Brucella spp. in Wildlife of the Lombardy Region, Northern Italy.

Surveillance data collected in the period 2017-20 for Brucella spp. in wildlife of the Lombardy Region in northern Italy were used to describe the exposure of the wildlife species to Brucella spp. in wild boar (Sus scrofa), European brown hare (Lepus europaeus), fallow deer (Dama dama), red deer (Cervus elaphus), and roe deer (Capreolus capreolus). Among the tested species, wild boar (n=6,440) showed the highest percentage of seropositive samples (5.9%). Notably, wild boars of perifluvial area of the Po River showed higher percentages of positivity than those of the pre-Alpine district. In addition, during the hunting season in 2018, 95 organs (uterus or testes, spleen, and submandibular lymph nodes) from wild boar of the perifluvial area of the Po River were collected for bacteriological examination. Brucella suis was isolated in culture from 18.9% of tested lymph nodes. These serological and microbiological results highlight the presence of B. suis in wild boar and suggest the importance of wild boar as a reservoir for B. suis. Comparison of the spatial distribution of Brucella-seropositive wild boars with the location of backyard swine farms revealed a higher chance of contact between the two populations only in the areas where the lower percentage of seropositive samples was observed. Conversely, the high percentage of seropositive samples observed in the Po River area coupled with positive microbiological cultures suggest a greater risk of infection for the humans directly or indirectly involved in wild boar hunting activity. These results may serve as a basis to establish sound wildlife management and to adopt education campaigns aimed at reducing the risk of human infection in people involved in wild boar hunting related activities.

Factors related to longevity and mortality of dogs in Italy.

Besides its translational value, an improved understanding of dog longevity and mortality is necessary to guide health management decisions, breed selection, and improve dog welfare. In order to analyse the lifespan of dogs in Italy, identify the most common causes of death, and evaluate possible risk factors, anonymised medical records were collected from 9 veterinary teaching hospitals and 2 public health institutions. Data regarding breed, sex, neuter status, age, diagnosis, and mechanism of death were retrieved. Cause of death (COD) was classified by pathophysiologic process (PP) and organ system (OS). Of the 4957 dogs that died between 2004 and 2020 included in the study, 2920 (59.0%) were purebred, 2293 (46.2%) were female, 3005 (60.6%) were intact, 2883 (58.2%) were euthanised. Overall median longevity was 10.0 years. Median longevity was significantly longer for crossbreds, females, neutered dogs, and small-sized breeds. The breeds with the highest median age at death were the Yorkshire terrier, English cocker spaniel, West Highland white terrier, Italian volpino, and Shih Tzu, whilst the American bulldog, English bulldog, American pit bull terrier, Bernese mountain dog and the Maremma and the Abruzzes sheepdog had the lowest median age at death. The most frequent COD by PP was neoplasia (34.0%), which occurred more frequently in large breeds, namely German shepherd, Labrador retriever and Boxer. Degenerative diseases mostly affected small-sized dogs like Miniature pinscher and Dachshund. Regarding the OS involved, diseases of the renal/urinary system were most frequently responsible for COD (15.0%), prevalently degenerative and inflammatory/infectious. Substantial variation in median longevity according to causes of death by PP and OS was observed. These data are relevant for breeders, veterinary practitioners, and owners, to assist breed selection, facilitate early diagnosis, guide choice when purchasing a purebred dog and making health management decisions, and ultimately improve dog welfare.

Assessing pig farm biosecurity measures for the control of Salmonella on European farms.

Salmonella spp. is a common zoonotic pathogen, causing gastrointestinal infections in people. Pigs and pig meat are a major source of infection. Although farm biosecurity is believed to be important for controlling Salmonella transmission, robust evidence is lacking on which measures are most effective. This study enrolled 250 pig farms across nine European countries. From each farm, 20 pooled faecal samples (or similar information) were collected and analysed for Salmonella presence. Based on the proportion of positive results, farms were categorised as at higher or lower Salmonella risk, and associations with variables from a comprehensive questionnaire investigated. Multivariable analysis indicated that farms were less likely to be in the higher-risk category if they had '<400 sows'; used rodent baits close to pig enclosures; isolated stay-behind (sick) pigs; did not answer that the hygiene lock/ anteroom was easy to clean; did not have a full perimeter fence; did apply downtime of at least 3 days between farrowing batches; and had fully slatted flooring in all fattener buildings. A principal components analysis assessed the sources of variation between farms, and correlation between variables. The study results suggest simple control measures that could be prioritised on European pig farms to control Salmonella.

The role of co-infections in M. hyopneumoniae outbreaks among heavy fattening pigs: a field study.

Little is known about how co-infections and genotype dynamics affect Mycoplasma hyopneumoniae infection in fattening pigs. This study was aimed at assessing the role of co-infections in M. hyopneumoniae outbreaks, their influence on the presence of M. hyopneumoniae genotypes and their impact on consequent lung lesions. Tracheobronchial swabs (TBS) from 300 finishers were collected from 10 farms at the onset of enzootic pneumonia outbreaks and 1 month later, sampling of 3 groups per farm: Group A showed clinical signs first, Group B was housed near Group A, and Group C was located in a different building. Pigs' lungs were scored at the slaughterhouse. TBS were tested for the main pathogens involved in respiratory diseases, and samples positive for M. hyopneumoniae were genotyped by multiple-locus variable-number tandem repeat analysis (MLVA). Pigs in Group A showed the highest prevalence and load of M. hyopneumoniae. A positive association was detected between M. hyopneumoniae and Mycoplasma hyorhinis, whereas Actinobacillus pleuropneumoniae was more frequent when the M. hyopneumoniae load was higher. Nevertheless, co-infection had no effect on lung lesion scores. The presence of multiple MLVA types (mixed infections) increased in time only in pigs from Group C and was positively associated with porcine reproductive and respiratory syndrome virus infection. Lung lesions were more severe in pigs with at least one TBS positive for M. hyopneumoniae and in pigs with a history of mixed infections. The central role of M. hyopneumoniae and relevance of mixed infections suggest that increased biosecurity might be beneficial for lung lesion sequelae.

Genomic variability of Mycoplasma hyopneumoniae within pig lung lobes.

BACKGROUND: Genotypic variability in M. hyopneumoniae has been reported within and among herds. However, information regarding VNTR types within single lung lobes is lacking. The objective of his study was to analyse M. hyopneumoniae infections and their association with VNTR types and lung lesions at the lobe level. Lungs from 300 pigs from 10 farms experiencing an enzootic pneumonia outbreak were collected and scored. M. hyopneumoniae was detected by real-time PCR and genotyped by MLVA assay in all samples. RESULTS: The results showed genotypic variability within single pigs and among lung lobes. At the lobe level, infection with one VNTR type (SN infection) was dominant. Lobes with lesion scores > 0 were associated with positive results for real-time PCR. At the lobe level, no relationship was observed between infections with more than one genotype (MX infections) and the proportion of Mycoplasma-like lesions. Lesion-free lobes presented a higher proportion of MX infections than lobes scored > 0. M. hyopneumoniae was detected more frequently in the right lobe of the lung (p < 0.05), with a similar distribution within lobes for SN and MX infections. The anatomic conformation of swine lungs led to a higher prevalence of infections in the right lobe. However, this study showed that this condition did not affect the distribution of infections with multiple VNTR types. Nevertheless, careful consideration of sample selection should be practised for M. hyopneumoniae genotype analyses, including lung lobes with no visible lesions. CONCLUSION: The results did not show a significant association between the number of detected genotypes and the severity of the lesions at the lung lobe level, but revealed the unexpected detection of M. hyopneumoniae genotypes in lesion-free lobes. These results imply that a representative sampling of all lobes may lead to an accurate identification of the VNTR-type distribution. Further studies including factors that can affect pathogenetic evolution of this bacterium could shed light on the complexity of the relationship between genotypes and the lung lesions magnitude.

The Use of Antimicrobials in Italian Heavy Pig Fattening Farms.

Data on antimicrobial use (AMU) in heavy pig production (>150 kg) are limited. The aim of this study was to investigate the AMU in this production. Data from 2015 were collected for 143 fattening farms. The AMU was estimated through a treatment index per 100 days (TI100) using the defined daily dose animal for Italy (DDDAit). When possible, a comparison with the European Medicines Agency's defined daily doses for animals (DDDvet) was performed. The median TI100 was 10.7 (range, 0.2-49.5). Group treatments represented 94.6% of overall consumption. The AMU calculated using DDDAit and DDDvet were strongly correlated (ρ = 0.976; p < 0.001). The AMU was negatively correlated with injectables use (ρ = -0.46, p < 0.001) and positively correlated with oral products (ρ = 0.21, p = 0.014), premixes (ρ = 0.26, p = 0.002), and mortality (ρ = 0.18; p = 0.027). Farm size was negatively correlated with AMU (ρ = -0.29, p < 0.001). Smaller farms were more frequently above the median TI100 (odds ratio = 2.3, 95% confidence interval = 1.2-4.7), suggesting that they may have lower biosecurity and management standards. The results of this study should provide useful insights for the development of an Italian monitoring system.

Effect of welfare standards and biosecurity practices on antimicrobial use in beef cattle.

Antimicrobial use (AMU) in livestock species and the associated antimicrobial resistance are a global concern, thus strategies for their reduction and a more judicious use are needed. Previous research has revealed a link between improved animal welfare, biosecurity and AMU reduction in pig and dairy sectors, however, little is known about the beef sector. This study aimed to investigate the impact of welfare standards and biosecurity on AMU in beef cattle. Data on performance traits and AMU were collected over a 3.5 year time from 27 specialised beef farms and a treatment incidence was calculated using the defined daily dose for animals. An on-farm assessment was carried out by assigning a score from 0 (very poor) to 100% (very good) to 3 sections: welfare, biosecurity and emergency management. The highest average score was obtained for the welfare section (76%) followed by emergency management (39%) and biosecurity (24%). This suggests that major focus on strategies for the implementation of biosecurity measures and emergency management is needed, due to the low scores reported. A statistically significant lower AMU was observed with improved level of welfare. These results may be helpful for farm benchmarking and highlight the importance of improved animal welfare for an efficient antimicrobial stewardship.

Monitoring of Farm-Level Antimicrobial Use to Guide Stewardship: Overview of Existing Systems and Analysis of Key Components and Processes.

The acknowledgment of antimicrobial resistance (AMR) as a major health challenge in humans, animals and plants, has led to increased efforts to reduce antimicrobial use (AMU). To better understand factors influencing AMR and implement and evaluate stewardship measures for reducing AMU, it is important to have sufficiently detailed information on the quantity of AMU, preferably at the level of the user (farmer, veterinarian) and/or prescriber or provider (veterinarian, feed mill). Recently, several countries have established or are developing systems for monitoring AMU in animals. The aim of this publication is to provide an overview of known systems for monitoring AMU at farm-level, with a descriptive analysis of their key components and processes. As of March 2020, 38 active farm-level AMU monitoring systems from 16 countries were identified. These systems differ in many ways, including which data are collected, the type of analyses conducted and their respective output. At the same time, they share key components (data collection, analysis, benchmarking, and reporting), resulting in similar challenges to be faced with similar decisions to be made. Suggestions are provided with respect to the different components and important aspects of various data types and methods are discussed. This overview should provide support for establishing or working with such a system and could lead to a better implementation of stewardship actions and a more uniform communication about and understanding of AMU data at farm-level. Harmonization of methods and processes could lead to an improved comparability of outcomes and less confusion when interpreting results across systems. However, it is important to note that the development of systems also depends on specific local needs, resources and aims.

Detection of a New Genetic Cluster of Influenza D Virus in Italian Cattle.

Influenza D virus (IDV) has been increasingly reported all over the world. Cattle are considered the major viral reservoir. Based on the hemagglutinin-esterase (HEF) gene, three main genetic and antigenic clusters have been identified: D/OK distributed worldwide, D/660 detected only in the USA and D/Japan in Japan. Up to 2017, all the Italian IDV isolates belonged to the D/OK genetic cluster. From January 2018 to May 2019, we performed virological surveillance for IDV from respiratory outbreaks in 725 bovine farms in Northern Italy by RT-PCR. Seventy-four farms were positive for IDV. A full or partial genome sequence was obtained from 29 samples. Unexpectedly, a phylogenetic analysis of the HEF gene showed the presence of 12 strains belonging to the D/660 cluster, previously unreported in Europe. The earliest D/660 strain was collected in March 2018 from cattle imported from France. Moreover, we detected one viral strain with a reassortant genetic pattern (PB2, PB1, P42, HEF and NP segments in the D/660 cluster, whilst P3 and NS segments in the D/OK cluster). These results confirm the circulation of IDV in the Italian cattle population and highlight the need to monitor the development of the spreading of this influenza virus in order to get more information about the epidemiology and the ecology of IDV viruses.

Salmonella Typhimurium infection primes a nutriprive mechanism in piglets.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important cause of acute food- borne zoonoses worldwide, typically carried by pigs. It is well known that Salmonella has evolved a wide array of strategies enabling it to invade the host, but little information is available on the specific host responses to Salmonella infections. In the present study, we used an in vivo approach (involving piglets infected with a virulent or an attenuated S. Typhimurium strain) coupled to histological and proteomic analysis of the cecum mucosa, to highlight the host pathways activated during S. Typhimurium infection. We confirm the complex host-pathogen interaction. Our data showed that the metabolic and the cytoskeleton organization functions were the most significantly altered. In particular, the modifications of energy metabolic pathway could suggest a "nutriprive" mechanism, in which the host reduce its metabolic and energetic status to limit Salmonella infection. This study could represent a preliminary approach, providing information useful to better understand the host-Salmonella interaction.

Salmonella Typhimurium exploits inflammation to its own advantage in piglets.

Salmonella Typhimurium (S. Typhimurium) is responsible for foodborne zoonotic infections that, in humans, induce self-limiting gastroenteritis. The aim of this study was to evaluate whether the wild-type strain S. Typhimurium (STM14028) is able to exploit inflammation fostering an active infection. Due to the similarity between human and porcine diseases induced by S. Typhimurium, we used piglets as a model for salmonellosis and gastrointestinal research. This study showed that STM14028 is able to efficiently colonize in vitro porcine mono-macrophages and intestinal columnar epithelial (IPEC-J2) cells, and that the colonization significantly increases with LPS pre-treatment. This increase was then reversed by inhibiting the LPS stimulation through LPS antagonist, confirming an active role of LPS stimulation in STM14028-intracellular colonization. Moreover, LPS in vivo treatment increased cytokines blood level and body temperature at 4 h post infection, which is consistent with an acute inflammatory stimulus, capable to influence the colonization of STM14028 in different organs and tissues. The present study proves for the first time that in acute enteric salmonellosis, S. Typhimurium exploits inflammation for its benefit in piglets.

Microencapsulated sorbic acid and pure botanicals affect Salmonella Typhimurium shedding in pigs: a close-up look from weaning to slaughter in controlled and field conditions.

The aim of this study was to assess the efficacy of a combination of sorbic acid, thymol, and carvacrol in reducing the prevalence and shedding level of Salmonella Typhimurium in pigs either in a controlled challenge environment or in a production setting. In the first study, 24 weaned piglets were separated in 4 isolation units (6 piglets/isolation unit). Each unit received either a basal diet (no treatment) or a microencapsulated mixture of sorbic acid, thymol, and carvacrol at 1, 2, or 5 g/kg of feed. After 21 d, pigs were orally challenged with 6 log10 colony-forming units of Salmonella Typhimurium. Blood samples and feces from rectal ampullae were collected every week. On d56 of the study, pigs were euthanized and necropsied to collect intestinal contents (jejunum through colon) and ileocecal lymph nodes. Samples were analyzed for Salmonella Typhimurium and serological analysis was also conducted. In the second study, an all-in-all-out multisite pig farm that was positive for monophasic Salmonella Typhimurium was followed throughout a production cycle from weaning to slaughter. Pigs received either a basal diet or the basal diet including 5 g/kg of the microencapsulated additive. Environmental, fecal, and blood samples were collected monthly, and cecal contents and ileocecal lymph nodes were collected at slaughter to isolate and enumerate Salmonella. The results indicate that the additive at 5 g/kg tended to reduce Salmonella fecal prevalence in both a controlled challenge (p=0.07) and in production conditions (p=0.03). Nevertheless, the additive did not reduce the number of pigs seropositive for Salmonella, nor it reduced the Salmonella prevalence at slaughter. The data indicate that these additives are not effective alone but must be used in conjunction with appropriate containment measures at lairage in order to prevent reinfection in pigs and to reduce the number of pigs carrying Salmonella entering the food chain.

Salmonella enterica Serovar Typhimurium Exploits Inflammation to Modify Swine Intestinal Microbiota.

Salmonella enterica serovar Typhimurium is an important zoonotic gastrointestinal pathogen responsible for foodborne disease worldwide. It is a successful enteric pathogen because it has developed virulence strategies allowing it to survive in a highly inflamed intestinal environment exploiting inflammation to overcome colonization resistance provided by intestinal microbiota. In this study, we used piglets featuring an intact microbiota, which naturally develop gastroenteritis, as model for salmonellosis. We compared the effects on the intestinal microbiota induced by a wild type and an attenuated S. Typhimurium in order to evaluate whether the modifications are correlated with the virulence of the strain. This study showed that Salmonella alters microbiota in a virulence-dependent manner. We found that the wild type S. Typhimurium induced inflammation and a reduction of specific protecting microbiota species (SCFA-producing bacteria) normally involved in providing a barrier against pathogens. Both these effects could contribute to impair colonization resistance, increasing the host susceptibility to wild type S. Typhimurium colonization. In contrast, the attenuated S. Typhimurium, which is characterized by a reduced ability to colonize the intestine, and by a very mild inflammatory response, was unable to successfully sustain competition with the microbiota.

Attenuated Salmonella enterica serovar Typhimurium lacking the ZnuABC transporter: an efficacious orally-administered mucosal vaccine against salmonellosis in pigs.

We have recently demonstrated that an attenuated strain of Salmonella enterica serovar Typhimurium unable to synthesize the zinc transporter ZnuABC (S. Typhimurium ΔznuABC), is able to protect mice against systemic and enteric salmonellosis and is safe in pigs. Here, we have tested the protective effects of S. Typhimurium ΔznuABC in pigs. Resistance to challenge with the fully virulent strain S. Typhimurium ATCC 14028 was assessed in animals vaccinated with S. Typhimurium ΔznuABC (two dosages tested), in controls vaccinated with a formalin-inactivated virulent strain and in unvaccinated controls. Clinical signs of salmonellosis, faecal shedding and bacterial colonization of organs were used to assess vaccine-induced protection. After the challenge, pigs vaccinated with the attenuated S. Typhimurium ΔznuABC strain did not display clinical signs of salmonellosis (fever or diarrhoea). The vaccine also reduced intestinal tract colonization and faecal shedding of the fully virulent Salmonella strain, as compared to control groups. S. Typhimurium ΔznuABC represents a promising candidate vaccine against salmonellosis in pigs.

Effects of DNA extraction method on Porcine circovirus-2 real-time polymerase chain reaction quantification in swine lymph node samples.

Quantitative real-time polymerase chain reaction (PCR) has become an important tool for Porcine circovirus-2 (PCV-2) research and diagnosis. However, significant differences in detection limit and quantification data, among laboratories and quantitative real-time PCR methods, have been demonstrated. New efforts are required for providing more accurate and comparable results. The current study is an evaluation of the effects of DNA extraction procedures on PCV-2 quantification in lymph node samples. Differences, greater than 1 log(10) copies/g, were shown among PCV-2 loads detected after different extraction procedures. The work highlighted the critical role of the DNA extraction method in PCV-2 quantification by quantitative real-time PCR. This important aspect should be evaluated when comparing data from different laboratories or different studies. The PCV-2 quantification data should not be considered comparable before demonstrating the equivalence of the DNA extraction methods performed.