Matteo Cuccato

The authorship list of the published article has been corrected to include Dr

Background: Actinobacillus pleuropneumoniae (APP) remains a major respiratory pathogen in swine production worldwide. The limited cross-protection of available vaccines and the increasing occurrence of antimicrobial resistance (AMR) highlight the need for integrated phenotypic and genomic surveillance to support effective local control strategies. Methods: Twelve APP isolates previously collected in Piedmont (Italy) were investigated. Antimicrobial susceptibility was assessed by minimum inhibitory concentration (MIC) testing against a panel of antimicrobials commonly used in swine medicine. Whole-genome sequencing was performed using Illumina technology, followed by resistome profiling and comparative genomic analyses. Virulence-associated genes, including apx toxin genes and capsular polysaccharide loci, were also characterized. Results: Phenotypic testing revealed resistance to tetracycline, macrolides, β -lactams, tiamulin, and sulfamethoxazole/trimethoprim in a subset of strains. Genomic analysis identified tetracycline- and phenicol-resistance genes ( tet , flor ) in one isolate, while genes associated with macrolide and elfamycin resistance were widespread across the dataset. All isolates carried major apx toxin genes, with variable distribution of apxII and apxIII operons. A serovar 9/11 isolate showed a partial deletion within the capsular polysaccharide biosynthesis locus, confirmed by PCR and sequencing. Comparative genomics also suggested distinct genomic lineages among serovar 6 isolates. Conclusion: The combined phenotypic and genomic approach provides valuable insights for evidence-based antimicrobial use and provides additional genomic information on clinically relevant APP field isolates and supports future studies on AMR and genomic diversity.

Nocardia spp. and Mycobacterium spp. are known etiological agents of granulomatous pulmonary infections in humans and animals; however, co-infections involving these pathogens have not previously been reported in veterinary medicine. This paper describes the first documented case of co-infection with Mycobacterium bovis and Nocardia spp. in a captive roan antelope (Hippotragus equinus). The animal was a 9-year-old female roan antelope from a safari park in northern Italy that died suddenly with a one-month history of weight loss. Post-mortem examination revealed severe, diffuse, chronic granulomatous pneumonia associated with fibrino-granulomatous pleuritis and granulomatous pericarditis. Histologically, multifocal to coalescing necrotizing granulomas were observed, with intralesional acid-fast bacteria. Microbiological culture and biomolecular analyses allowed the identification of M. bovis and Nocardia spp. in lung tissue samples. The Nocardia genome sequence was 98.5% similar to N. tengchongensis, a recently discovered species. The findings emphasize the importance of comprehensive diagnostic approaches in animal granulomatous lung disease. Mixed infections in captive wildlife represent a One Health concern, as the potential for zoonotic adaptation and transmission to humans cannot be excluded. Therefore, pathogen surveillance is of particular importance within zoological collections.

Ochratoxin A (OTA), Zearalenone (ZEA), and Fumonisins (FB) are common contaminants of poultry feed associated with oxidative damage and potentially dangerous residues in products from exposed animals. We investigated the molecular effects in broilers of a short-term (10 days) dietary exposure to OTA (0.26 mg/kg), ZEA (2.9 mg/kg), or FB (60 mg/kg) on cytochrome P450 enzymes (CYP), drug transporters (DT) and the antioxidant defence system. OTA markedly decreased serum antioxidant capacity, while all mycotoxins depressed reduced glutathione content and increased lipid peroxidation in the liver, indicating a hepatic pro-oxidant effect. All the tested mycotoxins also reduced both the activities and the gene expression of selected antioxidant enzymes in the liver and duodenum as a result of the modulation of the Nrf2/Keap1 pathway. Moreover, mycotoxins differentially altered the hepatic and intestinal gene expression of CYP enzymes (i.e., CYP2A6, CYP2C45, CYP3A4, and CYP1A isoforms). Finally, the transcription of selected DT (i.e., ABCB1, ABCC2 and ABCG2) was generally enhanced in both the liver and duodenum. In conclusion, short-term exposure to OTA, ZEA, or FB at dietary concentrations higher than those recommended in the EU, but occurring in third countries, not only disrupt the antioxidant defence but also affect the expression of CYP and DT, which might potentially alter the kinetics of drugs and toxicants. Our results provide new insights into mycotoxin adverse effects in the light to assess the effectiveness of new mitigation strategies that contribute to food and feed safety.

Introduction: The misuse of antimicrobials (AMs) in poultry production contributes significantly to the global threat of antimicrobial resistance. Despite legislative efforts within the European Union (EU) to reduce AM use, Italy remains among the countries with the highest consumption levels. Effective and accessible screening tools for AMs residues in poultry meat are crucial for food safety monitoring and regulatory compliance. This study evaluates the performance of the Beadyplex flow cytometric assay as a field-applicable method to detect residues of thiamphenicol, sulphadiazine, and amoxicillin in broiler chicken skeletal muscle. Methods: Pectoralis major muscle samples were collected from 41 male broiler chickens (Ross 308) previously treated with thiamphenicol ( n = 11), sulphadiazine ( n = 6), amoxicillin ( n = 12), or untreated (controls, n = 12). Beadyplex assays were performed following manufacturer instructions, and results were acquired using a standard flow cytometer. Sensitivity, specificity, predictive values, and kappa agreement were calculated to assess the performance of the test in recognizing the AMs treatment. The agreement with the current reference method for detecting AM residues (high-performance liquid chromatography–high-resolution mass spectrometry, or HPLC-HRMS) was also calculated. Results: The Beadyplex assay demonstrated high sensitivity and specificity for thiamphenicol (Se = 1.00; Sp = 0.83; k = 0.791) and sulphadiazine (Se = 1.00; Sp = 0.91; k = 0.813), with substantial to almost perfect agreement with HPLC-HRMS results ( k = 0.706 and 0.827, respectively). Amoxicillin residues were not detected in any treated sample by Beadyplex. Discussion: The Beadyplex assay offers a promising, cost-effective, and rapid screening complementary to confirmatory analysis for detecting selected AM residues in poultry meat. Its substantial agreement with reference methods and ease of use with standard cytometers support its potential application in field-based veterinary surveillance and food safety programs.

On the beach of Linosa Island (Italy), 43 loggerhead sea turtle (Caretta caretta) unhatched eggs were recovered from nests, formalin-fixed and necropsied. The tissue samples were stained with hematoxylin-eosin (HE), Grocott, von Kossa, periodic acid-Schiff (PAS), and Movat pentachrome stains. Histologically, vacuolar degeneration (100.0%) and increased numbers of melanomacrophages (18.6%) in the liver, and edema (14.0%) in the lungs were observed. Twenty-five kidneys (58.1%) showed deposition of blue amorphous material with HE staining, which also appeared PAS-positive and black with von Kossa staining, allowing a diagnosis of calcium oxalate, confirmed by transmission electron microscopy (TEM). The hepatic lesions may be indicative of toxicosis, infection, or a defense mechanism. A statistically significant association between the nest position and renal oxalosis (renal calcium oxalate deposition) was observed. Renal oxalosis was probably due to the exceptionally high summer temperatures, which were statistically higher compared to the temperatures recorded in the previous two years.

On the beach of Linosa Island (Italy), forty-three loggerhead sea turtle (Caretta caretta) unhatched eggs were recovered from nests, formalin-fixed and necropsied. The tissue samples were stained with Haematoxylin-Eosin (HE), Grocott, von Kossa, Periodic Ac-id-Schiff (PAS), and Movat pentachrome stains. Histologically, vacuolar degeneration (100.0%) and increased numbers of melanomacrophages (18.6%) in the liver, and edema (14.0%) in the lungs were observed. Twenty-five kidneys (58.1%) showed deposition of blue amorphous material with HE staining, which also appeared PAS-positive and black with von Kossa staining, allowing a diagnosis of calcium oxalate, confirmed by transmis-sion electron microscopy. The hepatic lesions may be indicative of toxicosis, infection, or a defense mechanism. A statistically significant association between the nest position and renal calculosis was observed. Renal calculosis was probably due to the exceptionally high summer temperatures, which were statistically higher compared to the temperatures recorded in the previous two years.

Aflatoxin B1 (AFB1) can impair the growth of chickens and reduce the quality of eggs and meat, resulting in significant economic losses. The inclusion of mycotoxin detoxifying agents (MyDA) with binding properties in the diet is an efficient tool to reduce their absorption rate in the gastrointestinal tract. Our aim was to investigate the ability of two innovative MyDA (SeOX, a feed additive featuring a tri-octahedral smectite mixed with lignocellulose, and CHS, a di-octahedral smectite functionalized with an organic non-toxic modifier) in both reducing the bio-accessibility and mitigating the adverse effects of AFB1 in broilers exposed for 10 days to concentrations approaching the European Union maximum limits in feed (0.02 mg/kg). The amount of AFB1 in the excreta of birds, collected over four consecutive days (starting on day 7), was significantly lower (p < 0.001) in the group exposed to AFB1 alone compared to the groups treated with either SeOX or CHS. The calculated bio-accessibility was decreased by nearly 30% with both MyDA. This positive effect was reflected by a significant reduction (p < 0.001) in the oxidative stress (measured as serum antioxidant capacity and hepatic lipid peroxidation) induced by AFB1. Although antioxidant enzyme activities and glutathione levels were unaffected by any treatment, AFB1 significantly induced (p < 0.001) the upregulation of CYP2A6 and the downregulation of Nrf2; the latter was reverted by each MyDA. Overall, these results demonstrate that the selected MyDA are effective in limiting the AFB1 absorption rate, thereby mitigating or even reverting the oxidative stress induced by AFB1 in broilers.

Porcine pleuropneumonia (PPP) is one of the main causes leading to massive losses in the pig industry, with high economic impacts. Among different etiological agents, Actinobacillus pleuropneumoniae (APP) is responsible for severe fibrinous-necrotizing pleuropneumonia. A total of 19 different APP serotypes are currently recognized. This study aimed to identify APP serotypes isolated from pneumonic lesions in naturally infected and dead pigs in the Piedmont Region and to describe lesions. A total of 107 dead pigs with a suspected PPP diagnosis were included in this study. Lungs were evaluated using gross-pathology scoring systems, histopathology, and APP isolation and serotypes identification by multiplex PCR were conducted. Gross lung lesions were mainly represented by fibrinous pneumonia and pleuropneumonia. APP was isolated in 20/107 (18.7%) samples. PCR indicated APP DNA presence in 53/107 (49.5%) of lung samples. The most observed serotypes were serotype 2 in 24/53 (45.3%) and serotype 6 in 13/53 (24.5%) samples. Moreover, multiplex PCR results suggested a coinfection of different serotypes in five samples. This study emphasizes the importance of an integrated approach, utilizing various techniques, such as gross- and histopathology, and bacteriological culture and PCR, to enhance the diagnosis of APP infections.

Porcine pleuropneumonia (PPP) is one of the main causes leading to massive losses in the pig industry, with high economic impacts. Among different etiological agents, Actinobacillus pleuropneumoniae (APP) is responsible for severe fibrinous-necrotizing pleuropneumonia. A total of 19 different APP serotypes are currently recognized. This study aimed to identify APP serotypes isolated from pneumonic lesions in naturally infected and dead pigs in the Piedmont Region and to describe lesions. A total of 107 dead pigs with a suspected PPP diagnosis were included in this study. Lungs were evaluated using gross pathology scoring systems, APP isolation and serotypes identification by multiplex PCR were conducted. Gross lung lesions were mainly represented by fibrinous pneumonia and pleuropneumonia. APP was isolated in 21/107 (19.6%) samples. On the contrary, PCR indicated APP DNA presence in 53/107 (49.5%) of lung samples. The most observed serotypes were serotype 2 in 24/53 (45.3%) and serotype 6 in 13/53 (24.5%) samples. Moreover, multiplex PCR results suggested a coinfection of different serotypes in 5 samples. This study highlights the relevance of the different techniques application (i.e. bacteriological culture, PCR, gross and histopathology) for improving the identification of APP infections.