SARS-CoV-2 in Animal Companions: A Serosurvey in Three Regions of Southern Italy.

Several animal species have been found to be susceptible to SARS-CoV-2 infection. The occurrence of infection in dogs and cats living in close contact with owners deserves particular attention from public health authorities in a One Health approach. In this study, we conducted serological screening to identify SARS-CoV-2 exposure in the sera from dogs and cats in three regions of southern Italy sampled during the years 2021 and 2022. We collected 100 serum samples in 2021 (89 from dogs and 11 from cats) and 640 in 2022 (577 from dogs and 63 from cats). Overall, the ELISA positivity rate was found to be 2.7% (20/740), with higher seroprevalence in dogs. Serum neutralization tests confirmed positivity only in two samples collected from dogs, and the assays, performed with serologically distinct SARS-CoV-2 variants, showed variant-specific positivity. This paper shows that monitoring SARS-CoV-2 exposure in animals might be affected by the viral antigenic evolution, which requires continuous updates to the serological tests used. Serological surveys are useful in understanding the true extent of exposure occurring in specific animal populations, not suffering the same limitations as molecular tests, and could help in identifying the infecting virus if tests able to characterize the immune response are used. The use of variant-specific validated serological methods should always be considered in serosurvey studies in order to determine the real impact of emerging variants on animal populations and its implications for veterinary and human health, as well as to identify potential reservoirs of the virus and its evolutionary changes.

High Genetic Diversity and Virulence Potential in Bacillus cereus sensu lato Isolated from Milk and Cheeses in Apulia Region, Southern Italy.

The Bacillus cereus group includes species that act as food-borne pathogens causing diarrheal and emetic symptoms. They are widely distributed and can be found in various foods. In this study, out of 550 samples of milk and cheeses, 139 (25.3%) were found to be contaminated by B. cereus sensu lato (s.l.). One isolate per positive sample was characterized by Multilocus Sequence Typing (MLST) and for the presence of ten virulence genes. Based on MLST, all isolates were classified into 73 different sequence types (STs), of which 12 isolates were assigned to new STs. Virulence genes detection revealed that 90% and 61% of the isolates harboured the nheABC and the hblCDA gene cluster, respectively. Ninety-four percent of the isolates harboured the enterotoxin genes entS and entFM; 8% of the isolates possessed the ces gene. Thirty-eight different genetic profiles were identified, suggesting a high genetic diversity. Our study clearly shows the widespread diffusion of potentially toxigenic isolates of B. cereus s.l. in milk and cheeses in the Apulia region highlighting the need to adopt GMP and HACCP procedures along every step of the milk and cheese production chain in order to reduce the public health risk linked to the consumption of foods contaminated by B. cereus s.l.

Isolation and characterization of Yersinia enterocolitica from foods in Apulia and Basilicata regions (Italy) by conventional and modern methods.

Yersiniosis is the third most reported food-borne zoonosis in Europe. The aim of the present study was to perform the search for Yersinia enterocolitica in food samples collected from Apulia and Basilicata regions (Southern Italy) and to characterize any isolates by classical and modern analytical methods. A total of 130 samples were analyzed between July 2018 and July 2019: most of them were raw milk and dairy products made from it. Furthermore, 8 out of 130 samples were individual milk samples collected from bovines reared in a Brucella-free farm which showed false positive serological reaction for brucellosis due to the presence of pathogenic Y. enterocolitica O:9 biotype 2 in faeces. The Real Time PCR targeting the ail gene and the culture method were performed to detect pathogenic Y. enterocolitica. Isolates were subjected to API 20E (Biomerieux) and MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization Time-of-Flight) for species identification. All samples were negative for the ail gene. The culture method allowed to isolate suspicious colonies from 28 samples. The API 20E system and the MALDI-TOF MS technique identified 20 Y. enterocolitica and 1 Y. intermedia in a concordant way. The remaining 7 strains were all identified as Y. enterocolitica by the API 20E system, while the MALDI-TOF MS recognized 4 Y. intermedia, 1 Y. bercovieri and 2 Y. massiliensis. Genotypic characterization of the discordant strains was performed by rMLST and it confirmed the MALDI-TOF MS' results. Only non-pathogenic Y. enterocolitica biotype 1A strains were found, although with a non-negligible prevalence (P = 0.15 with CI 95% = ± 0.06). This study indicates a poor circulation of pathogenic Y. enterocolitica in food products made and marketed in the investigated areas. However, the small number of samples, insufficient for some food categories such as meat and vegetable, does not allow to exclude the presence of pathogenic strains at all.

Multi-locus sequence typing and virulence profile in Bacillus cereus sensu lato strains isolated from dairy products.

Members of Bacillus cereus group are important food contaminants and they are of relevant interest in food safety and public heath due to their ability to cause two distinct forms of food poisoning, emetic and diarrhoeal syndrome. In the present study, 90 strains of B. cereus isolated from dairy products, have been typed using Multilocus Sequence Typing (MLST) analysis and investigated for the occurrence of 10 enterotoxigenic genes (hblA, hblC, hblD, nheA, nheB, nheC, cytK, entFM, entS and bceT) and one emetogenic gene (ces), to determine their genetic diversity. A total of 58 sequence types were identified and among these 17 were signalled as new profiles. Among the virulence genes, the majority of our strains carried the entS (92%), entFM (86%), nhe (82%) and cytK (72%) genes. All remaining genes were identified in at least one strain with different prevalence, stressing the genetic diversity, how even the different grade of pathogenicity of B. cereus isolated from dairy products.

Antimicrobial Susceptibility and Multilocus Sequence Typing of Listeria monocytogenes Isolated Over 11 Years from Food, Humans, and the Environment in Italy.

Due to the increasing number of studies reporting the detection of antimicrobial-resistant isolates of Listeria monocytogenes, we sought to determine the antimicrobial susceptibility of L. monocytogenes isolates collected in Italy and find potential correlations to their serotypes and multilocus sequence types (MLST). The antimicrobial susceptibility of 317 L. monocytogenes isolates collected from food, humans, and the environment from 1998 to 2009 was assessed by minimum inhibitory concentration (MIC). Serotyping and MLST was also performed on all isolates. Potential correlations among antimicrobial resistance profiles, serotyping, and MLST were statistically evaluated. Twenty-four percent of L. monocytogenes isolates were resistant to oxacillin, 28.7% intermediate to clindamycin, and 24.3% to ciprofloxacin. The majority of isolates with elevated MIC to oxacillin was of environmental origin and belonged to serotype 4b/4e and ST2. Isolates with intermediate MIC values to clindamycin and ciprofloxacin were mostly of food and human origin and belonged to serotype 4b/4e and ST9. Regarding the time frame of isolate collection, comparing the last 3 years (2007-2009) to previous years (1998-2006), an increase was observed in the percentage of resistant and intermediate isolates per year. This trend strongly suggests the need for increasing attention on the prevalence of antimicrobial resistance in L. monocytogenes in Italy. To predict future resistance trends, the monitoring of clinical intermediate resistance might represent a useful tool especially for antibiotics associated to multiple-step mechanisms of acquired resistance. A specific focus should be addressed to antimicrobial-resistant isolates of serotype 4b, repeatedly associated with food-borne outbreaks.

MRSA in swine, farmers and abattoir workers in Southern Italy.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important medical issue, since it causes serious and sometimes fatal infections in humans. Intensively reared swine may serve as reservoirs for MRSA that can infect swine workers, and also consumers (via contaminated meat). In this study, MRSA strains were isolated from 55 of the 85 (64.7%) intensive pig farms surveyed, and prevalence was greater on pig fattening farms than on breeding farms. In addition, we included in the study 63 foreign pigs imported for slaughter. Overall, the prevalence of MRSA in the 418 sampled swine was 59.1%; 12 genotypes were identified among the isolates; ST398 (96.4%) was most prevalent, followed by ST97 (2%), ST9 (0.8%) and ST1 (0.8%). MRSA isolates were also detected in 26 (17.3%) of the 150 operators included in the study; the genotypes detected were ST398 (85%), ST9 (7.6%), ST5 (3.8%) and ST1 (3.8%). All the strains were pvl negative and pia positive. Both swine and human strains displayed a multi-resistance pattern, and almost all were resistant to tetracycline. The results obtained in this study confirm the high prevalence of MRSA in swine reared and slaughtered in Italy, and underline the public health risk linked to the spread of antimicrobial-resistant Staphylococcus aureus among intensively reared pigs.

Arcobacter spp. in bovine milk: An emerging pathogen with potential zoonotic risk.

The aim of the present study was to assess the prevalence and genetic characteristics of Arcobacter spp. in bovine bulk tank milk produced in Apulia Region (Italy). Samples collected from 396 dairy farms, after enrichment in a selective broth, were subjected to an Arcobacter genus - specific Real Time PCR. Positive broths, previously filtered, were seeded on Karmali, MCCD and Columbia Blood Agar plates; presumptive Arcobacter spp. colonies were identified using an amplification and sequencing method and then characterized by Multi-Locus Sequence Typing (MLST). Prevalence of Arcobacter spp. in bovine milk samples was 5% (20/396); A. butzleri was the only isolated species, in agreement with previous studies that reported A. butzleri as the most commonly recovered species in milk and dairy products. MLST analysis of the 20 A. butzleri strains identified 81 alleles and 16 STs. Consistent with previous studies, MLST revealed a high level of heterogeneity between the A. butzleri isolates and confirmed the high discriminatory power of this method and its suitability for epidemiological investigations. This study confirmed the importance of raw milk as a possible source of Arcobacter spp. for humans.

High Occurrence of Methicillin-Resistant Staphylococcus aureus in Horses at Slaughterhouses Compared with Those for Recreational Activities: A Professional and Food Safety Concern?

The epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in horses and its zoonotic potential is poorly understood. The objective of this study is to provide data on the prevalence and genetic characteristics of MRSA isolated from horses on farms, at racecourses, and at slaughterhouses in Italy, using standard and molecular methods. In addition, we report the prevalence of MRSA in horse handlers. Among 388 horses tested by nasal swabs, 27 (7%) were positive for MRSA ST398 (t011, t899, t1255) and ST1 (t127). The prevalence of MRSA in horses tested at slaughterhouses was significantly higher (p < 0.001) compared with those tested on farms and racecourses. Five (7%) out of 67 staff members working in close contact with horses (2 from slaughterhouse, 2 from riding stable, and 1 from racecourse) were carriers of MRSA ST398 (t011, t034) and ST1 (t127). The isolates from horses and humans carried SCCmec IVa or V and were pvl negative and pia positive. All the isolates from both horses and humans were resistant to at least two antimicrobial classes. The circulation of MRSA in horses and in humans working in close contact with them should be considered an emerging public health issue. In fact, it represents a potential risk for people who work in close contact with horses, and for horse meat consumers.

Comparison between Salmonella enterica Serotype Enteritidis Genotyping Methods and Phage Type.

A quantitative comparison between discriminatory indexes and concordance among multilocus variable-number tandem-repeat analysis (MLVA), pulsed-field gel electrophoresis (PFGE), automated ribotyping, and phage typing has been performed, testing 238 Salmonella enterica serotype Enteritidis isolates not epidemiologically correlated. The results show that MLVA is the best choice, but each typing method provides a piece of information for establishing clonal relationships between the isolates.

Comparative Genomics of Listeria Sensu Lato: Genus-Wide Differences in Evolutionary Dynamics and the Progressive Gain of Complex, Potentially Pathogenicity-Related Traits through Lateral Gene Transfer.

Historically, genome-wide and molecular characterization of the genus Listeria has concentrated on the important human pathogen Listeria monocytogenes and a small number of closely related species, together termed Listeria sensu strictu. More recently, a number of genome sequences for more basal, and nonpathogenic, members of the Listeria genus have become available, facilitating a wider perspective on the evolution of pathogenicity and genome level evolutionary dynamics within the entire genus (termed Listeria sensu lato). Here, we have sequenced the genomes of additional Listeria fleischmannii and Listeria newyorkensis isolates and explored the dynamics of genome evolution in Listeria sensu lato. Our analyses suggest that acquisition of genetic material through gene duplication and divergence as well as through lateral gene transfer (mostly from outside Listeria) is widespread throughout the genus. Novel genetic material is apparently subject to rapid turnover. Multiple lines of evidence point to significant differences in evolutionary dynamics between the most basal Listeria subclade and all other congeners, including both sensu strictu and other sensu lato isolates. Strikingly, these differences are likely attributable to stochastic, population-level processes and contribute to observed variation in genome size across the genus. Notably, our analyses indicate that the common ancestor of Listeria sensu lato lacked flagella, which were acquired by lateral gene transfer by a common ancestor of Listeria grayi and Listeria sensu strictu, whereas a recently functionally characterized pathogenicity island, responsible for the capacity to produce cobalamin and utilize ethanolamine/propane-2-diol, was acquired in an ancestor of Listeria sensu strictu.

Prevalence in bulk tank milk and epidemiology of Campylobacter jejuni in dairy herds in Northern Italy.

Thermotolerant Campylobacter spp. are frequently the cause of human gastroenteritis and have assumed more importance in Italy following the increased consumption of raw milk. Our objectives were to determine the prevalence and genotypes of Campylobacter spp. in dairy herds and to investigate the possible sources of bulk milk contamination. Bulk milk from dairy herds (n = 282) was cultured for Campylobacter spp. and Enterobacteriaceae. At three Campylobacter jejuni-positive farms, bovine feces, pigeon intestines, milk, and water points were also investigated. Isolates were identified by PCR and genotyped using multilocus sequence typing (MLST). C. jejuni was detected in 34 (12%) bulk milk samples. The strains belonged to 14 sequence types, and the most common clonal complexes were CC-21, CC-48, and CC-403. No association was demonstrated between the presence of C. jejuni and high levels of Enterobacteriaceae in bulk milk. At the three farms examined, C. jejuni was isolated from bovine feces (25/82 [30.5%]), pigeon intestines (13/60 [21.7%]), bulk milk (10/24 [41.7%]), and water points (4/16 [25%]). MLST revealed lineages that were common between milk and bovine feces but distinct between cattle and pigeons. In one herd, C. jejuni with the same genotype was isolated repeatedly from bulk milk and a cow with an udder infection. Our results showed a high prevalence of C. jejuni in bulk milk and suggested that udder excretion, in addition to fecal matter, may be a route of bulk milk contamination. MLST analysis indicated that pigeons are probably not relevant for the transmission of C. jejuni to cattle and for milk contamination.

Amplified Fragment Length Polymorphism and Multi-Locus Sequence Typing for high-resolution genotyping of Listeria monocytogenes from foods and the environment.

Standardized tools for typing Listeria monocytogenes isolates are required in epidemiological surveys investigating food-borne disease outbreaks and in the food-processing environment to identify the sources of contamination and routes by which the organisms are spread. In this survey Amplified Fragment Length Polymorphism (AFLP) and Multi-Locus Sequence Typing (MLST) have been used to study 103 L. monocytogenes isolates from food and environmental sources. A total of 62 AFLP types and 66 MLST Sequence Types were identified. AFLP and MLST produced similar results in terms of discriminating power. The Discrimination Index calculated for the two techniques was 0.976 for AFLP and 0.972 for MLST. These values were appreciably higher compared to serotyping (0.739). A good congruence was observed between AFLP and MLST. The present study demonstrated that AFLP and MLST subtyping are suitable tools for studying the epidemiology of L. monocytogenes. The great advantage of MLST over AFLP and other molecular typing methods based on fragment fingerprinting lies in the unambiguity of sequence data while AFLP is less costly and highly processive. In conclusion the two methods can be perfectly integrated for high-resolution genotyping of L. monocytogenes.

Genetic analysis of canine parvovirus type 2c.

The sequence of the full-length gene encoding for the main capsid protein VP2 of 58 canine parvovirus (CPV) type 2c strains, along with recent CPV-2a/2b strains, was determined and analysed in comparison with reference CPV isolates. The CPV-2c strains displayed a low genetic variability and shared amino acid changes already detected in recent CPV-2a/2b isolates, with a phylogenetic clustering accounting for their geographical distribution. Analysis of the selection pressure driving CPV evolution confirmed that the VP2 gene is under purifying selection. The emergence and global spread of the new CPV variant provides an interesting model to better understand virus evolution.

Diagnosis of Coxiella burnetii-related abortion in Italian domestic ruminants using single-tube nested PCR.

Coxiella burnetii, an obligate intracellular parasite with a worldwide distribution, is the causative agent of acute and chronic Q fever in humans. Although infection is often unapparent in cattle, sheep and goats, there is increasing evidence that C. burnetii infection in these species is associated with abortion and stillbirth. This paper describes the introduction of a single-tube nested PCR protocol for the diagnosis of C. burnetii-related abortion in domestic ruminants in Italy. A total of 514 aborted foetuses from cattle (n = 138) and sheep and goat (n = 376), collected from 301 farms, were analyzed from January 2001 to March 2005. Ninety-seven of 514 (18.9%) animals tested PCR-positive, with 16/138 (11.6%) cattle and 81/376 (21.5%) sheep and goat. Eleven of 102 (10.8%) farms with reproductive disorders in cattle and 37/199 (18.6%) farms with reproductive disorders in sheep and goats were infected with C. burnetii. A greater incidence was observed in three of the seven investigated provinces (p < 0.01), with rates of infected farms of up to 23.8%. Data showed that almost all the C. burnetii-related abortions were recorded between October and April (p < 0.01). These findings suggest that Q fever in humans is largely underestimated in Italy, probably because its occurrence is obscured by flu-like symptoms in acute forms.