Molecular differentiation of Sarcocystis miescheriana and Sarcocystis suihominis using a new multiplex PCR targeting the mtDNA cox1 gene in wild boars in southern Italy.

The increase of wild boar populations density and their meat consumption across Europe could expose humans to a plethora of foodborne diseases as sarcocystosis, caused by the zoonotic protozoan Sarcocystis suihominis. Humans become infected by eating raw or undercooked pig (Sus scrofa domesticus) containing S. suihominis sarcocysts. Despite this, to date very few data are available on the risk of infection by this parasite to wild boar (Sus scrofa) meat consumers. Thus, the present study aimed to assess the occurrence of Sarcocystis spp. in wild boars from southern Italy, applying both histology and a new multiplex PCR assay targeting the cox1 gene. Between 2019 and 2020, 997 muscle tissues (i.e., n = 269 oesophagus, n = 277 diaphragms, n = 298 hearts, n = 153 tongues) from 311 wild boars were collected and screened by a combined histological and molecular approach. Overall, 251 (80.7%) animals tested were positive for Sarcocystis spp., and S. miescheriana whose definitive hosts are canids, was the only molecularly identified species. A statistically significant difference (p < 0.05) in the prevalence of Sarcocystis infection was found according to the wild boar age and muscle tissue. Findings outlined the low zoonotic potential of infection to humans via wild boar meat consumption in Italy and the importance of the application of new molecular methods in distinguishing different Sarcocystis species.

Trichinella britovi in wild boar meat from Italy, 2015-2021: A citizen science approach to surveillance.

As a result of the increase of game meat intended for human consumption through Europe, a plethora of food-borne diseases, including trichinellosis, may occur in consumers, posing a relevant public health threat. Thus, this study aims to a citizen science approach to monitor the occurrence of Trichinella spp. in wild boar meat intended for human consumption, evaluating the risk of infection for consumers. Following the European Regulation 2015/1375 (laying down specific rules on official controls for Trichinella in meat), from 2015 to 2021, hunters (n = 478) were involved to collect diaphragm pillar samples of wild boars from mainland southern Italy, which were tested for Trichinella spp. L1 larvae via HCl-pepsin digestion and Multiplex PCR. Overall, 139,160 animals were collected (average of 19,880 per year), being 14 (i.e., 0.01%) tested positive to Trichinella britovi by the combined biochemical and molecular approach. An average larval burden of 28.4 L1 per gram of meat was found (minimum 3.2 - maximum 132.6). A statistically significant difference was found in the prevalence according to hunting seasons (p < 0.01, with higher values in 2016 and 2021) and regions of the study area (p < 0.01). No statistically significant decrease in the prevalence of T. britovi throughout the study period was found (p = 0.51), except in Apulia region (p < 0.01). These findings revealed a stable prevalence of T. britovi in wild boar meat intended for human consumption, suggesting a risk of infection for consumers, especially hunters and local markets users. Citizen science surveillance models could be promoted to improve trichinellosis control and prevention in a One Health perspective.

First description of Eucoleus garfiai (Gallego and Mas-Coma, 1975) in wild boar (Sus scrofa) in Italy.

Eucoleus garfiai (syn. Capillaria garfiai) is a nematode infecting lingual tissue of domestic and wild swine. Prevalence data for this parasite are scant and often related to accidental findings, occurring only in Japan and a few European countries. In this study, an epidemiological survey was performed in order to identify E. garfiai in wild boar from the Campania region, southern Italy. A total of 153 wild boar carcasses were inspected over the course of two hunting seasons (2019-2020). Histological examinations were performed on tongue samples fixed and stained with haematoxylin and eosin. The scraping of dorsal tongue tissue was carried out to collect adult worms for parasitological examination. Out of 153 wild boars, 40 (26.1%, 95% CI: 19.8-33.6%) tested positive for helminths and/or eggs in tongue tissues. Parasites were identified morphologically and identification was confirmed by molecular analysis of the 18S rRNA gene, showing a 99% nucleotide match with E. garfiai sequences available in literature. No statistically significant differences were found according to age, sex nor hunting province. Our findings agree with previous histopathological data confirming the low pathogenic impact of this nematode. The present study represents the first report of E. garfiai in wild boar from Italy.

Haematological and biochemical abnormalities in hunting dogs infected with Acanthocheilonema reconditum, associated risk factors, and a European overview.

Acanthocheilonema reconditum is a filarial parasite transmitted by arthropods (fleas, lice, and ticks) that infect dogs. There is minimal published data available to date on potential haematological and biochemical changes associated with this parasitic infection. Study aims were (i) provide an overview of A. reconditum in Europe, (ii) define A. reconditum prevalence and risk factors in a specific dog population (hunting) from southern Italy, and (iii) assess the frequency of haemato-biochemical abnormalities associated with infection. Blood samples collected from 3020 dogs were tested by a modified Knott's technique to count and identify microfilariae. Eighty-four dogs were infected by A. reconditum (2.78%; 95% CI 2.19-3.37%). Microfilariae ranged from 1 to 212/ml. Based on clinical examination, all but six dogs with non-specific symptoms were healthy. Haematological abnormalities included leucocytosis (n = 15), with eosinophilia (n = 14) and monocytosis (n = 13). Serum biochemical abnormalities included increased total serum proteins (n = 19), albumins (n = 7), total globulins (n = 14), ALT (n = 1), and ALP (n = 1); one dog was hypoalbuminemic, and BUN was mildly increased in 2 dogs. Risk factors included the province origin (Napoli, OR=5.4, 95%CI: 2.1-14.0; Caserta, OR=5.1, 95%CI: 2.5-10.6), hunting wild mammals (OR=2.8, 95% 95%CI: 1.6-4.8), and ectoparasite infestation (OR=1.9, 95%CI: 1.1-3.1). There was a negative correlation between microfilaraemic load and decreased albumin level (-0.37; p=0.021). Our results showed that A. reconditum circulates within the hunting dog population of southern Italy, with seemingly low pathogenic potential.

Genotyping of Toxoplasma gondii in wild boar (Sus scrofa) in southern Italy: Epidemiological survey and associated risk for consumers.

Toxoplasma gondii is a widespread protozoan parasite (phylum Apicomplexa), which causes a zoonotic parasitic disease, known as toxoplasmosis. The aim of this study was to evaluate the occurrence and genotypes of T. gondii in wild boars of southern Italy and thus to assess the risk of infection for consumers. The boars were inspected during the hunting season within the regional project 'Wild Boar Emergency Plan in Campania', and molecular analyses were performed on 338 boars analysing a total number of 884 matrices (263 brains, 310 hearts and 311 masseter muscles). Toxoplasma gondii was detected in 134 out of 338 boars (39.6%). No significant statistical difference between genders was found (χ2  = 0.15 p = .70). The prevalence was 47.1%, 39.3% and 39.2% in piglets, yearlings and adults, respectively (χ2  = 0.41; p = .81). The highest prevalence of T. gondii was found in masseter muscles (74/311, 23.8%), followed by the heart (70/310, 22.6%) and brain (58/263, 22.0%), respectively. Microsatellite (MS) analysis of 11 samples revealed eleven T. gondii genotypes (nine atypical, one belonging to type II one to type III). Most of the genotypes found were thus atypical and may be virulent in humans. Hierarchical clustering analysis showed the presence of three distinct clusters, with the majority of atypical genotypes in the GII-GIII cluster. The high prevalence of infection in masseters highlights the potential risk for public health, considering that this muscle is commonly used to prepare raw meat products ('guanciale' and sausages), which may be a source of T. gondii infection in humans. Wild boars may act as an interface role between wildlife, livestock and humans. Our data highlight the urgent need to minimize the risk of infection for animals and humans by setting up a surveillance programme and preventive strategies in a One Health approach to wildlife species.

Massive Taenia hydatigena Cysticercosis in a Wild Boar (Sus scrofa) from Italy.

PURPOSE: Taenia hydatigena cysticercosis, due to Cysticercus tenuicollis, is a parasitic disease infecting domestic and wild animals worldwide causing economic and productive losses. Nonetheless, little attention has been paid to the role of the wild ungulates in the epidemiology of this disease. In the last years, the increasing population of wild boars in Europe has raised the attention of researchers on their role in the spreading of several infections, including those caused by cestodes. Herein, we report the description of a massive infection due to T. hydatigena cysticercosis in a wild boar from southern Italy. METHODS: An adult female boar was examined during the hunting season 2018 within the regional project "Piano Emergenza Cinghiali in Campania". A complete necropsy was performed on the boar carcass and all viscera were examined to determine number and location of the cysts. Morphological and molecular analyses of the cysts were performed to confirm the C. tenuicollis identity. RESULTS: The boar examined has revealed an impressive massive infection with 265 cysts. Measurements of the large and small larval hooks showed a mean of length as 200.3 µm and 136.8 µm, respectively. Molecular analysis of Cox1 and ND1 mitochondrial genes confirmed the C. tenuicollis identity. CONCLUSIONS: Our findings suggest that wild boar could be involved in the epidemiology of T. hydatigena, due to the significant amount of boar raw offal available to definitive hosts (i.e., hunting dogs, foxes and wolves), during the hunting seasons.

Preliminary Observations of the Effect of Garlic on Egg Shedding in Horses Naturally Infected by Intestinal Strongyles.

Intestinal strongyles are the most common endoparasites of horses, and anthelmintic treatments are the main strategy to control these nematodes. However, the development of anthelmintic resistance has led to a decreased efficacy of synthetic drugs, and for this reason, there is a growing interest in alternative control strategies as the use of medicinal plants. The aim of the present study was to determine the in vivo efficacy of garlic (Allium sativum) in horses naturally infected by intestinal strongyles. The field trial was conducted in a horse trotter farm in Southern Italy. Fifteen mares were selected based on fecal egg count >200 eggs per gram and allocated into three groups of five animals: fresh garlic group (FG group), animals received 40 g of fresh crushed garlic once daily for 15 days; dry garlic group (DG group), animals received 40 g of commercial dry garlic flakes food supplement once daily for 15 days; and control group (C group), not treated. Two weeks after the first administration of garlic, fecal egg count reduction test showed failure of garlic to reduce intestinal strongyles egg shedding (-11.7% and -19.4% for FG and DG groups, respectively). Red blood cell count values were in the normal ranges over the entire period of garlic administration. In our study model, the oral administration of garlic formulations has no effect on reducing the egg shedding of intestinal strongyles, and the garlic supplementation over a short period of time is not responsible for hematological changes in horses.

Distribution and risk factors associated with Babesia spp. infection in hunting dogs from Southern Italy.

Canine babesiosis is caused by haemoprotozoan organisms of the genus Babesia which are transmitted by the bite of a hard tick. The aim of this survey was to determine the prevalence and risk factors associated with Babesia species infections in hunting dogs from Southern Italy. Blood samples were collected from 1311 healthy dogs in the Napoli, Avellino and Salerno provinces of the Campania region of Southern Italy. Serological testing was performed using two enzyme-linked immunosorbent assays (ELISA), with one designed to detect B. canis and B. vogeli antibodies, and the other designed to detect B. gibsoni antibodies. Blood samples were also tested by quantitative real-time polymerase chain reaction (qPCR) assays for amplification of B. canis, B. vogeli and B. gibsoni DNA. The overall seroprevalence for B. canis/B. vogeli was 14.0%, compared to 0.2% for B. gibsoni. B. canis and B. vogeli PCR positive rates were 0.15% and 1.1%, respectively. B. gibsoni DNA was not amplified by qPCR. Male gender (OR 1.85), increased age (OR 1.01), long hair coat (OR 1.61) and living in Salerno province (OR 1.71) represented risk factors for B. canis/B. vogeli seroreactivity. Hunting dogs in Southern Italy are often exposed to B. canis/B. vogeli, however Babesia spp. infection was infrequently detected using qPCR. Further studies are needed to determine the extent to which Babesia spp. cause clinical disease in hunting dogs, and to evaluate the potential epidemiological relationships between hunting dogs and wild animal populations sharing the same area.

Seroprevalence and risk factors associated with Ehrlichia canis, Anaplasma spp., Borrelia burgdorferi sensu lato, and D. immitis in hunting dogs from southern Italy.

Canine vector-borne diseases (CVBDs) are caused by a range of pathogens transmitted to dogs by arthropods. The present study investigates Ehrlichia canis, Anaplasma spp., Borrelia burgdorferi sensu lato, and Dirofilaria immitis seroprevalences in hunting dogs from southern Italy. Dogs (no. 1335) were tested using a commercial in-clinic enzyme-linked immunosorbent assay kit. Odds ratios (ORs) were calculated by logistic regression analysis to identify risk factors. Overall, 138/1335 dogs (10.3%) were seroreactive to at least one CVBD pathogen. E. canis, Anaplasma spp., B. burgdorferi s.l., and D. immitis seroprevalences were 7.6, 4.4, 0.3, and 0.2%, respectively. E. canis and Anaplasma spp. co-exposures were found in 30 dogs (2.2%), compared with Anaplasma spp. and B. burgdorferi s.l. co-exposures in 2 dogs (0.1%). Adult age was a risk factor for E. canis (OR 2.35) seroreactivity whereas hunting fur-bearing animals for E. canis (OR 4.75) and Anaplasma spp. (OR 1.87), respectively. The historical presence of tick infestation was identified as a risk factor for positivity to E. canis (OR 2.08) and Anaplasma spp. (OR 2.15). Finally, larger dog pack size was significantly associated with E. canis (OR 1.85) and Anaplasma spp. (OR 2.42) exposures. The results of the present survey indicated that hunting dog populations are at relative risk of CVBDs in southern Italy. Further studies are needed to evaluate the role of hunting dogs in the epidemiology of vector-borne organisms due to sharing common environments with wild, sympatric animal populations.