Fabrizio Bertelloni

Serological survey of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, and Ehrlichia canis infections in rural and urban dogs in Central Italy.

INTRODUCTION Borrelia burgdorferi sensu lato (s.l.) and Anaplasma phagocytophilum are well known zoonotic pathogens, whereas Ehrlichia canis is usually considered to be of veterinary concern, although on the basis of recent reports it also seems to be able to infect humans. OBJECTIVE The aim of the study was to determine the seroprevalence of B. burgdorferi s.l., A. phagocytophilum and E. canis in an Italian canine population, and to verify if there are differences between dogs living in urban areas and those from a rural environment. MATERIALS AND METHOD Blood sera of 1,965 dogs, 1,235 from cities and 730 from rural areas, were tested by indirect immunofluorescent assay (IFAT). RESULTS The overall seroprevalence was highest for E. canis (7.07%), followed by A. phagocytophilum (4.68%), and B. burgdorferi s.l. (1.47%). Rural dogs showed the highest seroprevalence to B. burgdorferi s.l. and A. phagocytophilum. No significant differences were observed between rural and urban E. canis-positive dogs. A low percentage (1.32%) of dogs with dual seropositivity was detected, and no triple positive reactions were observed. No significant differences were detected in the seroprevalence of the three agents in relationship to the age and gender of the dogs. Seroprevalence in the five years considered were not statistically different, except for the lowest rate for E. canis observed in 2012. CONCLUSIONS The results confirm the presence of B. burgdorferi s.l., A. phagocytophilum and E. canis in Italian dogs in both urban and rural areas. Monitoring pet dogs, which share the same environment with their owners, is useful for identifying the presence of tick-borne disease agents of both veterinary and public health significance.

Molecular survey of tick-borne pathogens in Ixodid ticks collected from hunted wild animals in Tuscany, Italy.

OBJECTIVE To determine the prevalence of zoonotic tick-borne bacteria in feeding ticks removed from hunted wild animals. METHODS PCR was executed on DNA extracted from 77 tick pools to detect Anaplasma phagocytophilum, Bartonella spp., Borrelia burgdorferi sensu lato, Coxiella burnetii and Rickettsia spp. RESULTS A total of 432 ticks were collected: 30 (6.94%) Haemaphysalis punctata, 72 (16.7%) Dermacentor marginatus and 330 (76.38%) Ixodes ricinus. For each animal one or two pools of 3 ticks of the same species was constituted. Seventy-seven tick pools were examined by PCR: 58 (75.32%) resulted infected and among them 14 (18.18%) showed co-infections. In particular, 29 (37.66%) pools were positive for Bartonella spp., 23 (29.87%) for Anaplasma phagocytophilum, 16 (20.78%) for Rickettsia spp., and 5 (6.49%) for Borrelia burgdorferi s.l. All samples were negative for Coxiella burnetii. CONCLUSIONS The results demonstrate the presence of several zoonotic tick-borne pathogens in the studied area, and underline the risk of exposure to infections for hunters not only during the outdoor activity, but also when they manipulate hunted animals infested by infected ticks.

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.

Serological evidence of exposure to Ehrlichia canis and Anaplasma phagocytophilum in Central Italian healthy domestic cats.

The aim of the present survey was to estimate the seroprevalences of Ehrlichia canis and Anaplasma phagocytophilum in the Central Italian feline population. Serum samples of 560 healthy domestic cats were examined by indirect immunofluorescence assay (IFAT), considering an antibody titre of 1:40 as cut-off. Seroprevalences of 6.4% and 4.5% were found for E. canis and A. phagocytophilum, respectively. Adult, mixed breed cats showed seroprevalences higher than younger and purebred subjects, whereas no differences were observed in relation to gender and living conditions.

Isolation and identification of mycobacteria from captive reptiles

The occurrence of Mycobacterium species in clinically healthy pet reptiles was studied in Italy during the period 2004-2006. The feces samples of 223 animals were examined bacteriologically. Thirty-seven strains were isolated, in particular from 13/18 (72.2%) ophidians, 13/134 (9.7%) saurians and 11/71 (15.5%) chelonians. The isolates were classified, after HPLC analysis of bromophenacyl esters of cell wall mycolic acids, as Mycobacterium fortuitum (14 strains, 37.8%), Mycobacterium fortuitum-like (17, 45.9%), Mycobacterium peregrinum (4, 10.8%), and Mycobacterium chelonae (1, 2.7%). M. fortuitum was isolated from seven pythons, five saurians and two turtles; M. fortuitum-like from six saurians, six pythons and five turtles; M. peregrinum from four turtles; M. chelonae from one lizard. One isolate from an Iguana iguana could not be identified by HPLC analysis showing a previously unreported profile. Comparative 16S rDNA sequencing showed a low similarity with Mycobacterium triviale (97.2%) and Mycobacterium confluentis (97.1%). On the basis of such data the unidentified bacterium turned out to belong to a not yet described Mycobacterium species.

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.

Occurrence of Bartonella henselae types I and II in Central Italian domestic cats

Serological and molecular surveys were conducted to determine the occurrence of Bartonella henselae in domestic cats in Central Italy. Samples from 234 pet cats were tested for B. henselae antibodies by indirect immunofluorescence with 78 (33.3%) positive. A PCR assay specific for the Bartonella 16S rRNA gene was carried out on DNA samples extracted from blood of the 234 cats; 26 (11.1%) of the seropositive cats were positive. Two PCR protocols, which discriminate genotypes I and II of B. henselae, were performed on all DNA samples. Sixteen (6.8%) cats were infected by genotype I, 6 (2.5%) by genotype II, and two males (0.8%) by both genotypes. Two female (0.8%) cats which were Bartonella sp. PCR positive, gave negative results with the types I and II PCR. This protocol facilitates the direct and rapid detection of Bartonella DNA in feline blood samples, and differentiates B. henselae genotypes.

Antibacterial and Antifungal Activity of Essential Oils against Pathogens Responsible for Otitis Externa in Dogs and Cats

Background: Essential oils (EOs) are recommended by some veterinarians to treat otitis externa in pets, but data about their efficacy in scientific literature are very scant. Methods: Nine commercial EOs, from roman chamomile (Anthemis nobilis L.), star anise (Illicium verum), lavender (Lavandula hybrida), litsea (Litsea cubeba (Lour.) Pers.), basil (Ocimum basilicum L.), oregano (Origanum vulgare L. subsp. hirticum), rosemary (Rosmarinus officinalis L.), clary sage (Salvia sclarea L.), and thyme (Thymus vulgaris L.) were tested against bacterial and fungal pathogens previously isolated from dogs and cats with otitis externa. In particular, the analyses were carried out against Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus pseudointermedius, Aspergillus niger, Aspergillus fumigatus, Aspergillus terreus, Candida albicans, Candida tropicalis, Trichosporon sp., and Rhodotorula sp. Results: O. vulgare and S. sclarea showed superior antibacterial activity, even if not against all the strains. Trichosporon sp., C. albicans, and A. terreus were insensitive to most Eos, while other yeasts and molds showed different degrees of sensitivity. In particular, most fungi were inhibited by O. vulgare and R. officinalis. Conclusions: The obtained results suggest that some EOs could be included in treatment as an alternative therapeutic option in bacterial otitis complicated by fungi, in association with conventional drugs.

Salmonella infection in healthy pet reptiles: Bacteriological isolation and study of some pathogenic characters

The fecal samples from 213 captive reptiles were examined, and 29 (13.61%) Salmonella enterica isolates were detected: 14/62 (22.58%) from chelonians, 14/135 (10.37%) from saurians, and 1/16 (6.25%) from ophidians. The isolates were distributed among 14 different serotypes: Miami, Ebrie, Hermannsweder, Tiergarten, Tornov, Pomona, Poona, Goteborg, Abaetetube, Nyanza, Kumasi, Typhimurium, 50:b:z6, 9,12:z29:1,5, and a non-motile serotype with antigenic formula 1,4,[5],12:-:-. Salmonella typhimurium and 50:b:z6 isolates showed the spv plasmid virulence genes, responsible of the capability to induce extra-intestinal infections. In some cases, pulsed field gel electrophoresis revealed different profiles for the strains of the same serotypes, showing different origins, whereas a common source of infection was supposed when one pulsotype had been observed for isolates of a serovar. Twenty-seven (93.10%) isolates showed resistance to one or more antibiotics. Ceftazidime was active to all the tested isolates, whereas the highest percentages of strains were no susceptible to tigecycline (93.10%), streptomycin (89.66%), and sulfonamide (86.21%).