Ten different viral agents infecting and co-infecting children with acute gastroenteritis in Southern Italy: Role of known pathogens and emerging viruses during and after COVID-19 pandemic.

Acute gastroenteritis (AGE) represents a world public health relevant problem especially in children. Enteric viruses are the pathogens mainly involved in the episodes of AGE, causing about 70.00% of the cases. Apart from well-known rotavirus (RVA), adenovirus (AdV) and norovirus (NoV), there are various emerging viral pathogens potentially associated with AGE episodes. In this study, the presence of ten different enteric viruses was investigated in 152 fecal samples collected from children hospitalized for gastroenteritis. Real time PCR results showed that 49.3% of them were positive for viral detection with the following prevalence: norovirus GII 19.7%, AdV 15.8%, RVA 10.5%, human parechovirus (HPeV) 5.3%, enterovirus (EV) 3.3%, sapovirus (SaV) 2.6%. Salivirus (SalV), norovirus GI and astrovirus (AstV) 1.3% each, aichivirus (AiV) found in only one patient. In 38.2% of feces only one virus was detected, while co-infections were identified in 11.8% of the cases. Among young patients, 105 were ≤5 years old and 56.0% tested positive for viral detection, while 47 were >5 years old with 40.0% of them infected. Results obtained confirm a complex plethora of viruses potentially implicated in gastroenteritis in children, with some of them previously known for other etiologies but detectable in fecal samples. Subsequent studies should investigate the role of these viruses in causing gastroenteritis and explore the possibility that other symptoms may be ascribed to multiple infections.

In-depth immunophenotyping reveals significant alteration of lymphocytes in buffalo with brucellosis.

Water buffalo (Bubalus bubalis) has a prominent position in the livestock industry worldwide but still suffers from limited knowledge on the mechanisms regulating the immune against infections, including brucellosis (BRC), one of the most significant neglected zoonotic diseases of livestock. Seventy-three buffalo were recruited for the study. Thirty-five were naturally infected with Brucella spp. The aims of the study were to (i) verify the cross-reactivity of 16 monoclonal antibodies (mAbs) developed against human, bovine, and ovine antigens; (ii) evaluate lymphocyte subset alterations in BRC positive buffalo; (iii) evaluate the use of the canonical discriminant analysis (CDA), with flow cytometric data, to discriminate BRC positive from negative animals. A new set of eight mAbs (anti CD3e, CD16, CD18, CD45R0, CD79a; CD172a) were shown to cross-react with water buffalo orthologous molecules. BRC positive animals presented a significant (p < 0.0001) decrease in the percentage of PBMC (29.5 vs. 40.3), total, T and B lymphocytes (23.0 vs. 35.5, 19.2 vs. 28.9, 2.6 vs. 5.7, respectively). In contrast, they showed an increase in percentage of granulocytes (65.2 vs. 55.1; p < 0.0001) and B lymphocytes CD21neg (22.9 vs. 16.1; p = 0.0067), a higher T/B lymphocyte ratio (10.3 vs. 6.4; p = 0.0011) and CD3+ /CD21+ (14.7 vs. 8.3; p = 0.0005) ratio. The CDA, applied to 33 different flow cytometric traits, allowed the discrimination of all BRC positive from negative buffalo. Although this is a preliminary study, our results show that flow cytometry can be used in a wide range of applications in livestock diseases, including in support of uncertain BRC diagnoses.

Comprehensive phenotyping of peripheral blood monocytes in healthy bovine.

Monocytes are bone marrow derived innate myeloid cells that circulate in the blood and play important roles in infection and inflammation. As part of the mononuclear phagocytic system, monocytes provide innate effector functions, support the adaptive immune response, and play a role in the maintenance of tissue homeostasis. In addition to their role in sensing pathogen-associated molecular patterns using several pattern recognition receptors, monocytes are characterized by their ability to ingest and kill microbes, to produce cytokines and chemokines, and to present antigens to T cells. For a long time, monocytes have been considered as a homogenous cell population, characterized by the expression of CD14, the receptor of lipopolysaccharide. Studies in several species have shown that the monocyte population consists of phenotypically and functionally different cell subsets. In this review, we report a comprehensive phenotyping of monocyte subsets in cattle. In addition, the most characterizing cell markers and gating strategies for detailed immunophenotyping of bovine monocyte subsets are discussed.

The effects of intradermal M. bovis and M. avium PPD test on immune-related mRNA and miRNA in dermal oedema exudates of water buffaloes (Bubalus bubalis).

Tuberculosis (TB) is a zoonotic disease primarily caused by pathogens belonging to the genus of Mycobacterium. Programs of control and eradication for bovine TB include a screening using single intradermal tuberculin (SIT) test with Mycobacterium bovis (M. bovis)-purified protein derivatives (PPD-B) single or concurrent with Mycobacterium avium (M. avium)-purified protein derivatives (PPD-A). This study aimed to determine the effects of intradermal PPD-B and PPD-A test on immune-related mRNA and microRNAs in dermal oedema exudates of water buffaloes (Bubalus bubalis). The investigation was carried out on RNA extracted from dermal oedema exudates of 36 animals, of which 24 were M. bovis positive (M. bovis+) and 12 M. avium positive (M. avium+). The lymphocyte polarization toward Th1, Th2, TReg, and Th17 lineages was addressed by measuring the abundance of the respective cytokines and transcription factors, namely TBET, STAT4, IFNγ, and IL1ß for Th1; STAT5B, and IL4 for Th2; FOXP3 and IL10 for TReg; and RORC, STAT3, and IL17A for Th17. Due to the very low abundance of Th17-related genes, a digital PCR protocol was also applied. The abundance of microRNAs involved in the immune response against PPDs, including miR-122-5p, miR-148a-3p, miR30a, and miR-455-5p, was equally measured. Results showed that IFNγ (fold change = 2.54; p = 0.037) and miR-148a-3p (fold change = 2.54; p = 0.03) were upregulated in M. bovis+ as compared to M. avium+ samples. Our preliminary results supported the pivotal role of IFNγ in the local immune response related to PPD-B and highlighted the differential expression of miR-148a-3p, which downregulates the proinflammatory cytokines and the TLR4-mediated NF-κB activation, providing an anti-inflammation modulator in responses to mycobacterial infection.

Short communication: Milk microbiota profiling on water buffalo with full-length 16S rRNA using nanopore sequencing.

The identification of milk microbial communities in ruminants is relevant for understanding the association between milk microbiota and health status. The most common approach for studying the microbiota is amplifying and sequencing specific hypervariable regions of the 16S rRNA gene using massive sequencing techniques. However, the taxonomic resolution is limited to family and, in some cases, genus level. We aimed to improve taxonomic classification of the water buffalo milk microbiota by amplifying and sequencing the full-length 16S rRNA gene (1,500 bp) using Nanopore sequencing (single-molecule sequencing). When comparing with short-read results, we improved the taxonomic classification, reaching species level. We identified the main microbial agents of subclinical mastitis at the species level that were in accordance with the microbiological culture results. These results confirm the potential of single-molecule sequencing for in-depth analysis of microbial populations in dairy animals.

Cetacean strandings in Italy: an unusual mortality event along the Tyrrhenian Sea coast in 2013.

An unusual mortality event involving cetaceans, mainly striped dolphins Stenella coeruleoalba (Meyen, 1833), occurred along the Tyrrhenian Sea coast of Italy during the first 3 mo of 2013. Based on post-mortem analyses carried out according to body condition on 66 dolphins (54% of stranded animals), several hypotheses to explain the causes of this mortality event were proposed. Although no definitive conclusions can be drawn, dolphin morbillivirus was deemed the most likely cause, although other infectious agents (including Photobacterium damselae damselae and herpesvirus) or environmental factors may also have contributed to this recent mortality event.

Lightning deaths in three outdoor dogs: A case study.

This case report presents findings in three German Shepherd placed outdoor, dead after a night of thunderstorm.

Antibiotic Resistance of Bacteria Isolated from Clinical Samples and Organs of Rescued Loggerhead Sea Turtles (Caretta caretta) in Southern Italy.

Antimicrobial resistance affects all environments, endangering the health of numerous species, including wildlife. Increasing anthropic pressure promotes the acquisition and dissemination of antibiotic resistance by wild animals. Sea turtles, being particularly exposed, are considered sentinels and carriers of potential zoonotic pathogens and resistant strains. Therefore, this study examined the antibiotic resistance profiles of bacteria isolated from loggerhead sea turtles hospitalised in a rescue centre of Southern Italy over a 9-year period. Resistance to ceftazidime, doxycycline, enrofloxacin, flumequine, gentamicin, oxytetracycline and sulfamethoxazole-trimethoprim was evaluated for 138 strains isolated from the clinical samples or organs of 60 animals. Gram-negative families were the most isolated: Vibrionaceae were predominant, followed by Shewanellaceae, Pseudomonadaceae, Enterobacteriaceae and Morganellaceae. These last three families exhibited the highest proportion of resistance and multidrug-resistant strains. Among the three Gram-positive families isolated, Enterococcaceae were the most represented and resistant. The opportunistic behaviour of all the isolated species is particularly concerning for diseased sea turtles, especially considering their resistance to commonly utilised antibiotics. Actually, the multiple antibiotic resistance was higher when the sea turtles were previously treated. Taken together, these findings highlight the need to improve antimicrobial stewardship and monitor antibiotic resistance in wildlife, to preserve the health of endangered species, along with public and environmental health.

A decade of tuberculosis eradication programs in the Mediterranean water buffalo (Bubalus bubalis) in South Italy: Are we heading toward eradication?

The water buffalo (Bubalus bubalis) is susceptible to bovine tuberculosis (TB), which receives increased attention in areas where buffalo breeding is prevalent, such as in Southern Italy, especially in the Campania region, where 70% of the buffalo stock is bred. Since 2012, TB testing in buffalo herds has been conducted using the Single Intradermal Test (SIT), with the Comparative Intradermal test (CIT) used in cases of inconclusive results. From 2012 to 2016, the interferon-gamma (IFN-γ) test was occasionally employed experimentally in herds with TB outbreaks to expedite eradication efforts. A local TB eradication program was implemented in officially TB-free buffalo herds between 2017 and 2019. This program involves initial screening with SIT, followed by confirmatory tests, including CIT and IFN-γ, for positive reactions. Since June 2019, the IFN-γ test has replaced the CIT in officially TB-free herds upon positive SIT reactions. Additionally, in suspected and confirmed TB-outbreak herds, the IFN-γ test was used at the discretion of the competent authority. Between 2017 and 2019, approximately 295,000 buffaloes in Campania were screened annually with in vivo tests provided by TB eradication programs. During this period, 32,040 animals from 855 herds were tested using the IFN-γ test and 4,895 tested positive. Since 2020, the use of IFN-γ testing has increased, and has become a prerequisite for the acquisition of TB-free status and is being systematically applied for TB outbreak-extinction procedures. The test was performed in all breeding buffaloes in cases of doubtful SIT results in TB-free herds and when TB lesions are detected at slaughter in animals from TB-free herds. This combined approach helped detect more TB outbreaks, and thereby led to a reduction in the TB prevalence and incidence rates. By 2022, the prevalence had decreased to 1.56%, and the incidence had decreased to 0.73%, after the increased use of the IFN-γ test. This study highlights the effectiveness of implemented strategies in reducing TB in this region. Overall, the data demonstrate the successful impact of TB eradication measures and surveillance activities in reducing bubaline TB prevalence and incidence in the Campania region.

Identification of Aichivirus in a Pet Rat (Rattus norvegicus) in Italy.

We investigated the occurrence of eight potential zoonotic viruses in 91 exotic companion mammals from pet shops in southern Italy via real-time PCR and end-point PCR. The animals were screened for aichivirus, sapovirus, astrovirus, hepatitis A, noroviruses (GI and GII), rotavirus, circovirus, and SARS-CoV-2. Among the nine species of exotic pets studied, only one rat tested positive for aichivirus. The high sequence similarity to a murine kobuvirus-1 strain previously identified in China suggests that the virus may have been introduced into Italy through the importation of animals from Asia. Since exotic companion mammals live in close contact with humans, continuous sanitary monitoring is crucial to prevent the spread of new pathogens among domestic animals and humans. Further investigations on detecting and typing zoonotic viruses are needed to identify emerging and re-emerging viruses to safeguard public health.

Biochemical profile differences during the transition period based on different levels of non-esterified fatty acids at 7 weeks before parturition in Mediterranean Italian dairy buffaloes (Bubalus bubalis).

Metabolic adaptations to negative energy balance, as well as lipomobilization, influence inflammatory responses, immune function, and oxidative stress in animals. This study aimed to evaluate the biochemical profile of Mediterranean buffaloes with different levels of lipomobilization from the prepartum to the postpartum period. A total of 76 Mediterranean buffaloes were enrolled, and a weekly blood sample was taken from 7 weeks before to 6 weeks after calving. The concentration of non-esterified fatty acids (NEFAs) was determined in serum and was used to categorize buffaloes into three lipomobilization groups 7 weeks before calving: mild (NEFA-I; NEFA ≤ 0.29 mEq/L; n = 18), medium (NEFA-II; 0.29 < NEFA < 0.57 mEq/L; n = 20), and severe (NEFA-III; NEFA ≥ 0.57 mEq/L; n = 38). Two-way repeated measures ANOVA was used to assess changes within and between the groups and over time. Significant differences were found in the concentration levels of NEFA, ß-hydroxybutyrate, glucose, cholesterol, protein profile, oxygen radicals, antioxidants, lysozyme, complement, and minerals. These results suggest that both medium and severe lipomobilization groups are associated with metabolic alterations. In conclusion, buffaloes with higher NEFA levels (>0.29 mEq/L; NEFA-II and NEFA-III) at 7 weeks before calving should be monitored more closely to reduce the risk of metabolic diseases. Furthermore, the medium (NEFA-II) and severe (NEFA-III) lipomobilization groups could be associated with differences in the animals' ability to manage their metabolic status. Specifically, the severe mobilization group was most associated with a greater energy deficit during both the prepartum and postpartum periods without oxidative stress. On the contrary, the medium mobilization group was associated with a less severe energy deficit but was also associated with an inflammatory status and oxidative stress during the prepartum period. These distinctions highlight the need for tailored management strategies to address varying levels of metabolic stress in dairy buffaloes.

First description of Rhodococcus equi infection in common bottlenose dolphin (Tursiops truncatus).

Rhodococcus equi is a terrestrial bacterium and a common pathogen in foals (Equus caballus), in which causes pneumonia. This report describes for the first time the infection caused by R. equi in a common bottlenose dolphin (Tursiops truncatus) stranded in the Calabrian coast, Italy. The post mortem examination of the animal revealed lesions in lung and colon. The animal was also positive to dolphin morbillivirus. The histological study showed lesions attributable to R. equi infection, such as pyogranulomatous bacterial pneumonia and chronic granulomatous colitis. Whole genome sequencing of the isolated strain confirmed its identification as R. equi.

Detection and quantification of Brucella abortus DNA in water buffaloes (bubalus bubalis) using droplet digital polymerase chain reaction.

Brucellosis represents a major public health concern worldwide. Human transmission is mainly due to the consumption of unpasteurized milk and dairy products of infected animals. The gold standard for the diagnosis of Brucella spp in ruminants is the bacterial isolation, but it is time-consuming. Polymerase Chain Reaction (PCR) is a quicker and more sensitive technique than bacterial culture. Droplet digital PCR (ddPCR) is a novel molecular assay showing high sensitivity in samples with low amount of DNA and lower susceptibility to amplification inhibitors. Present study aimed to develop a ddPCR protocol for the detection of Brucella abortus in buffalo tissue samples. The protocol was validated using proficiency test samples for Brucella spp by real time qPCR. Furthermore, 599 tissue samples were examined. Among reference materials, qPCR and ddPCR demonstrated same performance and were able to detect up to 225 CFU/mL. Among field samples, ddPCR showed higher sensitivity (100%), specificity and accuracy of 93.4% and 94.15%, respectively. ddPCR could be considered a promising technique to detect B. abortus in veterinary specimens, frequently characterized by low amount of bacteria, high diversity in matrices and species and poor storage conditions.

Detection of Brucella abortus DNA and RNA in different stages of development of the sucking louse Haematopinus tuberculatus.

BACKGROUND: Brucellosis is considered the world's most widespread zoonotic infection. It causes abortion and sterility in livestock leading to serious economic losses and has even more serious medical impact in humans, since it can be a trigger to more than 500,000 infections per year worldwide. The aim of this study was to evaluate the role of Haematopinus tuberculatus, a louse that can parasitize several ruminants, as a new host of brucellosis. Louse specimens were collected from seropositive and seronegative water buffaloes and divided in 3 developmental stages: adults, nymphs and nits. All samples were separately screened for Brucella spp. DNA and RNA detection by Real Time PCR. In particular, primers and probes potentially targeting the 16S rRNA and the Brucella Cell Surface 31 kDalton Protein (bcsp31) genes were used for Real Time PCR and buffalo ß actin was used as a housekeeping gene to quantify host DNA in the sample. A known amount of B. abortus purified DNA was utilized for standard curve preparation and the target DNA amount was divided by the housekeeping gene amount to obtain a normalized target value. A further molecular characterization was performed for Brucella strain typing and genotyping by the Bruce-ladder, AMOS-PCR and MLVA assays. Data were statistically analysed by ANOVA. RESULTS: Brucella abortus DNA and RNA were detected in all developmental stages of the louse, suggesting the presence of viable bacteria. Data obtained by MLVA characterization support this finding, since the strains present in animals and the relative parasites were not always identical, suggesting bacterial replication. Furthermore, the detection of Brucella DNA and RNA in nits samples demonstrate, for the first time, a trans-ovarial transmission of the bacterium into the louse. CONCLUSIONS: These findings identified H. tuberculatus as a new host of brucellosis. Further studies are needed to establish the role of this louse in the epidemiology of the disease, such as vector or reservoir.

Effects of milk feeding, frequency and concentration on weaning and buffalo (Bubalus bubalis) calf growth, health and behaviour.

Growth, weight at birth and daily weight gain (DWG) on 12 water buffalo calves, starting from 6 days of age until completion of weaning, was investigated in this study. Different feeding regimens were given to two groups of animals with regard to daily milk replacer: (1) group 1 (G1) received a double concentration in single administration; whereas (2) group 2 (G2) received the same amount of milk replacer split twice daily. Blood samples were collected from each calf on days 6, 30, 60 and 90 to evaluate acute phase proteins (haptoglobin), bactericide activity, lysozime, total protein content and biochemical parameters. No differences were observed between the two groups in terms of dry matter intake, feed efficiency and live body weight at the end of the study. Interestingly, a significantly (P < 0.05) reduced DWG was observed earlier in G1 (day 45) than in G2 (day 60). Gastrointestinal disorders were not recorded throughout the experimental period, and no significant differences were recorded between the two groups for all considered parameters. This study confirms the possibility of utilising one daily administration of milk replacer in water buffalo calf during weaning. This new approach facilitates calves management, without interfering with calves growing performances.

First serological evidence of SARS-CoV-2 natural infection in small ruminants : Brief report.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) emerged in late December 2019 and spread worldwide, quickly becoming a pandemic. This zoonotic coronavirus shows a broad host range, including wildlife and domestic animals. Small ruminants are shown to be susceptible to SARS-CoV-2 but, to date, no natural infection has been reported. Herein, we performed a survey for SARS-CoV-2 among sheep and goats in the Campania region of Italy using an indirect multispecies ELISA. Next, positive sera were submitted to virus serum neutralization for the quantification of specific neutralizing antibodies. Out of 612 sheep and goats, 23 were found ELISA positive (3.75%) and 1 of them showed 1:20 neutralizing antibodies titer. No significant difference was found between the two species, as well as between male and female, geographical location and age. Our findings demonstrate that natural infection can occur in flocks in a field situation. Moreover, low susceptibility to SARS-CoV-2 is reported for sheep and goats, nevertheless, the continuous mutations of this virus open new scenarios on viral host range and tropism, highlighting the importance of investigating animal species that could represent ongoing or future possible hosts.

Possible etiological association of ovine papillomaviruses with bladder tumors in cattle.

INTRODUCTION: Bladder tumors of cattle are very uncommon accounting from 0.1% to 0.01% of all bovine malignancies. Bladder tumors are common in cattle grazing on bracken fern-infested pasturelands. Bovine papillomaviruses have a crucial role in tumors of bovine urinary bladder. AIM OF THE STUDY: To investigate the potential association of ovine papillomavirus (OaPV) infection with bladder carcinogenesis of cattle. METHODS: Droplet digital PCR was used to detect and quantify the nucleic acids of OaPVs in bladder tumors of cattle that were collected at public and private slaughterhouses. RESULTS: OaPV DNA and RNA were detected and quantified in 10 bladder tumors of cattle that were tested negative for bovine papillomaviruses. The most prevalent genotypes were OaPV1 and OaPV2. OaPV4 was rarely observed. Furthermore, we detected a significant overexpression and hyperphosphorylation of pRb and a significant overexpression and activation of the calpain-1 as well as a significant overexpression of E2F3 and of phosphorylated (activated) PDGFßR in neoplastic bladders in comparison with healthy bladders, which suggests that E2F3 and PDGFßR may play an important role in OaPV-mediated molecular pathways that lead to bladder carcinogenesis. CONCLUSION: In all tumors, OaPV RNA could explain the causality of the disease of the urinary bladder. Therefore, persistent infections by OaPVs could be involved in bladder carcinogenesis. Our data showed that there is a possible etiologic association of OaPVs with bladder tumors of cattle.

Risk Factors for the Spread of Brucellosis in Sheep and Goats in the Campania Region in the Years 2015-2020.

Brucella is a Gram-negative facultative intracellular pathogen that causes infection in sheep and goats (B. melitensis.); B. melitensis can also infect other animals. Sheep and goat brucellosis is still present in some regions of Italy, including Campania, and causes considerable economic losses and health threats. The aim of this study was to evaluate the possible risk factors influencing the spread of brucellosis among sheep and goat farms in the Campania region in order to provide the local veterinary services with practical support in evaluating and planning diagnostic, preventive and control interventions. The results of official controls for brucellosis carried out from 2015 to 2020 in the sheep and goat farms of the Campania Region were analyzed. Data were extracted from the National Veterinary Information Systems and the Laboratory Management System of the Experimental Zooprophylactic Institute of Southern Italy. Statistical analysis was carried out through the software R version 4.1.0; the dataset consisted of 37,442 observations, and 9 qualitative and quantitative variables were evaluated on 8487 farms, 248 of which were positive. The association between covariates and the outcome (presence/absence of the disease) was evaluated (Fisher and Wilcoxon tests). A logistic regression model with mixed effects was carried out. This study confirmed that brucellosis in sheep and goats in the Campania region mostly occurs through contact with infected animals imported from other farms (OR = 3.41-IC 95% [1.82-6.41]). Farms with a greater number of animals were seen to be at the greatest risk of infection (OR = 1.04-IC 95% [1.03-1.05]); previous suspension of healthy status also proved to be a risk factor (OR = 55.8-IC 95% [26.7-117]).

Prevalence of feline leukemia virus and feline immunodeficiency virus in cats from southern Italy: a 10-year cross-sectional study.

Introduction: Feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) are well-known retroviruses causing important infections in domestic cats worldwide. The goal of this study was to determine the prevalence of FeLV and FIV infections in cat living indoor and outdoor in southern Italy. Methods: The survey was conducted on 1322 stray and owned cats from the regions of Campania, Basilicata, and Calabria. It was carried out over a 10-year period to obtain a more realistic picture of the prevalence of these retroviral diseases in the country. FIV and FeLV status was determined by enzyme-linked immunosorbent assay (ELISA) using a commercial kit (SNAP Combo Plus FeLV/FIV, IDEXX). Risk factors were analysed by logistic regression. Results and Discussion: The results showed that 101/1322 (7.64%) cats were positive for FeLV antigen and 110/1322 (8.32%) cats were positive for FIV antibody. Twenty-six of the 1322 cats (1.97%) were positive for both FIV and FeLV infection. Our results are similar to those published in recent studies in Europe. A statistically significant association (p < 0.05) was found between year, province, region, lifestyle and risk of FeLV infection. FIV positivity was instead statistically associated only with year and lifestyle.