faecal samples according to the OIE procedure.

Paratuberculosis is a chronic enteric progressive disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP). Despite cultural methods being considered the gold standard for the diagnosis of paratuberculosis, quantitative PCR (qPCR) assays have been developed for this purpose. These assays showed sensitivity and specificity comparable to cultural method but provide more rapid analysis results. Aim of our work was the validation of an IS900-qPCR assay for detection of MAP in faeces according to the OIE guidelines relative to the validation of assays for infectious diseases. The analytical and diagnostic characteristics and the reproducibility of the qPCR method were assessed. The robustness of the assay was evaluated using two extraction methods (silica column and magnetic beads DNA capture) and two qPCR systems (STEPONE™ and CFX96™). According to our validation, the analytical specificity, inclusivity and exclusivity were found to be appropriate for the use of this qPCR assay as a diagnostic test. Specifically, the limit of detection was approximately 100 CFU/g or even less if binomial approaches were used for the determination of the 95 % probability of detection (logit and clog-log models) with sufficient repeatability and reproducibility. Estimation of test accuracy was performed using a Bayesian two latent class model, in various scenarios combining different priors for prevalence and accuracy of the two tests used. All models were run considering three different cut-offs for qPCR. Our validation study underlines the good performance of this IS900-qPCR assay for diagnosis of MAP representing a valid and robust alternative to culture. Moreover, coupled with the semiautomatic magnetic beads DNA extraction method, this assay allows the rapid processing of numerous samples.

Circulation of Streptococcus agalactiae ST103 in a Free Stall Italian Dairy Farm.

We report here on an outbreak of mastitis caused by Streptococcus agalactiae, or group B Streptococcus, in a northern Italy (Lombardy Region) free stall dairy farm. This outbreak was unusual because it occurred in a closed dairy herd and proved to be extremely difficult to resolve even after the application of the classical control procedures, which are specifically focused on the contagious nature of S. agalactiae. In order to better understand the potential origins of the pathogen and the critical points that could impair the eradication program and to investigate the possible presence of S. agalactiae in sources outside the mammary gland, we collected 656 individual composite milk samples, 577 samples from extramammary body sites (289 rectal, 284 vaginal, and four throat samples from milking cows, dry cows, heifers, and calves), and 81 samples from the cattle environment, including the milking parlor and the barn. Twenty-two S. agalactiae isolates were obtained from lactating cows or their environment. Of these, nine were isolated from milk, two were from rectal swabs, and two were from vaginal swabs, while nine were isolated from environmental samples. Based on molecular serotyping, pilus island (PI) typing and multilocus sequence typing, all isolates belonged to serotype III, pilus type PI-1/2b, and sequence type 103 (ST103), a type previously described to have an environmental transmission cycle and a potential human origin. Once the classical mastitis control measures were supplemented with environmental hygiene measures, herd monitoring using bulk tank milk revealed no further positive results for S. agalactiae, and the outbreak was considered resolved. IMPORTANCE Streptococcus agalactiae is an important pathogen in humans and cattle. Bovine mastitis caused by this bacterium and its control are generally associated with contagious transmission between animals. More recently, the presence of a fecal-oral transmission cycle in cattle has been proposed, linked to the ability of some S. agalactiae strains to survive in the bovine gastrointestinal tract and environment. Based on analysis of 1,316 specimens from cattle and their environment on a single dairy farm, we demonstrate the presence of sequence type 103 (ST103), which may have an environmental mode of transmission. This possibility was supported by the fact that the mastitis outbreak could not be controlled through measures to prevent contagious transmission alone and required additional environmental hygiene measures to be brought to a halt. This case study highlights that measures to control animal disease need to evolve alongside the microorganisms that cause them.

Goat Paratuberculosis: Experimental Model for the Evaluation of Mycobacterium Persistence in Raw Milk Cheese.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of chronic proliferative enteritis found in ruminants, known as paratuberculosis (PTB). The spread of PTB is increasing in countries with advanced animal husbandry practices, leading to significant economic losses. Moreover, a supposed zoonotic role of MAP in Crohn's disease (CD) in humans has been discussed by the scientific community; however, although the association between MAP and CD has generally been accepted, it is still up for debate if MAP is the main cause of CD, a contributing factor, or merely a commensal organism for the development of CD. The aim of this study was to assess the survival of MAP during the entire production process of a traditional Italian goat's raw milk fresh cheese, the "Robiola di Roccaverano", assessing the survival rate and persistence of MAP in the final product. A mix of MAP field isolates from goats of the Roccaverano area and a reference ATCC strain were used to carry out milk in experimental inoculation. Samples of milk, curd and cheese were taken in two consecutive batches of production. Microbiological challenge tests, evaluated by f57-qPCR, showed a significant decrease in MAP charge during the cheesemaking process for both batches, suggesting the productive process has an impact on MAP survival.

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 (>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.

Development of a reference standard for the detection and quantification of Mycobacterium avium subsp. paratuberculosis by quantitative PCR.

Quantitative PCR (qPCR) has become a frequently employed direct method for the detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP). The quantity of MAP determined by qPCR, however, may be affected by the type of qPCR quantification standard used (PCR product, plasmid, genomic DNA) and the way in which standard DNA quantity is determined (absorbance, fluorescence). In practice, this can be reflected in the inability to properly compare quantitative data from the same qPCR assays in different laboratories. Thus, the aim of this study was to prepare a prototype of an international MAP reference standard, which could be used to calibrate routinely used qPCR quantification standards in various laboratories to promote clinical data comparability. Considering stability, storage and shipment issues, a lyophilised fecal suspension artificially contaminated with a MAP reference strain was chosen as the most suitable form of the standard. The effect of five types of lyophilisation matrices on standard stability was monitored on 2-weeks interval basis for 4 months by F57 qPCR. The lyophilisation matrix with 10% skimmed milk provided the best recovery and stability in time and was thus selected for subsequent comparative testing of the standard involving six diagnostic and research laboratories, where DNA isolation and qPCR assay procedures were performed with the parallel use of the identical supplied genomic DNA solution. Furthermore, the effect of storage conditions on the standard stability was tested for at least 6 months. The storage at room temperature in the dark and under light, at + 4 °C, - 20 °C and - 80 °C showed no significant changes in the stability, and also no substantial changes in MAP viability were found using phage amplification assay. The prepared MAP quantification standard provided homogeneous and reproducible results demonstrating its suitability for utilisation as an international reference qPCR standard.

Residual SARS-CoV-2 RNA in nasal swabs of convalescent COVID-19 patients: Is prolonged quarantine always justified?

Real-time reverse transcription PCR is currently the most sensitive method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Defining whether a patient could be contagious or not contagious in the presence of residual SARS-CoV-2 RNA is of extreme importance in the context of public health. In this prospective multicenter study, virus isolation was prospectively attempted in 387 nasal swabs from clinically recovered patients showing low viral load (quantification cycle, Cq, value greater than 30). The median Cq value was 36.8 (range 30.0-39.4). Overall, a cytopathic effect was detected in nine samples, corresponding to a culture positivity rate of 2.3% (9/387). The results of this study help to dissect true virus replication and residual viral RNA detection in recovered patients.

Paratuberculosis in Captive Scimitar-Horned Oryxes (Oryx dammah).

Paratuberculosis, a chronic disease caused by Mycobacterium avium subsp. paratuberculosis (MAP), in ten scimitar-horned oryxes (SHOs) hosted in an Italian zoological park and originating from a Slovakian flock, was documented by pathology, molecular, cultural, and serological testing. The infection origin in this threatened species was also investigated by genomic analyses. Following the death of six of the 10 SHOs, serial investigations of dead and alive animals were performed. Necropsy, carried out on five out of six animals, identified intestinal thickening and mesenteric lymphadenomegaly in one of the animals. Histopathology (5/6) revealed lepromatous (2/5) and tuberculoid (2/5) intestinal forms or lack of lesions (1/5). Ziehl-Neelsen and immunohistochemistry stains identified two multibacillary, two paucibacillary forms, and one negative case. MAP was identified by quantitative PCR (qPCR) in tissue samples in five out of five SHOs and was microbiologically isolated from two of the three animals whose fresh tissue samples were available. Fecal samples were collected in four of the six dead animals: all four resulted positive to qPCR and in MAP was isolated in three. ELISA identified MAP-specific antibodies in three of the five dead animals whose serum was available. qPCR identified MAP in the freshly deposited feces of two out of the four alive animals. From the feces of these two animals, MAP was microbiologically isolated in one case. All isolates were classified as MAP type C and profiled as INMV2 and MVS27 by molecular analysis. Genomic analysis of a field isolate revealed clusterization with a European clade but was more similar to Italian than East European isolates. Our findings underline that paratuberculosis should always be considered in zoological parks in which endangered species are hosted. Infection can be subclinical, and multiple combined testing techniques may be necessary.

Immunoinformatic-Based Prediction of Candidate Epitopes for the Diagnosis and Control of Paratuberculosis (Johne's Disease).

Paratuberculosis is an infectious disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). MAP is an intracellular pathogen with a possible zoonotic potential since it has been successfully isolated from the intestine and blood of Crohn's disease patients.Since no cure is available, after the detection of the disease, animal culling is the sole applicable containment strategy. However, the difficult detection of the disease in its subclinical form, facilitates its spread raising the need for the development of effective diagnosis and vaccination strategies. The prompt identification and isolation of the infected animals in the subclinical stage would prevent the spread of the infection.In the present study, an immunoinformatic approach has been used to investigate the immunogenic properties of 10 MAP proteins. These proteins were chosen according to a previously published immunoproteomics approach. For each previously-described immunoreactive protein, we predicted the epitopes capable of eliciting an immune response by binding both B-cells and/or class I MHC antigens. The retrieved peptide sequences were analyzed for their specificity and cross-reactivity. The final aim is to employ the discovered peptides sequences as a filtered library useful for early-stage diagnosis and/or to be used in novel multi-subunit or recombinant vaccine formulations.

Isolation of Mycobacterium avium Subsp. Paratuberculosis in the Feces and Tissue of Small Ruminants Using a Non-Automated Liquid Culture Method.

Paratuberculosis is a chronic disease of ruminants caused by Mycobacterium avium subsp. Paratuberculosis (MAP). Since isolation of MAP type I (S) is rarely reported in Italy, our research was aimed at isolating, by an inexpensive liquid culture manual method, this type of MAP isolates. At first, we used an ELISA to point out to serologically positive samples from five flocks. Secondly, we used a fecal direct IS900-qPCR on the ELISA positive samples, in order to detect shedder animals. Feces from IS900-qPCR positive samples were inoculated in solid and liquid culture media. IS900-qPCR was further used to test the growth of MAP isolates in liquid medium, which were further confirmed by f57-qPCR and submitted to typing by specific PCR in order to identify the MAP type. Twenty-eight samples (24 fecal and four tissutal samples) were processed by culture methods, resulting in the isolation of six type I MAP field isolates. Notably, no isolates were recovered by solid media, underlining the utility of this liquid method. Few data about this type of MAP are currently available in Italy, and further analyses should be carried out in order to study the origin and epidemiology of type I strains circulating in Italy.

Management of Mycobacterium avium subsp. paratuberculosis in dairy farms: Selection and evaluation of different DNA extraction methods from bovine and buffaloes milk and colostrum for the establishment of a safe colostrum farm bank.

The aim of this study was to develop and validate different innovative DNA extraction methods to detect Mycobacterium avium subsp. paratuberculosis (MAP) DNA from bovine and buffalo colostrum. Paratuberculosis is a chronic inflammatory infection of domestic and wild animals, especially ruminants, caused by MAP. The primary route of disease transmission is feces, but MAP can also be excreted in milk and colostrum. In 2015, the Italian Ministry of Health has issued a voluntary control plan of MAP in order to allow risk-based certification of bovine and buffaloes farms. In addition to the annual diagnostic screening and to the clinical surveillance of animals the plan includes the adoption of biosecurity and management measures to progressively mitigate the incidence of MAP. To achieve this goal it is crucial to ensure the accuracy of the methods used to detect the presence of MAP in bovine and buffaloes milk and colostrum, in order to: (1) support a "safe colostrum farm-bank" set-up and thus prevent the main within-farm MAP transmission route and (2) to allow the MAP-free certification of milk products for export purposes. To achieve these goals, seven different DNA extraction protocols were identified from bibliography, out of which three methods were finally selected after the adoption of an evaluation procedure aimed at assessing the efficiency of extraction of DNA, the purity of DNA and the adaptability of the DNA amplification: NucleoSpin® Food Kit (Macherey-Nagel), NucleoSpin® Food Kit (Macherey-Nagel) combined with the magnetic beads, and QIAamp Cador Pathogen Mini kit (QIAGEN). In particular, the NucleoSpin® Food Kit (Macherey-Nagel) and the QIAamp Cador Pathogen Mini kit (QIAGEN) were tested on bovine and buffalo colostrum, showing a LOD between 4 × 104 (2.6 × 106  cfu/ml) and 4.08 (26.7 cfu/ml) IS900 target copies and a LOD between 5.3 × 105 (4.1 × 106  cfu/ml) and 53 (4.1 × 103  cfu/ml) IS900 target copies, respectively.