Microbial contamination pathways in a poultry abattoir provided clues on the distribution and persistence of Arcobacter spp.

The consumption of contaminated poultry meat is a significant threat for public health, as it implicates in foodborne pathogen infections, such as those caused by Arcobacter. The mitigation of clinical cases requires the understanding of contamination pathways in each food process and the characterization of resident microbiota in the productive environments, so that targeted sanitizing procedures can be effectively implemented. Nowadays these investigations can benefit from the complementary and thoughtful use of culture- and omics-based analyses, although their application in situ is still limited. Therefore, the 16S-rRNA gene-based sequencing of total DNA and the targeted isolation of Arcobacter spp. through enrichment were performed to reconstruct the environmental contamination pathways within a poultry abattoir, as well as the dynamics and distribution of this emerging pathogen. To that scope, broiler's neck skin and caeca have been sampled during processing, while environmental swabs were collected from surfaces after cleaning and sanitizing. Metataxonomic survey highlighted a negligible impact of fecal contamination and a major role of broiler's skin in determining the composition of the resident abattoir microbiota. The introduction of Arcobacter spp. in the environment was mainly conveyed by this source rather than the intestinal content. Arcobacter butzleri represented one of the most abundant species and was extensively detected in the abattoir by both metataxonomic and enrichment methods, showing higher prevalence than other more thermophilic Campylobacterota. In particular, Arcobacter spp. was recovered viable in the plucking sector with high frequency, despite the adequacy of the sanitizing procedure.IMPORTANCEOur findings have emphasized the persistence of Arcobacter spp. in a modern poultry abattoir and its establishment as part of the resident microbiota in specific environmental niches. Although the responses provided here are not conclusive for the identification of the primary source of contamination, this biogeographic assessment underscores the importance of monitoring Arcobacter spp. from the early stages of the production chain with the integrative support of metataxonomic analysis. Through such combined detection approaches, the presence of this pathogen could be soon regarded as hallmark indicator of food safety and quality in poultry slaughtering.

Rapid hollow fiber-coating device for thin film composite membrane preparation.

Aligned with the recent trend and imperative to reduce separation layer thickness in gas separation membranes to the nanometer scale in order to raise permeance to levels that can render them competitive with respect to other gas separation technologies, a novel approach and device for fabricating defect-free composite hollow fiber (HF) membranes by dip-coating is described. The presented method avoids the fundamental drawbacks of state-of-the-art techniques for applying a thin gas separation layer onto a porous HF substrate, providing a safe but, at the same time, easily up-scalable way of producing HF membranes at a relatively high production rate. As a basic concept, hanging HF substrates are coated by allowing the coating solution to flow and drip along their external surface. The adaptability of this method, stemming from the array of available coating solutions (a plethora of dispersed nanofillers) and the multitude of substrate options, holds great promise for the fabrication of highly selective and defect-free composite HF membranes.

Detection of Sarcocystis hominis, Sarcocystis bovifelis, Sarcocystis cruzi, Sarcocystis hirsuta and Sarcocystis sigmoideus sp. nov. in carcasses affected by bovine eosinophilic myositis.

Bovine eosinophilic myositis is an inflammatory myopathy characterized by multiple focal or diffuse grey to green patches leading to condemnation of affected carcasses. Although its etiology is still uncertain, there is evidence that Sarcocystis species may play a role in the development of eosinophilic myositis. The goal of the present study was to identify Sarcocystis spp. in intralesional and extralesional tissues of condemned cattle carcasses, in order to evaluate the possible role of different bovine Sarcocystis spp. in the etiology of bovine eosinophilic myositis. Muscle samples (n = 100) of 26 affected carcasses were collected in Northern Italy. One to five samples with lesions and two aliquots of tissue without lesions were collected from each carcass; lesions were grossly categorized in green focal lesions and green diffuse patches. Genomic DNA was extracted and analyzed by multiplex-PCR targeting different Sarcocystis spp. Unidentified species were characterized morphologically (light microscopy, histology), ultrastructurally (scanning and transmission electron microscopy) and on the molecular level (complete 18S rRNA gene and partial cox1 gene sequencing). A bovine eosinophilic myositis prevalence of 0.017% was visually assessed by routine carcass inspection between 2014 and 2019 in Italy (184/1,108,150 slaughtered cattle). Out of 26 carcasses, 25 revealed the presence of at least one Sarcocystis species (96.2%). The presence of Sarcocystis spp. DNA was significantly more frequent in intralesional than in extralesional samples. Considering the different species, Sarcocystis bovifelis and Sarcocystis hominis were significantly more frequent in intralesional (41.7% and 50%, respectively) than in extralesional samples (1.9% and 15.4%, respectively), while there was no significant difference between the presence of Sarcocystis cruzi and Sarcocystis hirsuta in intralesional (27.1% and 2.1%, respectively) and extralesional (30.8% and 1.9%, respectively) samples. The presence of an unnamed Sarcocystis sp. showing thick-walled (3.7-5.4 µm) cysts with densely packed, flattened, undulating and narrow protrusions, which showed an S-shape in side view, was recorded in the diaphragm of two carcasses. Genomic DNA from individual sarcocysts isolated from the diaphragm was successfully amplified and further sequenced. Sequence comparison revealed <94.6% and 83.4% identity at 18S rRNA and cox1 genes, respectively, with other named Sarcocystis spp., while the phylogenetic analysis clearly separated the unnamed Sarcocystis sp. from the other Sarcocystis spp. using cattle as intermediate hosts. The present study contributes to the understanding of the importance of different Sarcocystis spp. in the pathogenesis of bovine eosinophilic myositis. The results emphasize the association of Sarcocystis hominis and Sarcocystis bovifelis with bovine eosinophilic myositis and highlight the presence of a new Sarcocystis sp. using cattle as intermediate hosts. The name Sarcocystis sigmoideus sp. nov. is proposed for the newly described Sarcocystis species.

Molecular differentiation of Sarcocystis miescheriana and Sarcocystis suihominis using a new multiplex PCR targeting the mtDNA cox1 gene in wild boars in southern Italy.

The increase of wild boar populations density and their meat consumption across Europe could expose humans to a plethora of foodborne diseases as sarcocystosis, caused by the zoonotic protozoan Sarcocystis suihominis. Humans become infected by eating raw or undercooked pig (Sus scrofa domesticus) containing S. suihominis sarcocysts. Despite this, to date very few data are available on the risk of infection by this parasite to wild boar (Sus scrofa) meat consumers. Thus, the present study aimed to assess the occurrence of Sarcocystis spp. in wild boars from southern Italy, applying both histology and a new multiplex PCR assay targeting the cox1 gene. Between 2019 and 2020, 997 muscle tissues (i.e., n = 269 oesophagus, n = 277 diaphragms, n = 298 hearts, n = 153 tongues) from 311 wild boars were collected and screened by a combined histological and molecular approach. Overall, 251 (80.7%) animals tested were positive for Sarcocystis spp., and S. miescheriana whose definitive hosts are canids, was the only molecularly identified species. A statistically significant difference (p < 0.05) in the prevalence of Sarcocystis infection was found according to the wild boar age and muscle tissue. Findings outlined the low zoonotic potential of infection to humans via wild boar meat consumption in Italy and the importance of the application of new molecular methods in distinguishing different Sarcocystis species.

Macroscopic sarcocystosis in a pig carcass from an Italian abattoir.

Different food-safety institutions, including the European Food Safety Authority, encourage monitoring and characterising Sarcocystis spp. in animals and foodstuffs; among meat-producing animals, domestic pigs (Sus scrofa domesticus) can host two different Sarcocystis spp., that is Sarcocystis miescheriana and the zoonotic Sarcocystis suihominis. Herein, we report for the first time the presence of macrocysts of Sarcocystis miescheriana in a domestic pig resulting in carcass condemnation. In North-West Italy, in June 2022 the carcass of a clinically healthy sow was condemned due to the detection of multifocal macroscopic whitish fusiform lesions. Affected muscle samples were submitted to histological and molecular analyses targeting the mtDNA cox1 and 18S rRNA genes. At gross examination and histology, well demarcated, oval or elongated macrocysts up to 8 mm in length characterized by a calcified central core surrounded by fibrosis were detected. The molecular amplification and sequencing of the cox1 mtDNA and 18S rRNA genes revealed the presence of Sarcocystis miescheriana DNA in all sampled macrocysts. Our study provides the first molecularly confirmed case of Sarcocystis miescheriana infection in a domestic pig in Italy. The present report highlights the need to increase data related to the occurrence and the prevalence of Sarcocystis spp. in meat-producing animals, and in wild and domestic pigs in particular, taking into account the zoonotic potential of Sarcocystis suihominis and the possible financial losses related to carcass discard due to macroscopic Sarcocystis spp. cysts.

Validation Parameters of the Magnetic Stirrer Method for Pooled Sample Digestion for Trichinella spp. in Horse Meat Based on Proficiency Tests Results.

Meat of horses may be infested with nematodes of the genus Trichinella spp. and can cause serious disease in humans. Rules for the carcasses sampling of species susceptible to Trichinella spp. infection and examination are laid down in Commission Regulation 1375/2015, where the magnetic stirrer method for pooled-sample digestion is recommended (Commission Regulation 1478/2020). All personnel involved in the examination should be properly trained and participate in quality control programs. Proficiency tests (PTs) play a key role in the quality verification process. This paper presents the results of PTs organized for 68 Polish laboratories in 2014-2019. Results were assessed qualitatively at three levels of sample contamination (0, 3, 5 larvae) and quantitatively at one level (5 larvae). The laboratories have achieved the average correct qualitative results 100%, 96.2% and 96.8% for the samples contaminated with 0, 3 and 5 larvae, respectively. In the quantitative evaluation, an average 94.1% of the reported results were correct. The data from PTs enabled us to define, for the first time, validation parameters of the digestion method for the horse meat matrix in a large-scale experiment including: specificity (100%), sensitivity (95.6%), accuracy (97.1%), the limit of detection (LOD) (1.14 ≈ 1) and the limit of quantification (LOQ) (3.42 ≈ 3).

Understanding the Effect of Ozone on Listeria monocytogenes and Resident Microbiota of Gorgonzola Cheese Surface: A Culturomic Approach.

The occurrence of Listeria monocytogenes on Gorgonzola cheese surface was reported by many authors, with risks arising from the translocation of the pathogen inside the product during cutting procedures. Among the novel antimicrobial strategies, ozone may represent a useful tool against L. monocytogenes contamination on Gorgonzola cheese rind. In this study, the effect of gaseous ozone (2 and 4 ppm for 10 min) on L. monocytogenes and resident microbiota of Gorgonzola cheese rind stored at 4 °C for 63 days was evaluated. A culturomic approach, based on the use of six media and identification of colonies by MALDI-TOF MS, was used to analyse variations of resident populations. The decrease of L. monocytogenes was less pronounced in ozonised rinds with final loads of ~1 log CFU/g higher than controls. This behaviour coincided with a lower maximum population density of lactobacilli in treated samples at day 28. No significant differences were detected for the other microbial determinations and resident microbiota composition among treated and control samples. The dominant genera were Candida, Carnobacterium, Staphylococcus, Penicillium, Saccharomyces, Aerococcus, Yarrowia, and Enterococcus. Based on our results, ozone was ineffective against L. monocytogenes contamination on Gorgonzola rinds. The higher final L. monocytogenes loads in treated samples could be associated with a suppressive effect of ozone on lactobacilli, since these are antagonists of L. monocytogenes. Our outcomes suggest the potential use of culturomics to study the ecosystems of complex matrices, such as the surface of mould and blue-veined cheeses.

Shotgun metagenomic sequencing of bulk tank milk filters reveals the role of Moraxellaceae and Enterobacteriaceae as carriers of antimicrobial resistance genes.

In the present context of growing antimicrobial resistance (AMR) concern, understanding the distribution of AMR determinants in food matrices such as milk is crucial to protect consumers and maintain high food safety standards. Herein, the resistome of different dairy farms was investigated through a shotgun metagenomic sequencing approach, taking advantage of in-line milk filters as promising tools. The application of both the reads-based and the assembly-based approaches has allowed the identification of numerous AMR determinants, enabling a comprehensive resolution of the resistome. Notably most of the species harboring AMR genes were predicted to be Gram-negative genera, namely Enterobacter, Acinetobacter, Escherichia, and Pseudomonas, pointing out the role of these bacteria as reservoirs of AMR determinants. In this context, the use of de novo assembly has allowed a more holistic AMR detection strategy, while the reads-based approach has enabled the detection of AMR genes from low abundance bacteria, usually undetectable by assembly-based methods. The application of both reads-based and assembly-based approaches, despite being computationally demanding, has facilitated the comprehensive characterization of a food chain resistome, while also allowing the construction of complete metagenome assembled genomes and the investigation of mobile genetic elements. Our findings suggest that milk filters can successfully be used to investigate the resistome of bulk tank milk through the application of the shotgun metagenomic sequencing. In accordance with our results, raw milk can be considered a source of AMR bacteria and genes; this points out the importance of properly informing food business operators about the risk associated with poor hygiene practices in the dairy production environment and consumers of the potential microbial food safety risks derived from raw milk products consumption. Translating these findings as risk assessment outputs heralds the next generation of food safety controls.

Effect of gaseous ozone treatment on biofilm of dairy-isolated Pseudomonas spp. strains.

Microbial biofilms existing in food industries have been implicated as important contamination sources of spoilage and pathogenic microorganisms in the finished products. Among the innovative strategies proposed to contrast biofilms in food environments, ozone is recognised as an environmentally friendly technology but there are few studies about its effect against bacterial biofilms. The objective of this study was to evaluate the effect of gaseous ozone (50 ppm for 6 h) in inhibition and eradication of biofilm formed by twenty-one dairyisolated Pseudomonas spp. strains. Before ozone treatments, all isolates were screened for biofilm formation according to a previously described method. Strains were then divided in four groups: weak, weak/moderate, moderate/strong, and strong biofilm producers based on the biofilm biomass value of each isolate determined using the optical density (OD - 595 nm). Inhibition treatment was effective on the strain (C1) belonging to the weak producers' group, on all strains classified as weak/moderate producers, on two strains (C8 and C12) belonging to the group of moderate/strong producers and on one strain (C13) classified as strong producer. Conversely, eradication treatments were ineffective on all strains tested, except for the strain C4 which reduced its biofilm-forming abilities after exposure to ozone gas. In conclusion, gaseous ozone may be used to enhance existing sanitation protocols in food processing environments, but its application alone not seems sufficient to contrast Pseudomonas spp. established biofilms.

Comparison Between Full-Length 16S rRNA Metabarcoding and Whole Metagenome Sequencing Suggests the Use of Either Is Suitable for Large-Scale Microbiome Studies.

Since the number of studies of the microbial communities related to food and food-associated matrices almost completely reliant on next-generation sequencing techniques is rising, evaluations of these high-throughput methods are critical. Currently, the two most used sequencing methods to profile the microbiota of complex samples, including food and food-related matrices, are the 16S ribosomal RNA (rRNA) metabarcoding and the whole metagenome sequencing (WMS), both of which are powerful tools for the monitoring of foodborne pathogens and the investigation of the microbiome. Herein, the microbial profiles of 20 bulk tank milk filters from different dairy farms were investigated using both the full-length 16S (FL-16S) rRNA metabarcoding, a third-generation sequencing method whose application in food and food-related matrices is yet in its infancy, and the WMS, to evaluate the correlation and the reliability of these two methods to explore the microbiome of food-related matrices. Metabarcoding and metagenomic data were generated on a MinION platform (Oxford Nanopore Technologies) and on a Illumina NovaSeq 6000 platform, respectively. Our findings support the greater resolution of WMS in terms of both increased detection of bacterial taxa and enhanced detection of diversity; in contrast, FL-16S rRNA metabarcoding has proven to be a promising, less expensive, and more practical tool to profile most abundant taxa. The significant correlation of the two technologies both in terms of taxa diversity and richness, together with the similar profiles defined for both highly abundant taxa and core microbiomes, including Acinetobacter, Bacillus, and Escherichia genera, highlights the possible application of both methods for different purposes. This study allowed the first comparison of FL-16S rRNA sequencing and WMS to investigate the microbial composition of a food-related matrix, pointing out the advantageous use of FL-16S rRNA to identify dominant microorganisms and the superior power of WMS for the taxonomic detection of low abundant microorganisms and to perform functional analysis of the microbial communities.

Biofilm formation and genomic features of Listeria monocytogenes strains isolated from meat and dairy industries located in Piedmont (Italy).

Listeria monocytogenes is considered a major challenge for the food industry as it can persist for long periods in food processing plants by forming biofilms. The aims of this study were: i) to assess the biofilm producing ability of 57 Listeria monocytogenes isolates previously subjected to whole-genome sequencing (WGS); ii) to compare the levels of biofilm formation with the presence or absence of biofilm associated genes. To determine the presence or absence of a known set of biofilm associated genes, a comparative genomic analysis was performed on each strain. Among Listeria monocytogenes isolates, 58 %, 38.5 % and 3.5 % exhibited weak, moderate or strong biofilm production, respectively. No difference in biofilm production was observed between food and environmental isolates. The percentage of Listeria monocytogenes strains isolated from meat products (57 %) classified as moderate or strong biofilm producers was higher than the percentage obtained for strains isolated from dairy products (28 %). The presence of the Stress Survival Islet 1, the arsD stress gene and the truncated inlA protein was significantly associated with increased levels of biofilm. Combining biofilm phenotype with molecular and genotyping data may provide the opportunity to better understand the relationship between genes linked to biofilm formation in Listeria monocytogenes.

Correction: Rubiola et al. Risk Factors for Bovine Cysticercosis in North-West Italy: A Multi-Year Case-Control Study. Animals 2021, 11, 3049.

Error in Institutional Review Board Statement [...].

PHA Production from Cheese Whey and "Scotta": Comparison between a Consortium and a Pure Culture of Leuconostoc mesenteroides.

It is urgent to expand the market of biodegradable alternatives to oil-derived plastics owing to (i) increasingly limited oil availability/accessibility, and (ii) the dramatic impact of traditional plastics on aquatic life, the food chain, all Earth ecosystems, and ultimately, human health. Polyhydroxyalkanoates (PHAs) are promising biodegradable polymers that can be obtained through microbial fermentation of agro-industrial byproducts, e.g., milk and cheese whey. Here, the PHA-accumulating efficiency of a mixed microbial culture (MMC, derived from activated sludges) grown on dairy byproducts (cheese and scotta whey) was measured. Bioreactor tests featuring temperature and pH control showed that both scotta and pre-treated Toma cheese whey could be used for PHA production by MMC, although scotta cheese whey supported higher PHA yield and productivity. The advantages of open MMCs include their plasticity and versatility to fast changing conditions; furthermore, no growth-medium sterilization is needed prior to fermentation. However, the use of pure cultures of efficient PHA producers may support better metabolic performances. Therefore, PHA-producing strains were isolated from a MMC, leading to the satisfactory identification of two bacterial strains, Citrobacter freundii and Leuconostoc spp., whose ability to accumulate PHAs in synthetic media was confirmed. A more detailed investigation by mass spectrometry revealed that the strain was L. mesenteroides. Although the validation of L. mesenteroides potential to produce PHA through fermentation of agro-industrial byproducts requires further investigations, this is the first study reporting PHA production with the Leuconostoc genus.

Risk Factors for Bovine Cysticercosis in North-West Italy: A Multi-Year Case-Control Study.

Taenia saginata is the causative agent of bovine cysticercosis, a zoonotic parasitic disease with a worldwide distribution. Bovine cysticercosis is considered to be an important food safety issue responsible for human taeniasis and a major economic concern since infected carcasses undergo condemnation, freezing and downgrading. The aim of the current investigation was to assess the presence of farm-level risk factors for bovine cysticercosis in an endemic area in North-West Italy. A questionnaire was designed to collect information relating to several farm structural features, as well as management practices, environmental characteristics and attitudes of farmers. The questionnaire was administered in two separate time intervals by direct interview to previously selected case and control farms. Overall, 32 case farms and 131 control farms were included between 2005 and 2011 and 50 case farms and 192 control farms were included between 2014 and 2020. The present survey showed a significant association between the detection of bovine cysticercosis cases at slaughter and farm proximity to picnic spots, closeness of wastewater treatment plant effluents, loose-housing systems and presence of employees along with the family members, highlighting the need for targeted awareness raising policies.

A Survey on One Health Approach in Colombia and Some Latin American Countries: From a Fragmented Health Organization to an Integrated Health Response to Global Challenges.

The "One Health" (OH) approach has been recognized by world health authorities such as FAO/OIE/WHO, advocating for effective, multi-sectoral, and transdisciplinary collaboration. However, there is a lack of published evidence of the awareness of the OH concept in Colombia and other countries in the Latin American Region. In order to explore existing collaboration amongst the animal health, human-public health, environmental health sectors, and to describe the perception, knowledge, and barriers on OH in Colombia and other countries of Latin America, an online questionnaire-based survey was distributed among key professionals representing the three OH pillars (August 2018-August 2020). Overall, 76 key respondents from 13 countries (Colombia, México, Chile, Brazil, Argentina, Bolivia, Costa Rica, Ecuador, Perú, Guatemala, Nicaragua Uruguay, and Venezuela) completed the questionnaire. Respondents worked in institutions of animal (59%), public (20%), human (7%), and environmental health (7%); they mainly belonged to higher academic institutions (59%), followed by ministries (11%), and research organizations (9%). Most participants (92%) were familiar with the OH term and 68% were aware of the formal cooperation among sectors in their countries, mostly on zoonoses; in 46% of the cases, such connections were established in the last 5 years. The main reported limiting factors to intersectorality were the lack of commitment of policy-makers, resources, and budget for OH (38%) and the "siloed approach" of sectors and disciplines (34%). Respondents ranked a median score of 3.0 (1-5 scoring) in how good OH activities are implemented in their countries, and a median score of 2.0 in the citizen awareness on OH as regards their countries. The most important OH issues were identified in vector-borne diseases, rabies, wrong and/or improper use of antimicrobials, emerging viral diseases, food-borne diseases, neglected parasitic diseases, deforestation, and ecosystem fragmentation. Although there is a high-perceived importance on conjoint cooperation, OH implementation, and operationalization remain weak, and the environmental component is not well-integrated. We consider that integration and implementation of the OH Approach can support countries to improve their health policies and health governance as well as to advocate the social, economic, and environmental sustainability of the Region.

Effect of Gaseous Ozone on Listeria monocytogenes Planktonic Cells and Biofilm: An In Vitro Study.

Among food-borne pathogens, Listeria monocytogenes continues to pose concerns to food business operators due to its capacity to form biofilm in processing environments. Ozone may be an eco-friendly technology to control microbial contaminations, but data concerning its effect on Listeria monocytogenes biofilm are still limited. In this study, the effect of gaseous ozone at 50 ppm on planktonic cells and biofilm of reference and food-related Listeria monocytogenes strains was evaluated. Ozone caused a reduction in microbial loads of 3.7 ± 0.4 and 3.9 ± 0.4 Log10 CFU/mL after 10 and 30 min, respectively. A complete inactivation of planktonic cells after 6 h of treatment was observed. Biofilm inhibition and eradication treatments (50 ppm, 6 h) resulted in a significant decrease of the biofilm biomass for 59% of the strains tested, whilst a slight dampening of live cell loads in the biofilm state was observed. In conclusion, gaseous ozone is not sufficient to completely counteract Listeria monocytogenes biofilm, but it may be useful as an additional tool to contrast Listeria monocytogenes free-living cells and to improve the existing sanitization procedures in food processing environments.

Molecular detection of cattle Sarcocystis spp. in North-West Italy highlights their association with bovine eosinophilic myositis.

BACKGROUND: Cattle are intermediate hosts of six Sarcocystis species, among which Sarcocystis hominis and Sarcocystis heydorni can infect humans through the consumption of raw or undercooked meat. In addition to the zoonotic potential, there is increasing interest in these protozoa because of the evidence supporting the role of Sarcocystis spp. in the occurrence of bovine eosinophilic myositis (BEM), a specific inflammatory myopathy which leads to carcass condemnation and considerable economic losses. Actually, all the prevalence studies carried out on cattle in Italy have been based on either morphological or 18S rDNA-based molecular techniques, most likely leading to misidentification of closely related species. Therefore, there is a strong need for new data on the prevalence of the different Sarcocystis spp. in cattle in Italy and their association with bovine eosinophilic myositis. METHODS: To reach our aim, individual striated muscle samples from BEM condemned carcasses (N = 54) and diaphragm muscle samples from randomly sampled carcasses (N = 59) were obtained from Northwest Italy slaughterhouses. Genomic DNA was extracted and analyzed by multiplex-PCR targeting 18S rDNA and cox1 genes. PCR products amplified using the genus-specific primer set in absence of the specific fragment for S. hirsuta, S. cruzi, S. hominis or S. bovifelis were sequenced to achieve species identification. RESULTS: Sarcocystis DNA was detected in 67.8% of the samples from slaughter cattle and in 90.7% of the samples from BEM condemned carcasses. S. cruzi was identified as the most prevalent species in slaughter cattle (61%), followed by S. bovifelis (10.2%), S. hominis (8.5%) and S. hirsuta (1.7%). Notably, among the different Sarcocystis spp. detected, the presence of S. bovifelis and S. hominis was significantly higher in samples isolated from BEM condemned carcasses (46.3% and 40.7% respectively), while there was no statistically significant difference between the presence of S. cruzi or S. hirsuta in BEM condemned carcasses (42.6% and 1.8%, respectively) and randomly sampled carcasses. Furthermore, DNA sequence analysis revealed the presence of a putative new species in two carcasses. CONCLUSIONS: Our study contributes to updating the data on the prevalence of the different Sarcocystis spp. in cattle in Italy, highlighting the presence of three Sarcocystis spp., S. cruzi, S. hominis and S. bovifelis, in BEM lesions and allowing us to speculate on the possible role of S. hominis and S. bovifelis as the major sarcosporidian species involved in bovine eosinophilic myositis.

A Survey on One Health Perception and Experiences in Europe and Neighboring Areas.

A questionnaire survey was promoted under the COST Actions "Network for Evaluation of One Health-NEOH" and "European Network for Neglected Vectors and Vector-Borne Infections-EURNEGVEC", from June 2016 to April 2017, to collect information on the existence of One Health (OH) collaboration and implementation of OH initiatives in 37 EU COST Countries. The questionnaire was to be answered by key respondents representing the three major OH components: (i). Animal Health; (ii). Human Health/Public Health; (iii). Environmental Health. A target respondent rate of nine respondents/country was aimed for, representing the following categories: (i). ministries; (ii). academia-research; (iii). private sector and NGOs, associations and scientific societies. The questionnaire, composed of 27 questions organized in six sections, was circulated to target respondents by Committee Members of the two COST actions. A total of 171 respondents from 34 countries completed the questionnaire, mainly belonging to academic and research institutions (55.5%), and to Animal Health/Animal Science fields (53.8%). Although the majority (57.9%) declared they had heard about OH, few respondents (10.7%) provided a complete definition. The "human" and "animal" elements prevailed over other key elements of OH definition (ecosystem, intersectoral, transdisciplinary, holistic, collaboration). Overall, 62.6% respondents declared to take part in OH initiatives. Antimicrobial resistance, avian influenza and environmental pollution were cited as the top three OH issues over the past 5 years. Limitations and gaps in intersectoral collaboration included communication and organizational problems resulting in poor networking, differing priorities and a lack of understanding between sectors. Regarding control and monitoring of zoonotic diseases, respondents from different sectors preferentially selected their own directorates/ministries while actually in most countries both Ministry of Health and Ministry of Agriculture are engaged. According to respondents, the level of awareness of OH amongst the general public is limited. Similarly, a dearth of opportunities of collaborations at different institutional and/or professional levels was described. Our survey provided an overview of how respondents in COST countries perceived and experienced OH and current limits to OH implementation. Identifying how initiatives are currently working and knowing the promoting and hindering factors allowed suggesting strategies to promote efficiency and effectiveness of OH implementation in the future.

Molecular identification of four Sarcocystis species in cattle from Lithuania, including S. hominis, and development of a rapid molecular detection method.

BACKGROUND: Six Sarcocystis species are known to use cattle (Bos taurus) as the intermediate host, two of which, S. hominis and S. heydorni, are zoonotic. There is a need for a method that will enable rapid identification of the Sarcocystis species in cattle. METHODS: The diaphragm muscles of 102 cattle from Lithuania were examined for the presence of Sarcocystis spp., using two different methods for species identification. Individual sarcocysts were isolated from squash preparations of the diaphragm muscle under the light microscope, followed by genetic characterisation of excised cysts using sequence analysis of the 18S rRNA (18S rRNA) and cytochrome c oxidase subunit I (cox1) genes. The same cattle muscle samples were digested and species-specific PCR analyses targeting cox1 were developed to identify the Sarcocystis isolates to the species level. RESULTS: Under the light microscope, sarcocysts were detected in 87.3% of animals, and Sarcocystis infection was verified in all digested samples. Three species, namely S. cruzi (n = 20), S. bovifelis (n = 23) and S. hirsuta (n = 6), were identified by DNA sequence analysis of isolated sarcocysts. Based on sequence analysis of cox1, the level of genetic variability depended on Sarcocystis species and geographical location. Four Sarcocystis species, S. cruzi (96.1%), S. bovifelis (71.6%), S. hirsuta (30.4%) and S. hominis (13.7%), were confirmed in the digested samples. In individual samples, the most common finding was two species of Sarcocystis (44.1%), followed by three species (26.5%), a single species (24.5%) and four species (4.9%). CONCLUSIONS: Although examination of tissue preparations under the light microscrope did not detect any sarcocysts belonging to S. hominis, this species was identified in the digested samples subjected to a cox1-specific PCR analysis. These results demonstrate the need for effective molecular diagnosis techniques to detect Sarcocystis spp., which may be present at a lower prevalence and not detectable among the limited number of sarcocysts identified individually under the light microscope.

Analysis of the Antibiotic Resistance Profiles in Methicillin-Sensitive S. aureus Pathotypes Isolated on a Commercial Rabbit Farm in Italy.

The breeding of meat rabbits is an important sector in the livestock industry in Italy. The focus of this study was to describe the antibiotic resistance profile distribution among the Methicillin-sensitive Staphylococcus aureus isolated in a rabbit farm. From 400 animals of different ages and three farm workers, 96 randomly selected strains isolated from various anatomical sites and lesions were analysed. According to spa typing and the resistance profiles towards veterinary and human antibiotics, 26 pathotypes were identified. The highest resistance was observed against Tetracyclines (92.3%) and Macrolides (80.8%), while almost all were susceptible to Penicillins, according to the limited use of ß-lactams on the farm. In total, 92.3% of pathotypes were multidrug resistant (MDRs). Two MDR pathotypes belonging to the t2802 spa type were isolated from both farmers and rabbits. Age categories harboured significantly different pathotypes (p = 0.019), while no association was found between pathotypes and lesions (p = 0.128) or sampling sites (p = 0.491). The antibiotic resistance was observed to increase with the time spent in the farm environment (age category). The selective pressure exerted by antibiotic use acted by giving advantage to more resistant strains rather than by lowering susceptibility to various drug categories within strains.