Guido Rocchigiani

Molecular detection of tick-borne pathogens in wild red foxes (Vulpes vulpes) from Central Italy

Spleen samples from 153 red foxes, shot during regular hunting season in the province of Pisa (Central Italy), were examined to detect DNA of Anaplasma phagocytophilum, Ehrlichia canis, Coxiella burnetii, Francisella tularensis, Hepatozoon canis and Babesia sp./Theileria sp. DNA of vector-borne pathogens was detected in 120 (78.43%; 95% CI: 71.06-84.66%) foxes. Specifically, 75 (49%; 95% CI: 40.86-57.22%) animals scored PCR-positive per H. canis, 68 (44.44%; 95% CI: 36.42-52.69%) for E. canis, 35 (22.88%; 95% CI: 16.48-30.35%) for piroplasms (Theileria annae), 3 (1.96%; 95% CI: 0.41-5.62%) for C. burnetii and 1 (0.65%; 95% CI: 0.02-3.59%) for A. phagocytophilum. No positive reaction was observed for F. tularensis. Fifty-six animals (36.6%; 95% CI: 28.97-44.76%) were positive for two or three pathogens. Red foxes result to be involved in the cycle of vector-borne pathogens that are associated to disease in dogs and humans.

Behavioral asymmetries in ticks - Lateralized questing of Ixodes ricinus to a mechatronic apparatus delivering host-borne cues

Ticks are considered among the most dangerous arthropod vectors of disease agents to both humans and animals worldwide. Lateralization contributes to biological fitness in many animals, conferring important functional advantages, therefore studying its role in tick perception would critically improve our knowledge about their host-seeking behavior. In this research, we evaluated if Ixodes ricinus (L.) (Ixodiidae) ticks have a preference in using the right or the left foreleg to climb on a host. We developed a mechatronic device moving a tuft of fox skin with fur as host-mimicking combination of cues. This engineered approach allows to display a realistic combination of both visual and olfactory host-borne stimuli, which is prolonged over the time and standardized for each replicate. In the first experiment, the mechatronic apparatus delivered host-borne cues frontally, to evaluate the leg preference during questing as response to a symmetrical stimulus. In the second experiment, host-borne cues were provided laterally, in an equal proportion to the left and to the right of the tick, to investigate if the host direction affected the questing behavior. In both experiments, the large majority of the tested ticks showed individual-level left-biased questing acts, if compared to the ticks showing right-biased ones. Furthermore, population-level left-biased questing responses were observed post-exposure to host-mimicking cues provided frontally or laterally to the tick. Overall, this is the first report on behavioral asymmetries in ticks of medical and veterinary importance. Moreover, the mechatronic apparatus developed in this research can be exploited to evaluate the impact of repellents on tick questing in highly reproducible standardized conditions.

Occurrence of Toxoplasma gondii in Carcasses of Pigs Reared in Intensive Systems in Northern Italy

To evaluate the occurrence of Toxoplasma gondii and to genetically characterize its isolates in carcasses of industrial fattening pigs, blood, diaphragm, and heart samples were collected from 375 carcasses of pigs slaughtered to be processed for Parma ham production. Pigs had been bred on approved farms (n = 75) located in the so-called Food Valley in Italy. Sera were examined for immunoglobulin G antibodies to T. gondii by modified agglutination test (MAT). Both heart and diaphragm samples from seropositive carcasses were processed for the presence of T. gondii DNA (B1 locus) by real-time PCR and high resolution melting (HRM) assay. Anti-Toxoplasma antibodies were detected in 2.1% of pig carcasses, with titers from 1:10 to 1:320. T. gondii DNA was detected in all (eight) seropositive carcasses and in 11 (5 heart and 6 diaphragm samples) of 16 samples; that is, it was detected in heart tissue in two subjects, in diaphragm tissue in three subjects, and in both muscle tissues in three subjects. Toxoplasma genotypes were determined in seven of eight carcasses: type III was identified in four carcasses, type II in two, and both III and II in one carcass. The serological findings and the molecular detection of T. gondii strains suggest that cured meat products obtained from industrially bred pigs may be potential sources of toxoplasmosis for humans. Our results provide novel, important information regarding the seroprevalence and molecular prevalence of T. gondii in intensively reared pigs within this specific region of Italy, particularly because Parma ham from this region is known and consumed worldwide. On-farm preventive measures combined with slaughterhouse monitoring of carcasses of pigs bred for cured meat production should never be overlooked to prevent the introduction of T. gondii into the food chain and to ensure safety for consumers of these products.

Molecular survey on the presence of zoonotic arthropod-borne pathogens in wild red deer (Cervus elaphus).

To estimate the prevalence of some zoonotic tick-borne pathogens in red deer (Cervus elaphus) living in Italian areas with high risk of arthropod exposure, blood samples from 60 red deer were tested by PCR for A. phagocytophilum, Borrelia burgdorferi s.l., Coxiella burnetii, Francisella tularensis, and piroplasms. Thirty-four (56.67%) animals resulted positive for one or more pathogens. In particular, 24 (40%) red deer were positive for A. phagocytophilum, 16 (26.67%) for Babesia divergens, 6 (10%) for C. burnetii, 2 (3.33%) for B. burgdorferi s.l. No positive reaction was observed for F. tularensis. Thirteen (21.67%) animals resulted co-infected by two or three pathogens. Red deer is confirmed as competent reservoir of A. phagocytophilum and B. divergens, but not of B. burgdorferi. This is the first report of C. burnetii-positive red deer in central Italy. Hunters may be at risk of infection both through infected ticks and during the infected cervids carcasses dressing.

Molecular detection of vector-borne bacteria and protozoa in healthy hunting dogs from Central Italy

ABSTRACT Objective: To determine the prevalence of vector-borne bacteria and protozoa in hunting dogs living in Central Italy. Methods: Molecular testing was executed on DNA which was extracted from blood specimens collected from 117 asymptomatic dogs to detect Anaplasma phagocytophilum, Babesia canis ( B. canis ) , Bartonella spp., Coxiella burnetii ( C. burnetii ) , Ehrlichia canis, Hepatozoon canis, and Leishmania infantum . Results: A total of 48 dogs (41.0%) were infested by Ixodes ricinus and Rhipicephalus sanguineus ticks. Tick-borne infections were observed in 64 (54.7%) animals. More in detail, 38 dogs (32.5%) screened positive for Hepatozoon canis , 24 (20.5%) for Bartonella vinsonii subsp. berkhoffii , 20 (17.1%) for Leishmania infantum , 6 (5.1%) for C. burnetii , 5 (4.3%) for B. canis (3 B. canis vogeli and 2 B. canis canis ), 3 (2.5%) for Anaplasma phagocytophilum , and 2 (1.7%) for Ehrlichia canis . Mixed infection by 2 agents occurred in 17 (14.5%) subjects, by 3 agents in 7 (6.0%) dogs, and by 4 agents in 1 (0.9%) animal. Conclusions: The results demonstrated that several vector-borne pathogens were circulating in this region and dogs infected by these agents were usually asymptomatic. A relevant finding was the presence of DNA of C. burnetii , a severe zoonotic agent, in the 5.1% of tested dogs, which can be source of infection for their owners not only through tick bites, but also directly with urine, feces and birth products.

An open-field study comparing an essential oil-based shampoo with miconazole/chlorhexidine for hair coat disinfection in cats with spontaneous microsporiasis

Objectives The goal of the present study was to compare the antifungal efficacy of an essential oil (EO) shampoo proven to be effective against Microsporum canis with miconazole/chlorhexidine for topical haircoat disinfection in cats treated concurrently with oral itraconazole. Methods Cats received treatment with oral itraconazole (Itrafungol) at a dose of 5 mg/kg/day pulse administration for 1 week, every 2 weeks for at least 6 weeks and were washed twice a week with a neutral shampoo with added EOs of Thymus serpyllum (2%), Origanum vulgare and Rosmarinus officinalis (5% each) for the period of systemic treatment. This protocol was compared with a conventional treatment (oral itraconazole + 2% miconazole/2% chlorhexidine shampoo). Results The treatment was well tolerated and adverse effects were not recorded. All cats were clinically negative at week 11. With respect to animals with extensive lesions, the speed of resolution was higher in cats with focal lesions. The animals showing diffuse lesions required more than a course of treatment to achieve a mycological cure. There was no significant difference between the number of weeks to obtain mycological cure for cats treated with EOs and animals treated conventionally. Conclusions and relevance The treatment appeared to be effective and well appreciated by the owners. The use of shampoo with the added EOs of T serpyllum, O vulgare and R officinalis would seem an interesting, natural alternative to conventional topical treatment.

Occurrence of moulds from bee pollen in Central Italy--A preliminary study.

The present study aimed to preliminary evaluate the occurrence of fungi in 40 specimens of trapped pollen collected from April-September 2015 in 40 apiaries from Tuscany (Central Italy). Cultural and microscopical examinations allowed the recognition of Cladosporium sp., Alternaria sp., Humicola sp. Mucoraceae and Acremonium sp. Penicillium brevicompactum, Penicillium chrysogenum, Aspergillus flavus , Aspergillus nidulans, Aspergillus niger and Aspergillus terreus were also identified. Yeasts and Fusarium spp. were not isolated. All pollen specimens were positive for at least one fungal isolate. Total CFU per gram ranged from 4-568. Aspergillus and Penicillium were obtained from 8 (20%) and from 22 (55%) pollen samples, respectively, associated in 4 cases (10%). The recovery of storage fungi such as Aspergillus and Penicillium in trapped pollen presents a potential risk for human health and attention should be paid to all stages of the post-harvest process.

Neospora caninum in Wild Waterfowl: Occurrence of Parasite DNA and Low antibody titers

Abstract Thirty-five adult waterfowl (14 males and 21 females) representing various orders and species were sampled during the hunting season from 2015 to 2016. Antibodies to Neospora caninum were detected by IFAT on blood samples, while heart and brain were subjected to molecular analysis for the detection of Neospora caninum DNA. Twelve birds (34.3%) (6 Anas crecca, 3 Anas platyrhynchos, 2 Anas penelope, 1 Anas acuta) showed antibodies versus N. caninum, while 10 animals out of 35 (4 A. crecca, 2 A. platyrhynchos, 2 A. penelope, 1 A. acuta, and 1 Vanellus vanellus) scored positive for at least 1 DNA sample, with an overall prevalence of 28.6%. The present report shows for the first time the occurrence of antibodies and N. caninum DNA in waterfowl. The avian species investigated in the present report, which feed directly from the soil and/or water, would be able to ingest oocysts excreted by final canid hosts and could contribute to parasite transmission in the sylvatic cycle. To achieve a definitive result about the role of these avian species in the epidemiology of this protozoan, the presence of viable parasites should be demonstrated by bioassay and/or culture, as well as histological evidence of N. caninum cysts in avian tissues.

Correlation Between Cyclo-oxygenase-2 and Vascular Endothelial Growth Factor Expression in Canine and Feline Squamous Cell Carcinomas

Overexpression of cyclo-oxygenase (COX)-2 is involved in tumour growth and spread by modulating the production of angiogenic factors such as vascular endothelial growth factor (VEGF). Expression of COX-2 and VEGF was investigated immunohistochemically in 51 canine and feline cutaneous and non-cutaneous squamous cell carcinomas (SCCs) and the correlation between expression of these molecules and clinicopathological variables was evaluated. COX-2 and VEGF expression was not observed in normal skin keratinocytes. COX-2 overexpression occurred in 53% and 61% of the canine and feline SCCs, respectively. The expression of both markers was higher in cutaneous compared with non-cutaneous SCCs. In both species COX-2 and VEGF expression was correlated with the progression of the disease, but not with the presence of lymphatic invasion, tumour grading or tumour classification in the cutaneous tumours. Further study will be required to understand the role of the COX-2 pathway in angiogenesis in SCC.

Stonebrood and chalkbrood in Apis mellifera causing fungi: in vitro sensitivity to some essential oils

Abstract Aim of the present study was to evaluate the in vitro antimycotic activity of 17 chemically defined essential oils (EOs) both alone and as a mixture, against agents responsible for stonebrood caused by Aspergillus flavus, and chalkbrood caused by Ascosphaera apis in European honeybees. Cinnamomum zeylanicum yielded the lowest MIC value against A. flavus, but was not effective against A. apis, while Litsea cubeba and Pelargonium graveolens appeared to be effective against all checked fungi. Aspergillus niger showed the lower sensitivity. Two mixtures composed by L. cubeba, C. zeylanicum and Cymbopogon flexuosus (M1) and by L. cubeba, C. zeylanicum, P. graveolens and C. flexuosus (M2), respectively, were tested, both resulting effective. The components of M1 showed a synergistic effect. The use of mixtures allowed to decrease the total amount of EOs. The use of these products could be of interest for an alternative natural approach in honeybee disease management.