Vincenzo Di Marco

Background: In urban settings, the presence of a high density of the human population and contact with domestic and/or stray animals such as dogs and cats can be risk factors for the transmission of zoonotic protozoa parasites. Toxoplasma gondii (T. gondii) and Leishmania spp. are zoonotic protozoon parasites with significant health burdens worldwide. Methods: A cross-sectional study was used to investigate the antibody prevalence and risk factors of T. gondii and Leishmania spp. infections in 385 randomly selected dogs of Ambo, Bako, and Gojo towns of West Shewa Zone, Oromia regional state, Ethiopia. A questionnaire survey was administered to households to collect data on potential risk factors. Dog sera samples were assayed for T. gondii IgG antibodies using the direct agglutination test while Leishmania spp. specific antibodies tested using an indirect enzyme-linked immunosorbent assay (ELISA). Logistic regression was used for data analysis. Results: Overall, T. gondii and Leishmania spp. infection seroprevalence was found to be 82.86% (95% confidence interval (CI): 78.71–86.49%) and 92.47% (95% CI: 89.36–94.90%), respectively. Seropositivity for both T. gondii and Leishmania spp. was found in 82.58% of the dogs. None of the investigated factors were associated with Leishmania spp. seropositivity (p > 0.05). The seroprevalence of T. gondii was significantly different among the study towns (p = 0.003). The risk of T. gondii infection was 2.71 times higher in adult dogs than juvenile dogs (p = 0.043). Dogs kept simultaneously with other domestic animals had increased odds of T. gondii seropositivity compared to those with no other domestic animals (Adjusted Odds ratio: 1.96, p = 0.021). However, altitude, sex, breed, housing, feeding, educational level of head of the household, and dog’s living area were not significantly associated with T. gondii seropositivity (p > 0.05). Conclusions: The high seropositivity and the simultaneous presence of antibodies of T. gondii and Leishmania spp. in dogs suggest the widespread nature of these parasites in the environment and the high potential of transmission to other animals and humans. Further epidemiological studies, isolation and molecular characterization of the parasites, and educational campaigns are suggested.

The main objective of this study was to evaluate the efficacy of thymol in controlling environmental contamination in food processing facilities. The effect of thymol was tested as an agent to prevent planktonic and bacterial biofilm growth of twenty-five Listeria monocytogenes isolates from a variety of foods and five Escherichia coli isolates from a farm. The E. coli isolates were positive for extended spectrum β-lactamase (ESBL) genes. All isolates and reference strains were susceptible to thymol at Minimum inhibitory concentration (MIC) values ranging from 250 to 800 μg/mL. An interesting activity of interference with biofilm formation of L. monocytogenes and E. coli was found for thymol at sub-MIC concentrations of 200, 100, 75, and 50 μg/mL. Anti-biofilm activity ranging from 59.71% to 66.90% against pre-formed 24-h-old L. monocytogenes biofilms at concentrations of 500 or 800 µg/mL, corresponding to 2× MIC, was determined against free-living forms of six isolates chosen as the best or moderate biofilm producers among the tested strains. The property of thymol to attack L. monocytogenes biofilm formation was also observed at a concentration of 100 µg/mL, corresponding to 1/4 MIC, by using a stainless-steel model to simulate the surfaces in food industries. This study gives information on the use of thymol in food processing setting.

Polymorphisms of the prion protein gene may influence scrapie susceptibility in small ruminants through modified protein conformation. At least 47 amino acid substitutions and 19 silent polymorphisms have been described in goat PRNP reported from several countries. The objective of this study was to investigate PRNP polymorphisms of native Ethiopian goat breeds and compare the results with other goat breeds. The analysis of the prion protein gene PRNP in 229 goats belonging to three of the main Ethiopian native goat breeds showed a remarkably high frequency (> 34.6%) of p.(Asn146Ser) in these breeds, a variant involved in scrapie resistance in Cyprus. In addition, two novel amino-acid substitutions p.(Gly127Ala) and p.(Thr193Ile), with frequencies ranging from 1.5 to 7.3% were detected. Both amino acids are well conserved in prion proteins (PrP) of most species and these changes have never been reported before in goats worldwide. Residue 127 is within the N–terminal domain of PrP and is probably involved in the recruitment of neural cell adhesion molecules (NCAM). Residue 193 is within the highly conserved string of 4 threonines that plays a role in determining the efficiency of prion protein conversion towards its pathological form. Two novel coding polymorphisms and a high frequency of a scrapie protective variant indicate a high level of genetic diversity in PRNP of Ethiopian goats. This finding increases the interest in exploring PRNP polymorphisms of native goat breeds in areas where cross breeding with foreign goats has rarely occurred.

Staphylococcus aureus is the major cause of foodborne diseases worldwide. In this retrospective study, 84 S. aureus strains were characterized. The collection comprises 78 strains isolated during 1998 and 2014 from dairy products and tissue samples from livestock bred for dairy production in Sicily. One isolate was obtained from a pet (dog), one from an exotic animal (a circus elephant), and four human isolates were obtained during a severe food poisoning outbreak that occurred in Sicily in 2015. All the strains were characterized by pulsed-field gel electrophoresis (PFGE), for antibiotic resistance and presence of toxin genes. PFGE results showed 10 different pulsotypes, with three relatively frequent and three unique. The antibiotic resistance profiling showed that penicillin G (35.7%) and tetracycline (20.2%) resistance is largely spread. Most isolates contained at least one toxin gene making them a potential threat for public health. Enterotoxin sec gene was observed in 28.6% and seg in 23.8% of the strains, respectively; the human isolates were the only ones to concurrently harbor both seg and sei genes. In addition, 24 isolates were randomly selected and analyzed by multilocus sequence typing. Interestingly, the analysis showed the presence of 12 sequence types (STs), of which 6 were novel. One of them, ST700, was detected in 29% of the isolates and was found to be spread throughout Sicily. ST700 has been present in the island for almost 16 years (1998–2014) and it shows no host preference since it was isolated from different ruminant species. Four human isolates shared both the pulsotype (PT10) and the sequence type (ST9), as well as the virulence genes (seg-sei); this observation suggests that the isolates originated from a single clone, although they were obtained from two different individuals.

Goats are adapted to live in harsh areas of the world ensuring minimum levels of milk and meat production without much economic input. Scrapie is a fatal prion disease of small ruminants detected in different countries worldwide. Polymorphisms in the prion protein gene (PRNP) modify the degree of susceptibility/resistance to scrapie. PRNP polymorphisms were analysed in the four main Algerian goat breeds (Naine de Kabylie, Arbia, Mozabite, Mekatia), and in two native breeds of South Italy (Aspromontana from Calabria and Cilentana from Campania). The results were compared also with two previously described Sicilian native breeds (Girgentana and Rossa Mediterranea). Seven amino acid substitutions were detected in Naine de Kabylie goat which represents the original native Berber breed. All other breeds presented no more than six variants and common polymorphisms were present at codons, 154 and 240. Isoleucine at 137 was present in Algerian breeds only. The Italian Cilentana shared more variants with Algerian breeds whereas the Aspromontana breed was the only one to show a Serine at 127. Exclusive new variants were not detected since all polymorphisms were already described in other goats worldwide. The scrapie protective allele encoding lysine (K) at codon 222 was detected in the Naine de Kabylie and M’zabite breeds at low-frequency whereas is present with frequencies higher than 10% in all the Italian breeds reared in regions with elevated scrapie incidence. The overall results showed a substantial number of polymorphisms in PRNP, particularly in Naine de Kabylie breed, which carried also unique genotypes.

Bovine tuberculosis (bTB) is an important zoonosis, which has been re-emerging in different ecological scenarios. In Sicily, Italy, from 2004 to 2014, an anatomopathological survey for tuberculosis-like lesions both in farmed and wild animals was performed. The isolates were genotyped using spoligotyping and Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats (MIRU-VNTR) techniques. High prevalence of lesions was observed for cattle (4%), pigs (4.9%) and wild boars (6.8%), and a total of 625 Mycobacterium bovis isolates were identified. Genotyping analysis showed the presence of 37 different spoligotypes including fifteen spoligotypes not present in other Italian regions and 266 MIRU-VNTR profiles. Spoligotype SB0120 exhibited the highest prevalence in cattle (50%) and pigs (56%) and the highest genetic variety with 126 different MIRU-VNTR profiles. The isolation of M. bovis in a farmer underlines the importance of M. bovis identification during the human TB diagnostic processes. This study supported the use of the genotyping analysis as a valuable tool for the evaluation of the epidemiological role of pigs and other domestic reservoirs such as goats and the role of wildlife in the maintenance of bTB infection.

Typing of Toxoplasma gondii strains is important in epidemiological surveys, to understand the distribution and virulence of different clones of the parasite among human and animal populations. Stray dogs can be consider sentinel animals for contaminated environments playing an important but probably under- evaluated role in the epidemiology of T. gondii. We reported a rare case of acute toxoplasmosis in a stray dog due to clonal type I infection. The clonal type I, sporadic in Europe, is frequently associated with severe toxoplasmosis in humans and the control of its circulation is particularly relevant for public health. The symptomatology suggested a potential infection with the high similar parasite Neospora caninum but differential diagnosis showed that only T. gondii was involved highlighting the importance of multiple diagnostic methods beyond the clinical signs. A female stray dog approximately six-month of age presented muscular atrophy of the femoral region and hyperextension of hind limbs. Body condition score (BCS) was 20% below ideal weight, ribs had almost no fat and the sensor state was depressed. Haematological values were normal and the dog did not show any neurological abnormalities. Serological analysis showed a positive response for T. gondii immunoglobulin G (IgG) antibodies and exclude N. caninum infection. To confirm T. gondii infection, a muscle biopsy was performed and genomic DNA was extracted. PCR analysis resulted positive to T. gondii and strain genotyping reveals clonal type I infection. The dog recovered after 4 weeks of treatment with clindamycin hydrochloride and aquatic physiotherapy. Our study reports a rare and severe case of T. gondii clonal type I infection in a stray dog feeding in garbage containers. The data confirm the importance of an in vivo early diagnosis for toxoplasmosis in dog. Clinical signs are often related to specific T. gondii genotype and parasite genotyping is important in the epidemiological survey of toxoplasmosis in public health. The detection of parasitic DNA in the tissue could be an useful diagnostic method in facilitating early treatment of the disease, which is important for a timely clinical recovery.