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

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

A comparative study of the in vitro activity of iodopropynyl butylcarbamate and amphotericin B against Prototheca spp. isolates from European dairy herds.

ABSTRACT The objective of this study was to assess the in vitro effect of iodopropynyl butylcarbamate (IPBC) and amphotericin B (AMB) on Prototheca zopfii genotype 2 and Prototheca blaschkeae isolates recovered from dairy herds of Belgium, France, Italy, Germany, and Poland. The combination of IPBC with AMB on Prototheca isolates and toxicity of IPBC to the bovine mammary epithelial cells were also evaluated. The in vitro activity of IPBC and AMB against 96 isolates of P. zopfii genotype 2 and 42 isolates of P. blaschkeae was performed. Minimum inhibitory concentrations (MIC) and minimum algicidal concentrations (MAC) of IPBC and AMB were determined. To determine any synergistic, additive, or antagonistic effect of the combination of IPBC and AMB, 2-dimensional checkerboard combination tests were also performed to calculate fractional inhibitory concentrations. Cytotoxicity analysis of IPBC to the bovine mammary epithelial cell line was performed using a 3-(4,5-dimethyl-2-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The MIC for 50 and 90% of isolates (MIC 50 and MIC 90 , respectively) for IPBC were 4 and 8 mg/L versus 0.5 and 1 mg/L for AMB, respectively. The MIC profiles differed between P. zopfii genotype 2 and P. blaschkeae , with the latter species being more susceptible to both compounds. The MIC 50 and MIC 90 of IPBC were 4 and 8 mg/L for P. zopfii genotype 2 and 1 and 2 mg/L for P. blaschkeae , respectively. The MIC 50 and MIC 90 of AMB were both 1 mg/L for P. zopfii genotype 2 and 0.25 and 1 mg/L for P. blaschkeae , respectively. Both IPBC and AMB exhibited the ability to kill Prototheca spp. The MAC for 90% of isolates of IPBC was twice the MIC 90 , whereas an 8-fold increase of the MIC 90 was algicidal in the case of AMB. Overall, the combined use of IPBC and AMB exhibited an increased algicidal effect, albeit the fractional inhibitory concentration index showed synergistic activity only against 3 P. zopfii genotype 2 isolates. For all the remaining isolates (87.5%), this combination produced only an additive effect. The MTT assay results showed both IPBC and AMB, at the concentrations employed in the study, to be nontoxic to the epithelial mammary gland cells (cell viability >90%). Notably, only IPBC at the highest concentration (i.e., 8 mg/L) exerted a slight cytotoxic effect on the cell line tested (mean cell viability: 88.54 ± 3.88 and 90.66 ± 3.0, after 2 and 4 h of MTT treatment, respectively). The anti- Prototheca activity of IPBC was here demonstrated for the first time. In addition, the combined use of IPBC with AMB enhanced each other's effect, creating an additive rather than synergistic interaction. Both agents, used at concentrations corresponding to MIC values against Prototheca spp., showed no toxic effect for the mammary epithelial cells. In conclusion, IPBC, used either alone or in combination with AMB, can be considered a promising option in the treatment armamentarium for protothecal mastitis in dairy cows

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

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

"A Braccia Aperte". Une passerelle piétonne et une station maritime connectées aux infrastructures du MOSE de l’entrée de la lagune "Bocca Di Lido" à Venise

Suite aux nombreux dégâts causés par les marées, la commune de Venise a décidé de réagir en lançant un programme de protection et de sauvegarde de sa lagune. Le projet MOSE [MOdulo Sperimentale Elettromeccanico] a été conçu dans le but de protéger la ville des marées hautes. Des portes sous-marines ont été installées aux trois embouchures de la lagune nécessitant des moyens et des infrastructures très importants. L'ampleur des efforts mis en œuvre est particulièrement visible à l'embouchure Bocca di Lido où la construction d'une île artificielle a été nécessaire. Le but de ce projet est d'utiliser ces infrastructures et de s'y accrocher pour nouer un dialogue entre le MOSE et le grand public. L'île servira également d'arrêt pour les bateaux de croisières qui n’ont désormais plus le droit d’entrer dans la lagune. Le premier programme du projet est une passerelle piétonne permettant de lier le Lido à la Punta Sabbioni. Cette passerelle survole toute l'embouchure de la lagune, se développe en pente douce et permet à quiconque de s'y promener. En passant sur l'île, le pont se mue en balcon, sommet d'un grand gradin permettant d'observer le paysage. Ce gradin fonctionne comme une peau que l'on aurait décollée pour permettre aux visiteurs de découvrir les infrastructures du MOSE. Le front du gradin est composé d'une station maritime et autres services destinés aux visiteurs, des bureaux et des laboratoires de l'ISMAR [Istituto delle Scienze Marine] ainsi que de tous les services nécessaires au personnel travaillant sur place

Shiga toxin-producing Escherichia coli in meat and vegetable products in Emilia Romagna Region, years 2012-2013

In 2012-2013 Emilia-Romagna Region introduced a monitoring plan for Shiga toxin-producing <em>Escherichia coli</em> (STEC) in foodstuff. Six hundred eighty-nine meat samples and 273 fruit and vegetable products were analyzed according to ISOTS13136. Pre-enriched samples were tested by multiplex real time PCR targeting the virulence genes <em>eae</em>, <em>stx1</em> and <em>stx2</em>. <em>Stx2</em> positive samples were investigated for the presence of serogroup O104 associated gene. O103, O111, O145, O157, O26 associated genes were tested on samples positive for <em>stx</em> in association with <em>eae</em> gene. Isolation of E. coli strains was attempted from samples positive for serogroup-associated genes. Thirtyfour meat products (4.9%) resulted positive for <em>stx1</em> and/or <em>stx2</em> genes and 46 (6.7%) for <em>stx1</em> and/or <em>stx2</em> genes in association with <em>eae</em> gene. Forty-five (6.5%) samples resulted positive at least at one serogroup. Serogroup O103, O104, O111, O145, O157 and O26 genes were detected respectively in 1.3, 0.3, 0.1, 3.9, 2.9 and 2.5% samples; 0.6% samples resulted positive for STEC isolation (2 <em>E. coli</em> O103 and 2 <em>E. coli</em> O157). It is worth noting that STEC virulence genes were detected at high frequency (19%) in fresh pork meat sausages. Four (1.5%) vegetable samples were positive for <em>stx1</em> and/or <em>stx2</em> genes and 1 (0.4%) for <em>stx1</em> and/or <em>stx2</em> genes in association with <em>eae</em> gene; none resulted positive for the tested serogroups. Only a low number of samples positive by molecular methods were confirmed by cultural isolation. It is therefore of the uttermost importance for appropriate risk management, to be fully aware of the meaning of the analytical result