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Iron-molybdenum powders ground in a planetary ball mill under different operating conditions were studied by x-ray diffraction line profile analysis using a recently developed whole powder pattern modeling approach. The evolution of the microstructure, expressed in terms of size distribution of coherent scattering domains, average dislocation density, and edge/screw character, shows the importance of the main process parameters: the ratio between jar and main disk rotation speeds, and ball milling time. A characteristic three-stage process is observed, with work hardening followed by particle flattening/bending before nanocrystalline grains form by a fragmentation process triggered by localized deformation. The relation between lattice defect density and domain size suggests a progressive transition between statistically stored to geometrically necessary dislocations, with the latter mostly present as excess dislocations at the nanodomain boundary. [ABSTRACT FROM AUTHOR]

Leptospirosis is a widespread disease throughout the world, presenting in severe clinical forms in dogs. The pathogenicity of the different serovars in field infections is not fully documented, and clinical diagnosis is often limited to a combination of serological tests and molecular analyses. The latter, although a fundamental tool, cannot identify the infecting strain without further analysis. This study reports the use of various indirect (microscopic agglutination test, MAT) and direct (microbiological culture, real-time PCR) laboratory techniques, followed by typing protocols (Multi-locus Sequence Typing (MLST), Multiple Loci Variable number tandem repeat Analysis (MLVA), serotyping) that allowed for the identification of the Leptospira serovar Australis in a symptomatic and previously vaccinated dog (vaccine containing heterologous strains). This study reports long-term clinical follow-up (0-640 days) and describes the possible role of the infection in the development of chronic renal failure. This study aims to highlight how a combination of different techniques can be useful to better characterise the environmental circulation of zoonotic agents. Therefore, the identification and isolation of circulating L. strains would facilitate the updating of epidemiological data, enhance the knowledge of pathogenicity and long-term clinical effects, and provide a valuable resource for improving the efficacy of a specific serovar vaccination., Competing Interests: The authors declare no conflicts of interest.

The present study retrospectively analyzed the infectious agents associated with 829 abortion outbreaks occurring from 2011 to 2021 in northern Italy. Foetuses were subjected to necropsies, and organ samples were analyzed by direct PCR to screen for six swine pathogens. In 42.0% of the examined outbreaks, at least one infectious agent was found. Porcine reproductive and respiratory syndrome virus (PRRSV) (24.9%) and porcine circovirus-2 (PCV2) (11.5%) were the most frequently detected among the known abortion-inducing pathogens. Chlamydia spp. (5.6%), porcine parvovirus (PPV) (4.0%), and Leptospira spp. (2.6%) were less common. Although its role in swine reproductive disorders is still unclear, PCV3 was detected in 19.6% of the cases. Coinfections were detected in 25.0% of positive outbreaks, and the most frequent coinfection was represented by PRRSV and PCV2 (32.2%), followed by PRRSV and PCV3 (23%). PCV2 prevalence showed a slight but consistent reduction during the study period, while PCV3 increased in frequency. Our data suggest an overall reduction in abortion outbreaks during the study period. PRRSV was confirmed as the main abortion agent detected in the examined area, while PCV2 prevalence showed a decline. Conversely, PCV3 detection has been increasing, supporting its potential role as an abortion agent. Our results highlight the importance of implementing a consistent and standardized sampling procedure, as well as a thorough diagnostic protocol, to reduce the incidence of inconclusive diagnoses.

Leptospirosis is a widespread zoonosis recognised as a re-emerging infectious disease in both humans and dogs, yet the actual seroprevalence of Leptospira in pets in Italy is relatively unknown. The aim of this study was to evaluate Leptospira antibody prevalence in dogs and cats from a shelter by the microscopic agglutination test (MAT), the gold standard test in leptospiral serology, and to assess risk factors for Leptospira infection. This seroepidemiological study investigated the prevalence of leptospiral antibodies in a cohort of 106 dogs and 51 cats housed in a municipal shelter in Milan. Blood samples were collected from the animals during two sampling periods: spring/summer 2014 and autumn/winter 2016/2017. Eight serogroups were evaluated: L. Australis, L. Ballum, L. Canicola, L . Grippotyphosa, L. Icterohaemorrhagiae, L. Pomona, L . Sejroe, and L. Tarassovi. Antibody titres ranged from 1:100 to 1:6400. The results indicated that 21.7% of dogs had antibodies against serogroups L. Icterohaemorrhagiae and L. Australis, making them the most often found. Conversely, none of the cats showed any presence of antibodies. Seropositivity was higher in the spring/summer period (32.7%) than in autumn/winter (11.1%), and no statistically significant results were found regarding sex or age. These findings underscore the importance of ongoing serological surveillance and biosecurity measures in shelter environments to mitigate the zoonotic risk posed by leptospirosis.

Bovine tuberculosis (bTB) is a chronic inflammatory disease primarily caused by Mycobacterium bovis . The infection affects domestic animals and wildlife, posing a zoonotic risk to humans. To understand the dynamics of transmission and genetic diversity in Italy's M. bovis population, we conducted whole-genome sequencing (WGS) analysis on two prevalent genotypes, belonging to Spoligotype SB0120, identified in different geographical and temporal contexts. By comparing these genomes with international M. bovis isolates, we identified a distinct clade within the lineage La1.2, encompassing the Italian SB0120 isolates, indicating a genomic segregation of Italian M. bovis from other European isolates. Within Italy, a significant level of genetic variability emerged across regions, while isolates within epidemiologically linked outbreaks exhibited minimal genetic diversity. Additionally, isolates derived from cattle and wild boars within a tuberculosis hotspot in Central Italy and from cattle and black pigs in Sicily formed unified clonal clusters. This indicates the presence of persistent strains circulating in the examined regions. The genetic diversity within herds was limited, as specific clones endured over time within certain herds. This research enhances our comprehension of the epidemiology and transmission patterns of bTB in Italy, thereby aiding the development of precise control strategies and disease management. Using WGS and implementing standardized protocols and databases will be pivotal in combating bTB and promoting One-Health approaches to address this noteworthy public health concern., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Scaltriti, Iyad, Boniotti, Menozzi, Bolzoni, Ippolito, Ciarello, Loda, D’Incau, Zanoni, Presti, Mazzone, Gavaudan and Pacciarini.)

The presence of Salmonella spp. in dairy cattle farms poses a major risk to animal health and welfare. This study focused on Salmonella detection in dairy farms located in the Cremona and Mantua provinces (northern Italy) in samples collected and submitted to laboratories in 2021-2022. A total of 2710 samples from different sources, including calf carcasses/organs ( n = 128), rectal swabs ( n = 1937), feces ( n = 390), bulk milk ( n = 93), and overshoes/swabs ( n = 127) for environmental sampling, were analyzed for the presence of Salmonella spp. and were included in the present study. Our results indicate that Salmonella was most commonly firstly identified from calf carcasses and organs (61.67%) and that the serotypes most frequently detected in dairies were S . Dublin (38.33%), S . Typhimurium (23.33%), and S . Typhimurium monophasic variant (14.17%). The most common pathological findings in calf carcasses were enteritis, hepatosplenomegaly, and pneumonia. The antimicrobial resistance pattern analyzed using the MIC assay of 51 Salmonella isolates revealed the presence of multi-resistant strains, which pose a major risk to public and animal health.

Aims: Our study evaluates the capacity of direct real-time PCR for detecting Mycobacterium tuberculosis complex (MTBC), with a focus on diagnostic performances and the feasibility of implementing this protocol in an eradication campaign. Specifically, we compare the effectiveness of the direct PCR method to various culture systems used by the Italian National Reference Laboratory over the last decade to detect MTBC., Methods and Results: Bovine tissue samples were routinely tested and analyzed for bovine tuberculosis (bTB) confirmation using microbiological culture (solid and liquid media), histopathological analysis, and a direct PCR assay targeting IS6110, an insertion sequence specific to the MTBC that is widely used for tuberculosis diagnosis. The direct real-time PCR demonstrated a high concordance (K = 0.871) with microbiological culture, as well as good sensitivity (91.84%) and specificity (95.24%). In contrast, histopathology demonstrated lower concordance (K = 0.746) and performance levels (sensitivity 91.41%, specificity 82.88%). Liquid media promoted faster and more efficient growth of MTBC than solid media. M. bovis and M. caprae had the comparable ability to respond to the direct real-time PCR test and grow on the microbiological medium., Conclusions: This study confirms that direct real-time PCR can detect MTBC with high diagnostic accuracy within a few days. This study found no significant differences in performance between culture media and direct PCR for M. bovis and M. caprae., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

The microscopic agglutination test (MAT) assay is adopted as a world-wide reference test for the serodiagnosis of leptospirosis in humans and animals. One of the main limitations of MAT is the lack of sensitivity and serodiagnostic antigens should be periodically updated with locally circulating serovars in order to optimise its performance. The aim of this study was to determine the need to implement the antigen panel currently adopted in Northern Italy for the diagnosis of Leptospira infection in dogs. For this purpose, a group of 288 dogs with and without clinical signs potentially consistent with Leptospira infection or found to have an increased C-reactive protein (CRP) serum concentration, sampled in 2013-2016 in Northern Italy, were tested by MAT comparing the results obtained with a nine antigens panel (Australis-Bratislava, Ballum-Ballum, Canicola-Canicola, Grippotyphosa-Grippotyphosa, Icterohaemorrhagiae-Copenhageni, Icterohaemorrhagiae-Icterohaemorrhagiae, Sejroe-Hardjo, Pomona-Pomona and Tarassovi-Tarassovi serovars) routinely adopted and a panel expanded to 27 antigens. In general, the antigen panel currently adopted in Northern Italy for the routine MAT assay resulted adequate for the diagnosis of Leptospira infection in dogs. The main exception concerns the Sejroe serogroup, with the Saxkoebing and Sejroe serovars that were more effective than Hardjo for diagnosis in dogs and whose inclusion in the antigen panel is recommended. Among other antigens evaluated in this study, Cynopteri serovar was detected with high frequency but its pathogenic role in dogs and as public health threat deserve further investigation., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Leptospirosis is a worldwide widespread zoonosis caused by Leptospira genus. We report an acute leptospirosis case in a puppy housed at a municipal kennel and the subsequent diagnostic investigations carried out on all dogs housed in the kennel. Laboratory investigation included mainly a microagglutination test, real-time PCR, and multi-locus sequence typing (MLST) for Leptospira genus. Other agents of infection were excluded. The puppy resulted positive for Leptospira interrogans Icterohaemorrhagiae both with serological and molecular assays. All of the other 66 dogs in the kennel underwent clinical and laboratory investigations twice, 15 days apart. No other dog showed leptospirosis clinical signs. At the first sampling, eight dogs (12%) showed antibodies against Leptospira interrogans serogroup Icterohaemorragiae serovar Copenhageni. Real-time PCR on urine samples of seropositive dogs detected Leptospira spp. DNA in one sample, then identified as Leptospira interrogans serogroup Icterohaemorragiae by MLST. Fifteen days after, four of the previous seropositive dogs still showed antibodies against Leptospira spp. All urine samples collected from seropositive dogs were negative at real-time PCR. The study allowed the early confirmation of a Leptospirosis case and the identification of at least one asymptomatic carrier of pathogenic Leptospira spp. The prompt activation of all appropriate management measures allowed limiting and extinguishing the infection.

Campylobacter is one of the most common foodborne diseases worldwide with increasing rates of antibiotic resistance. Most cases of campylobacteriosis can be traced back to the consumption of poultry meat. Despite many efforts to reduce contamination in farms and in slaughterhouses, the persistence of this pathogen in poultry products remains a problem. This study aimed to evaluate the genetic diversity and antibiotic resistance of 542 C. jejuni and C. coli in Italian poultry, in the framework of two National Monitoring Programs. Genomes were screened for antibiotic resistance, virulence determinants and contextualized within a global collection of C. jejuni . ST2116, ST2863 and ST 832 were the most prevalent and significantly associated with Italian poultry. A worrying increase in resistance to quinolones, fluoroquinolones and tetracycline was observed in C. jejuni , while an increased occurrence of multidrug resistant (MDR) strains and strains resistant to macrolides was detected in C. coli . Low resistance rates were found for aminoglycosides. Molecular resistance determinants were consistent with the phenotypic resistance for tetracycline and quinolones. In silico analysis revealed 119 genes associated with virulence factors, with a notably higher prevalence of some genes in ST2863 genomes. This study highlights the increased resistance to macrolides and the emergence of MDR strains for C. coli , the genetic basis of AMR and the predominance of two genotypes among Campylobacter strains isolated from the Italian poultry farms.

In recent years, burial systems have covered increasingly higher costs due to the pollution caused by decomposition products. These products are understood as chemicals and microorganisms in the surrounding soil and groundwater and represent a topical issue. The purpose of this research was to ascertain the extent of decomposition when pig carcasses are buried in two different burial systems ("aerated" vs. "watertight") and catalogue the arthropods associated with burials at different time-points of removal from niches (after 6, 12, 24, 36, and 60 months). Thirteen taxa were collected in aerated niches, whereas five were collected in watertight niches. The initial access or exclusion of insect colonisers affected overall functional activity. Two Diptera species, Hydrotaea capensis and Megaselia scalaris , were the most abundant, supporting the hypothesis that insects can colonise carcasses in aerated burial systems. Furthermore, some species of bacteria have been documented as facilitators of the initial decomposition process of the carcass. Most bacterial colonies develop only in aerated niches. The trial showed that the first enzymatic-bacterial and insect actions helped promote the process of cadaveric decomposition and later skeletonisation, mainly when associated with aeration modes of the tomb/mound. The results obtained provide essential information on the process of human decomposition and taphonomy in cemeteries. Moreover, these data could benefit forensic science by adding information on insect colonisation and body modification in medico-legal investigations concerning the post-mortem interval in exhumed bodies and illegal burials.

Leptospirosis is a zoonosis of public health concern. Its prevalence in stray animals in the South of Italy is unknown. This study aimed to investigate Leptospira spp. prevalence in 1009 stray animals. Out of them, 749 were alive animals, including 358 dogs (316 from Palermo and 42 from Ragusa) and 391 cats (359 from Palermo and 32 from Ragusa), and 260 were corpses (216 dogs and 44 cats) randomly collected in Sicily. Dogs and cats underwent a serological screening by Microscopic Agglutination Test and a molecular investigation by Real-Time PCR targeting lipL32 . Corpses were subjected to Real-Time PCR. Serological analyses showed a prevalence of 1.12% (4/358) for dogs and 0.26% (1/391) for cats, with the only positive cat coming from Palermo. Serogroup Icterohaemorrhagiae serovar Icterohaemorrhagiae or Copenhageni, followed by Canicola and Bratislava, were the most spread among dogs, while the serological positive cat reacted with Hardjo serogroup. Two urine (2/32, 6.25%) and one blood (1/391, 0.26%) samples of cats, all from Ragusa, were positive at Real-Time PCR for pathogenic Leptospira spp. Sequencing analyses showed the presence of L. interrogans serogroup Icterohaemorrhagiae serovar Icterohaemorrhagiae or Copenhageni in one of the positive urine samples and in the positive blood sample. Analyses on corpses showed a prevalence of 1.85% (4/216) in Sicilian dog kidney samples, while all corpses of cats resulted in negative. Genotyping analysis showed a genetic relatedness between cat and human isolates. Results show Leptospira spp. circulation among Sicilian stray animals. The genetic relatedness between cat and human isolates suggests a possible common infection source.

Salmonella is a pathogen of considerable health concern, given its zoonotic potential, and, in Italy, is the most frequently reported causative agent for foodborne outbreaks. Wild animals and in particular wild carnivores may be carriers of different Salmonella enterica subspecies and serotypes. Given their potential role as reservoirs, surveillance activities are necessary. This study aims to investigate the presence of different Salmonella subspecies and serotypes in wild carnivores in the Emilia-Romagna Region. A total of 718 fox ( Vulpes vulpes ), 182 badger ( Meles meles ) and 27 wolf ( Canis lupus ) carcasses, submitted between 2016-2022, were included for the present work. Gender and age data were collected along with geographical coordinates of carcass' discovery site. Contents of the large intestine were sampled and cultured according to ISO 6579-1 and both serogroup and serotype identification were performed according to ISO/TR 6579-3:2014. Salmonella was retrieved from 42 foxes (6%), 21 badgers (12%) and 3 wolves (12%), respectively. Isolated Salmonella enterica strains belonged to 4 different subspecies and 25 different serotypes. S. veneziana and S. typhimurium were the most frequent serotypes found (11/67 and 10/67, respectively). In conclusion, zoonotic serotypes were found in all these species of wildlife, thus confirming their potential role in the ecology of Salmonella spp.

Colibacillosis is the most common bacterial disease in the poultry industry. The isolation of Escherichia coli ( E. coli ) strains with multiple resistance to various classes of antimicrobials has been increasing in recent years. In this study, antimicrobial resistance features, serotyping and the presence of avian pathogenic Escherichia coli (APEC) virulence genes were investigated on a total of 71 E. coli strains isolated during outbreaks of colibacillosis in laying hens. The correlation between these features was evaluated. The most frequently isolated serogroups were O2 and O88. Resistance was often detected with nalidixic acid (49%) and ampicillin (38%), while all strains were sensitive to ceftiofur and florfenicol. Overall, 25% of the isolates showed resistance to at least three or more antimicrobial classes (multidrug-resistant strains), and 56% of the isolates were defined as APEC strains (due to the presence of at least five virulence genes). Correlation between the different parameters (virulence genes, serogroup and antimicrobial resistance) did not reveal relevant associations. The comparison of the obtained results with those of similar studies highlighted the importance of continuous monitoring in order to have a better understanding of colibacillosis. An evaluation of the national epidemiological situation would allow, especially with regard to antimicrobial resistance, to focus on the right measures in order to prioritize the available resources for effective disease control.

Leptospirosis is a re-emerging zoonosis of worldwide significance; a wide spectrum of wild and domestic animal species act as natural or accidental hosts. Swine can act as maintenance or accidental hosts of pathogenic Leptospira spp. This study aimed at investigation of Leptospira spp. prevalence and diversity in slaughtered pigs in southern Italy (Sicily). In total, 55 samples of kidneys and blood were collected. Microscopic agglutination test and real-time PCR were performed to detect pathogenic and intermediately pathogenic Leptospira . Partial rpoB gene sequencing and multi-locus sequence typing (MLST) were performed to characterize Leptospira species. The analysis showed a seropositivity rate of 16.4%, with Australis representing the most frequently identified serogroup (63.64%); Pomona and Sejroe were detected with a prevalence of 27.27% and 9.09%, respectively. Pathogenic Leptospiral DNA was detected in 2 kidney samples (3.64%). Leptospira were identified through MLST as L. borgpetersenii serovar Tarassovi (serogroup Tarassovi). Obtained data confirmed the presence of Leptospira infection among pigs in southern Italy, suggesting that management of these animals may be considered an occupational risk for humans.

An unexpected high presence of Mycobacterium   microti in wild boar in Northern Italy (Garda Lake) has been reported since 2003, but the factors contributing to the maintenance of this pathogen are still unclear. In this study, we investigated the presence of M.   microti in wild rodents and in water and soil samples collected at wild boar aggregation areas, such as watering holes, with the aim of clarifying their role in M.   microti transmission. In total, 8 out of 120 captured animals tested positive for the Mycobacterium   tuberculosis complex (MTBC) as assessed by real-time PCR, and six samples were confirmed to be M.   microti . A strain with a genetic profile similar to those previously isolated in wild boars in the same area was isolated from one sample. Of the 20 water and 19 mud samples, 3 and 1, respectively, tested positive for the presence of MTBC, and spacer oligotype SB0118 (vole type) was detected in one sample. Our study suggests that wild rodents, in particular Apodemus   sylvaticus , Microtus sp. and Apodemus   flavicollis , play roles in the maintenance of M.   microti infections in wild boar through ingestion or by contact with either infected excreta or a contaminated environment, such as at animal aggregation sites.

Erysipelothrix rhusiopathiae (ER) is an old but still emerging zoonotic infection that is not yet completely understood. ER infects a wide range of species and wild boar is of significant interest because of their similarities to pigs, a known ER reservoir. Moreover, the increase of its densities and the limited data available about ER in this species should be considered. The need is to investigate whether wild boar could represent a risk of erysipelas at the wildlife-domestic-human interface. Here, 1067 sera and 149 tonsils of wild boar from five hunting districts in Northwest Italy were tested using ELISA and bacteriological culture, respectively. Using generalized linear models, we evaluated host and environmental factors influencing ER spread and dynamics. We found an ER seroprevalence of 69.4% among wild boar. Increased human density and pig farm density lead to an increase of ER seropositivity highlighting its association with anthropic environmental-related factors. The high ER percentage of isolation (34.2%) found in healthy wild boar suggests that this species can serve as a healthy carrier. This fact, together with the high seroprevalence, supports a role of wild boar as an ER reservoir. Potential zoonotic and economic risks should be considered in light of these data., (© 2021. EcoHealth Alliance.)

The health problem of antimicrobial resistance (AMR) involves several species. AMR surveillance is essential to identify its development and design control strategies; however, available data are still limited in some contexts. The AMR profiles of 2612 E. coli strains isolated over a period of 15 years (2002-2016) from calf enteric cases were analyzed to determine the presence of resistance and their temporal dynamics. Furthermore, the AMR profiles and the presence of the major virulence genes of 505 E. coli strains isolated from 1-week- and 2-week-old calves, 406 treated with antimicrobials and 99 untreated, were analyzed and compared to investigate the potential effects of treatment on AMR and strain pathogenicity. Resistance to tetracycline (90.70%) was the most common, followed by resistance to sulfamethoxazole/trimethoprim (77.70%) and flumequine (72.10%). The significantly higher percentage of AMR and virulence gene expression recorded in treated calves, combined with the statistically higher resistance to sulfamethoxazole/trimethoprim in E. coli with K99, corroborates the notion of resistance being induced by the frequent use of antimicrobials, leading to treatments potentially becoming ineffective. The significantly higher resistance to amoxicillin/clavulanic acid, enrofloxacin, and florfenicol in isolates from 1-week-old calves suggests the role of the environment as a source of contamination that should be investigated further.