Genomics insights into a Mycobacterium pinnipedii isolate causing tuberculosis in a captive South American sea lion (Otaria flavescens) from Italy.

Tuberculosis (TB) affects humans and other animals, and it is caused by bacteria within the Mycobacterium tuberculosis complex (MTBC). In this study, we report the characterisation of Mycobacterium pinnipedii that caused a TB case in a sea lion (Otaria flavescens) kept in an Italian zoo. The animal died due to severe, progressive disorders involving the respiratory and gastro-enteric systems and the skin. At necropsy, typical gross lesions referable to a TB generalised form were found. In particular, nodular granulomatous lesions were detected in the lungs and several lymph nodes, and colonies referable to Mycobacterium spp. were isolated from lung, mesenteric, and mediastinal lymph nodes. The isolate was identified by PCR as a MTBC, had a spoligotype SB 1480 ("seal lineage"), and was characterised and characterised by whole-genome sequencing analysis confirming that the MTBC involved was M. pinnipedii. The analysis of the resistome and virulome indicated the presence of macrolide and aminoglycoside resistance genes intrinsic in M. tuberculosis [erm-37 and aac(2')-Ic] and confirmed the presence of the region of difference 1 (RD1), harbouring the esxA and esxB virulence genes, differently from its closest taxon, M. microti. As for other MTCB members, M. pinnipedii infection can spill over into non-pinniped mammalian species; therefore, zoological gardens, veterinary practitioners, and public health officers should be aware of the hazard posed by tuberculosis from marine mammals. Since the isolate under study, as well as all available genomes of M. pinnipedii investigated in this study retains almost all the M. tuberculosis virulence genes, it could indeed cause infection, lesions, and disease in other animal species, including humans.

Mycobacterium microti Infection in Wild Boar (Sus scrofa): Histopathology Analysis Suggests Containment of the Infection.

The European wild boar (WB) (Sus scrofa) population has rapidly expanded over the years, raising public health concerns over the species reservoir of several pathogens, including Mycobacterium microti (Mm), a Mycobacterium tuberculosis complex member. In this study, we aimed to investigate the Mm natural infection in WB in Lombardy and Emilia Romagna Italian regions by statistically evaluating the granulomatous lesions' histological features and Mm microbiological isolation. We analyzed 103 WB retropharyngeal and submandibular lymph nodes (LNs) for Mm identified by gyrB PCR-restriction fragment length polymorphism, and were retrospectively selected and histologically assessed. For each sample, Hematoxylin-eosin and Ziehl-Neelsen stained slides were evaluated. Considered histological variables were: the number of granulomas, size and maturational stage of granulomas, granulomas completeness within the section, number of multinucleated giant macrophages (MGMs), and acid-fast (AF) bacilli per granuloma. Furthermore, Mm microbiological results were also considered. Mm microbiological isolation was negatively influenced by granulomas maturation and positively affected by AF bacilli's presence within the section. Granuloma maturation was positively influenced by granuloma size and granuloma incompleteness and negatively affected by the number of granulomas in the section and the number of MGMs within the granuloma. The results indicate that granuloma maturation should ensures an efficient containment of Mm infection in the WB, suggesting that the intra-species transmission of the disease might be an unlikely event.

Infection by Mycobacterium caprae in three cattle herds in Emilia-Romagna Region, Northern Italy.

Bovine tuberculosis (bTB) is a contagious chronic disease associated with progressive emaciation (starvation) and tubercles (granuloma) formation commonly caused by Mycobacterium bovis. In cattle, M. caprae may also be responsible for bTB. In EU, human tuberculosis due to M. bovis had a notification rate of 0.04 cases per 100,000 inhabitants in 2017, but data did not include M. caprae infections. From September 2018 to April 2019, bTB outbreaks were investigated in three neighbouring dairy cattle herds in Parma province, Northern Italy. Parma municipality belongs to an officially free of bovine tuberculosis (OTF) Italian region. Official testing on cattle herds, performed every three years as legally required, revealed no positive animals. Tubercular lesions were found during the post mortem (PM) examination of slaughtered cattle and M. caprae genotype SB0418/VNTR 4,3,5,3,4,5,2,2,4, 3,15,5 was isolated. This report confirms the crucial importance of PM veterinary inspection at slaughterhouse, despite the OTF status of cattle herds.

Acute lameness in a cat with disseminated mycobacteriosis.

Mycobacterium avium infection was diagnosed in an adult cat showing acute lameness of the right hind limb, enlargement of the right popliteal lymph node and two cutaneous nodular lesions of the right chest wall. Conventional radiography of the proximal tibia showed a proliferative osteolytic lesion. Cytological examination of the right popliteal lymph node and the nodular skin lesions fine needle aspiration smears, demonstrated granulomatous inflammation with many negative staining bacilli within macrophages or in smears background. The diagnosis was confirmed by Ziehl­Neelsen staining of the smears and the identification of mycobacteria was performed by microbiological and molecular methods. Histopathology performed after the necropsy revealed disseminated mycobacteriosis with granulomatous mesenteric lymphadenitis, granulomatous pneumonia, hepatitis and tibial osteomyelitis. M. avium is a well­known agent of gastro­enteric, respiratory or disseminated disease in immunocompromised cats but there are few cases reported in literature of bone involvement in systemic mycobacteriosis.

Types of pesticides involved in domestic and wild animal poisoning in Italy.

Data obtained from samples of poisoned domestic and wild animals sent for toxicological evaluation during the period between 2005 and 2014 have been analyzed. Among the 4606 tested samples, the majority of which were collected in the northern regions of Italy, 2006 (43.55%) were found positive for pesticides. Analytical detections were performed via solvent extraction followed by separation and chromatographic characterization and all the methods applied for the toxicological investigations were developed by IZSLER. Insecticides, mainly represented by acetylcholinesterase inhibitors (carbamates 17.55%, n = 352; organophosphates 15.15%, n = 304) and organochlorines (29.21%, n = 586), were found to be the first category of pesticides involved in intoxications, in both domestic and in wild animals, followed by rodenticides (anticoagulant rodenticides 21.09%, n = 423; zinc phosphide 2.59%, n = 52; chloralose 0.95%, n = 19 and thallium 0.15%, n = 3) and molluscicides (metaldehyde 6.63%, n = 133). Second and third generation anticoagulants (bromadiolone and brodifacoum) were the most represented (10.52%, n = 211) but also first generation compounds (i.e. coumatetralyl and warfarin) were still found responsible of intoxications. Even if some pesticides are frequently involved in domestic animal poisoning (i.e. metaldehyde and strychnine), they did not show the same diffusion in wild animals. In particular, unlike domestic species, cyanide and pyrethroids have not been found responsible of intoxications in wild animals. Interestingly, a great number of positive samples involved banned pesticides like α- (14.41%, n = 289) and ß- (14.16%, n = 284) endosulfan, carbofuran (5.73%, n = 115), methamidophos (9.47%, n = 190), strychnine (6.23%, n = 125) but, on the other hand, many positives were due to the exposure to commercially available products (i.e. metaldehyde and anticoagulant rodenticides). Thus, together with measures aimed to reduce illegal uses, educational campaigns and a wider range of compounds to detect would be beneficial in order to address the issue of animal poisoning, which besides has also repercussions on environmental and public health.

Mycobacterium genavense Infection in a Domestic Ferret (Mustela putorius furo).

Mycobacterium genavense infection was diagnosed in an adult ferret with ptosis of the left eye, a proliferative lesion of the conjunctiva of the nictitating membrane, conjunctival swelling, and tumefaction of the periorbital tissues with a watery ocular discharge and the presence of a retrobulbar mass. The diagnosis was based on characteristic cytology of the retrobulbar mass and left mandibular lymph node that revealed granulomatous inflammation. Ziehl-Neelsen staining showed the presence of positive acid-fast bacilli in the cytoplasm of the macrophages. The diagnosis was confirmed by sequence analysis of the 16S rRNA gene amplified by using a multiplex polymerase chain reaction from a fresh lymph node biopsy. Therapy with marbofloxacin, rifampicin, and clarithromycin was recommended for 6 months and after this period, the veterinarian who was treating the ferret reported the disappearance of clinical signs. Six months after the end of the antibiotic treatment, the symptoms described previously reoccurred. Confirmatory laboratory tests were not performed but a recurrence of M genavense infection was suspected and the veterinarian, in agreement with the owner, euthanized the ferret.

Identification and Characterization of Fringilla coelebs Papillomavirus 1 (FcPV1) in Free-living and Captive Birds in Italy.

A papillomavirus (PV) was identified by negative-staining electron microscopy in skin lesions of two bird species (Fringillidae) in Italy. Genetic analyses revealed an FcPV1 with a low genetic variability in the E6, E7, E1, E2, and L1 genes and the long control region when compared to the FcPV1 reference strain.

Lead, cadmium and organochlorine pesticide residues in hunted red deer and wild boar from northern Italy.

The objectives of the present study were to assess heavy metal cadmium (Cd), lead (Pb) and organochlorine pesticide concentrations in tissues of red deer (Cervus elaphus) and wild boar (Sus scrofa) from nine hunting areas and to evaluate related risk factors for the host animal. Over a period of 2 years, a total of 1055 and 210 masseters, 424 and 201 livers, 642 and 152 kidneys were collected from wild boar and red deer, respectively, and concentrations of Cd, Pb and organochlorine pesticides were determined. Comparing the two species, Cd concentration in the kidney (3.72 mg/kg), liver (0.67 mg/kg) and muscle (0.02 mg/kg) of wild boar was found to be significantly higher than in the organs of red deer (1.02 mg/kg in the kidneys, 0.07 mg/kg in the liver and 0.006 mg/kg in muscle). Mean Pb concentrations were found to be similar in both animals, with 0.39, 0.52 and 2.60 mg/kg detected in the wild boar kidney, liver and muscle, respectively, and 0.24, 0.21 and 2.04 mg/kg in the respective organs of the red deer. No difference in concentrations were found based on age class, location of tissue sample or contaminant in the case of wild boar. By contrast, a significantly lower Cd concentration was found in the kidney of the young red deer. The search for organochlorine pesticides in both red deer and wild boar produced negative results with values below the limits of detection. Due to the high levels of renal Cd and muscle Pb detected in wild boar and red deer, further research needs to be carried out in an effort to identify the source of contamination and preserve the health of animals and humans.