First Description of Mycoplasma agalactiae Anatomical Localization in Naturally Infected Hard Ticks (Rhipicephalus bursa).

Mycoplasma agalactiae (Ma) is considered the primary causative agent of contagious agalactia (CA) in sheep and goats, which causes severe losses to the small ruminant dairy industry. As early as 1816, it was thought that environmental factors played a role in pathogen maintenance in endemic areas. Specifically, recent studies hypothesized a vector role for arthropods in the epidemiology of disease. The aim of this study was to investigate the presence and anatomical localization of Ma in naturally infected Riphicephalus bursa ticks to better evaluate tick-pathogen interactions. Salivary glands and ovaries of confirmed Ma-positive R. bursa were analyzed to look for the Ma antigen using immunohistochemistry (IHC). IHC showed strong positivity to Ma in the cytoplasm of salivary cells as well as in cells from the ovary. Our work demonstrated for the first time the crossing of the tick midgut barrier by Ma and the subsequent infection of organs capable of spreading the infection, and this result represents an absolute novelty in disease-related knowledge. Our preliminary results provide conclusive evidence of the potential vector role represented by hard ticks in the epidemiology of CA. Further field and laboratory investigations are necessary to confirm the tick role in the transmission of clinical CA.

First Molecular Survey to Detect Mycoplasma gallisepticum and Mycoplasma synoviae in Poultry Farms in a Strategic Production District of Sicily (South-Italy).

Mycoplasmas are recognized as avian pathogens, which may cause both respiratory disease and synovial infections in poultry, resulting in severe economic losses. Our study aims to determine the occurrence of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) among commercial and rural laying hens located in Ragusa province (South Italy), using a duplex real time PCR. Four hundred tracheal swabs were collected from seven commercial (200 swabs) and 25 rural (200 swabs) farms without any clinical disease history. Out of 400 swabs collected, 50 (12.5%) and 93 (23.25%) were positive for MG and MS, respectively. In particular, 9 (18%) and 22 (23.65%) positive swabs for MG and MS, respectively, originated from commercial farms, compared to 41 (82%) and 71 (76.34%) obtained from rural farms. Data obtained show a lower prevalence of MG than MS in the studied farms. Moreover, both pathogens were spread in rural and commercial farms. PCR could be concluded as a rapid and sensitive method for the identification of MG and MS in areas where commercial farms that are declared Mycoplasma-free and rural flocks coexist. These data highlight the importance of surveillance also in rural poultry to monitoring the occurrence of mycoplasmas strains in strategic productive districts.

Preliminary Study on the Host Response to Bivalent and Monovalent Autogenous Vaccines against Mycoplasma agalactiae in Dairy Sheep.

In Italy, dairy sheep farming represents a vital agro-industry sector, but it is still challenged by contagious agalactia (CA), which is endemic there, and vaccination is the most economical and sustainable tool for control. This study aimed to evaluate the combined Mycoplasma agalactiae (Ma)-Staphylococcus aureus (Sa) vaccine (Ma-Sa) against the Ma monovalent vaccine in ewes. Twelve primiparous Ma-free ewes were randomly grouped into three equal groups: first, the control group injected with placebo, second, the group vaccinated with the Ma monovalent vaccine, and third, the group vaccinated with Ma-Sa combined vaccine, with two S/C doses at 45-day intervals. The animals were examined for serological, hematological, and somatic cell count (SCC) changes for 17 successive weeks. A significant increase in anti-Ma antibody mean titers, leukocytes, and platelets was observed in the vaccinated animals, with the highest values in those who received the combined vaccine. Neutrophils were high only in the animals who received the combined vaccine. SCC was lower in the vaccinated animals during the first six weeks. This study concludes that the combined Ma-Sa vaccines enhance immune response and potentiate its efficacy against Ma. This improvement might be attributed to the sensitization/activation effect of S. aureus on platelets, which are recoded to act as a key regulator for the coordination of all components of the innate immune system. Even though this study included a small number of animals, its findings about the potentialities of this inactivated vaccine in the control of CA are strongly encouraging. Further confirmation might be needed through additional replicates and a challenge study is needed before proceeding with widespread use.

Preliminary Results on the Prevalence of Salmonella spp. in Marine Animals Stranded in Sicilian Coasts: Antibiotic Susceptibility Profile and ARGs Detection in the Isolated Strains.

The presence of Salmonella spp. in marine animals is a consequence of contamination from terrestrial sources (human activities and animals). Bacteria present in marine environments, including Salmonella spp., can be antibiotic resistant or harbor resistance genes. In this study, Salmonella spp. detection was performed on 176 marine animals stranded in the Sicilian coasts (south Italy). Antibiotic susceptibility, by disk diffusion method and MIC determination, and antibiotic resistance genes, by molecular methods (PCR) of the Salmonella spp. strains, were evaluated. We isolated Salmonella spp. in three animals, though no pathological signs were detected. Our results showed a low prevalence of Salmonella spp. (1.7%) and a low incidence of phenotypic resistance in three Salmonella spp. strains isolated. Indeed, of the three strains, only Salmonella subsp. enterica serovar Typhimurium from S. coeruleoalba and M. mobular showed phenotypic resistance: the first to ampicillin, tetracycline, and sulphamethoxazole, while the latter only to sulphamethoxazole. However, all strains harbored resistance genes (blaTEM, blaOXA, tet(A), tet(D), tet(E), sulI, and sulII). Although the low prevalence of Salmonella spp. found in this study does not represent a relevant health issue, our data contribute to the collection of information on the spread of ARGs, elements involved in antibiotic resistance, now considered a zoonosis in a One Health approach.

Molecular Characterization of Antimicrobial Resistance and Virulence Genes of Bacterial Pathogens from Bovine and Caprine Mastitis in Northern Lebanon.

Mastitis is an infectious disease encountered in dairy animals worldwide that is currently a growing concern in Lebanon. This study aimed at investigating the etiology of the main mastitis-causing pathogens in Northern Lebanon, determining their antimicrobial susceptibility profiles, and identifying their antimicrobial resistance (AMR) genes. A total of 101 quarter milk samples were collected from 77 cows and 11 goats presenting symptoms of mastitis on 45 dairy farms. Bacterial identification was carried out through matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Antimicrobial susceptibility was tested by disc diffusion and broth microdilution methods. Molecular characterization included polymerase chain reaction (PCR) screening for genes encoding extended-spectrum beta-lactamases (ESBLs) and plasmid-mediated AmpC among Enterobacterales isolates, and virulence factors among Staphylococcus isolates. Escherichia coli isolates were subjected to phylogenetic typing by a quadruplex PCR method. The most frequently identified species were Streptococcus uberis (19.2%), Streptococcus agalactiae (15.1%), E. coli (12.3%), and Staphylococcus aureus (10.96%). Gram-positive bacteria were resistant to macrolides and tetracycline, whereas gram-negative bacteria displayed resistance to ampicillin and tetracycline. Two ESBL genes, blaTEM (83.3%) and blaOXA (16.7%), and one AmpC beta-lactamase gene, blaCMY-II (16.7%), were detected among six E. coli isolates, which mainly belonged to phylogenetic group B1. Among Staphylococcus spp., the mecA gene was present in three isolates. Furthermore, four isolates contained at least one toxin gene, and all S. aureus isolates carried the ica operon. These findings revealed the alarming risk of AMR in the Lebanese dairy chain and the importance of monitoring antimicrobial usage.

Detection of Mycoplasma agalactiae in Ticks (Rhipicephalus bursa) Collected by Sheep and Goats in Sicily (South-Italy), Endemic Area for Contagious Agalactia.

The aim of this preliminary study was to investigate the presence of Mycoplasma agalactiae (Ma) or other Contagious Agalactia (CA) causative organisms, in hard ticks infesting milking sheep and goats in endemic areas for CA in Sicily (South-Italy). Although there is accumulating evidence to support the role of ticks in the transmission of blood-borne haemoplasmas, information regarding their role in the transmission of CA, remains scarce. Ticks (n = 152) were collected from 25 lactating sheep and goats from three farms with previous outbreaks of CA. Microbiological and biomolecular, as well as serological analysis were performed on milk, tick, and serum samples, respectively. Rhipicephalus bursa species predominated, comprising 84.8% of the sampled ticks. Mycoplasma-like colonies were isolated from 5/56 (8.9%) tick pools and were identified as Ma by specific PCR and 16S rRNA gene sequencing. Unexpectedly, the organism was isolated from R. bursa ticks recovered only from animals whose milk tested negative for the pathogen. This preliminary demonstration suggests the potential role for ticks to act as a reservoir for the organisms, with potential involvement in the spread and maintenance of CA. Further work is required to determine the location of the organisms within the body of the ticks and to assess transmission potential.

Monitoring Campaign over an Edible Dormouse Population (Glis glis; Rodentia: Gliridae) in Sicily: First Report of Mesocestodiasis.

This study reports on the health status of the edible dormouse (Glis glis) living in Nebrodi Park (Sicily, Italy), responsible for nut crop damage in the area. In the frame of a monitoring campaign for potential zoonotic risk involving 30 dormice, rectal and conjunctival swabs and fur and nest content were collected for bacteriological and parasitological examinations, respectively. A large presence of fleas belonging to Monopsyllus sciurorum was found. Necropsy of a dead dormouse revealed an infection of Mesocestoides lineatus, whose cysts were found in the abdomen cavity and on the liver; this is the first report of this in this species. Further studies are necessary to identify their role in the environment, considering the limited knowledge of this species in Italy.

Diagnostic Findings in a Confirmed Outbreak of Brucella ovis Infection in a Traditional Sheep Farm in Sicily (South-Italy).

Aim of this study is to report a laboratory investigation performed following the isolation of Brucella ovis, causing ovine epididymitis, in a traditional sheep farm in Sicily (South Italy). This disease represents a newly emerging risk for Italian livestock and is listed among diseases of EU priority (EU Reg 2016/429). Blood samples from 56 rams and 143 ewes were analyzed by both Enzyme-Linked Immunosorbent Assay (ELISA) and Complement Fixation Test (CFT). Genital swabs from all rams and 15 lactating ewes were collected to perform real-time PCR. Eighteen serologically positive rams were slaughtered and postmortem-inspected. Samples of testicle, epididymis, lymph nodes, and urine were also collected in order to perform microbiological, molecular, and histopathological analysis. Twelve slaughtered rams showed anatomo-pathological lesions. Real-time PCR for B. ovis BOV_A0504 gene was positive for 13 testicles and epididymis and 11 urine while B. ovis was isolated from epididymis and testicles of 7 slaughtered rams. This is the first exhaustive laboratory report of a microbiological, molecular, and serological pattern of the disease in sheep in Italy. Despite the impact on health and animal welfare, the epidemiology of B. ovis infection is still unknown, particularly in our country where the disease is considered endemic.

Microbiological and Chemical Analysis of Land Snails Commercialised in Sicily.

In this study 160 samples of snails belonging to the species Helix aspersa maxima and Helix aspersa muller were examined for chemical and microbiological analysis. Samples came from Greece and Poland. Results showed mean concentration of cadmium (0.35±0.036 mg/kg) and lead (0.05±0.013 mg/kg) much higher than the limit of detection. Mercury levels in both species were not detected. Microbiological analysis revealed the absence of Salmonella spp. and Clostridium spp. in both examined species. E. coli and K. oxytoca were observed in Helix aspersa maxima and Helix aspersa muller. Furthermore, one case of fungi positivity in samples of Helix aspersa muller was found. The reported investigations highlight the need to create and adopt a reference legislation to protect the health of consumers.