Comparison of direct and indirect methods to maximise the detection of Babesia caballi and Theileria equi infections in Central Southern Italy.

Equine piroplasmosis is a disease of equids, caused by tick-borne apicomplexan protozoan pathogens Babesia caballi and Theileria equi, which, according to the World Organisation for Animal Health (OIE), can be diagnosed by enzyme-linked immunosorbent assay (ELISA), immunofluorescent antibody test (IFAT) and polymerase chain reaction (PCR). The present study was conducted to evaluate and compare the assays available for the diagnosis of equine piroplasmosis. Data employed were obtained from 1300 blood samples collected between 2012-2014 from asymptomatic and symptomatic equines (horses and donkeys) of central-southern regions of Italy and analyzed by ELISA, IFAT, PCR (one commercial and one from literature) and blood smear microscopic examination. Statistical differences of the proportions of positivity for each parasite and group (asymptomatic and symptomatic) among the methods were verified by the z test to identify the most sensitive. The concordance between each pair of methods - for each parasite and within the groups - and trends in detection of suspect samples of four hypothetical diagnostic algorithms using serological and biomolecular assays were evaluated to identify the most suitable laboratory diagnostic workflow. The results of this study highlighted a lower capacity to detect suspect samples of commercial ELISA for B. caballi in all groups when compared to biomolecular methods and IFAT; and of the commercial PCRs in asymptomatic animals, identifying a PCR from literature and IFAT as the best choice for a combined diagnosis. For T. equi, IFAT detected more suspect samples than ELISA, even if the latter showed good performance and some samples were positive only by the ELISA and PCR, indicating that their simultaneous employment is still advantageous. Host-parasite interaction, amino-acid/genetic diversity and differences in detection limits among the assays could be among the reasons in explaining the present results. In view of further studies, ELISA should be used in combination with PCR, that should regularly be included in the laboratory diagnosis to maximise the detection of early infections and support the evaluation of pharmacological treatment.

Giardia duodenalis in colony stray cats from Italy.

Giardia duodenalis is the most common intestinal protozoan in humans and animals worldwide, including eight morphologically identical assemblages, infecting pets, livestock, wildlife and human beings. Assemblages A and B are those with the higher zoonotic potential, and they have been detected in several mammals other than humans; the others (C to H) show a higher host specificity. Cats can harbour both the specific Assemblage F and the zoonotic ones A and B. Several studies have been carried out on G. duodenalis genotypes in cats; however, the role of this species in the epidemiology of giardiasis is still poorly understood. In this scenario, the present study carried out the detection and genetic characterization at sub-assemblage level of G. duodenalis from colony stray cats in central Italy. In the period 2018-2019, 133 cat faecal samples were analysed for the presence of G. duodenalis cysts by a direct immunofluorescence assay. Positive samples were subsequently subjected to molecular analyses for assemblage/sub-assemblage identification. Forty-seven samples (35.3%) were positive for G. duodenalis cysts by immunofluorescence. G. duodenalis DNA was amplified at SSU-rDNA locus from 39 isolates: 37 were positive for zoonotic Assemblage A and 2 showed a mixed infection (A + B). Positive results for the ß-giardin gene were achieved for 25 isolates. Sequence analysis revealed 16 isolates belonging to Sub-assemblage AII and 8 to Sub-assemblage AIII. One isolate resulted as ambiguous AI/AIII. Large sequence variability at the sub-assemblage level was detected, with several double peaks and mutations, making complex a proper isolate allocation. When compared with previous studies, the 35.3% prevalence of G. duodenalis in cats reported in the present article was surprisingly high. Moreover, all positive cats resulted to be infected with zoonotic assemblages/sub-assemblages, thus indicating stray cats as a possible source of human giardiasis and highlighting the sanitary relevance of cat colonies in the study area.

Seroprevalence and risk factors associated with exposure to Leishmania infantum in dogs, in an endemic Mediterranean region.

Canine leishmaniasis (CanL) is a zoonotic parasitic disease caused by Leishmania infantum in the Mediterranean area and transmitted by phlebotomine sand fly vectors. The domestic dog is the main reservoir host. The aim of this study was to assess the influence of different individual, environmental and spatial risk factors on the dog exposure to L. infantum and to estimate the seroprevalence among owned and kennel dogs, in the Lazio region (central Italy), where canine leishmaniasis is endemic. In the period 2010-2014, 13,292 sera from kennel and owned dogs were collected by official and private veterinarians. The presence of anti-Leishmania IgG was analysed by indirect fluorescent antibody test (IFAT), using a 1:80 titre cut-off. At the univariable analysis, CanL seropositivity was associated with sex, size, breed, coat length, living with other dogs and forest/semi-natural land cover. At the multivariable analysis, age, ownership and attitude were confirmed as risk factors, being more than 2 years old, owned, and hunting dogs at higher risk. Being a Maremma sheepdog was a protective factor. A true overall seroprevalence of 6.7% (95% CI: 6.2-7.2) was estimated in the whole population while 7.3% (95% CI: 6.8-7.8) was estimated in kennel dogs and 74.3% (95% CI: 70.8-77.6) in owned dogs. The role of kennels as a key component for CanL active and passive surveillance was also highlighted. This study confirmed the endemicity of CanL in the Lazio region and focused some factors that can influence the seropositivity of dogs in a Mediterranean region.

Scalp eschar and neck lymphadenopathy by Rickettsia slovaca after Dermacentor marginatus tick bite case report: multidisciplinary approach to a tick-borne disease.

BACKGROUND: Scalp Eschar and Neck LymphAdenopathy after Tick bite is a zoonotic non-pathogen-specific disease most commonly due to Rickettsia slovaca and Rickettsia raoultii. Diagnosis is mostly based only on epidemiological and clinical findings, without serological or molecular corroboration. We presented a clinical case in which diagnosis was supported by entomological identification and by R. slovaca DNA amplifications from the tick vector. CASE PRESENTATION: A 6-year-old child presented with asthenia, scalp eschar and supraclavicular and lateral-cervical lymphadenopathy. Scalp Eschar and Neck LymphAdenopathy After Tick bite syndrome following a Dermacentor marginatus bite was diagnosed. Serological test on serum revealed an IgG titer of 1:1024 against spotted fever group rickettsiae, polymerase chain reaction assays on tick identified Rickettsia slovaca. Patient was successfully treated with doxycycline for 10 days. CONCLUSIONS: A multidisciplinary approach including epidemiological information, clinical evaluations, entomological identification and molecular investigations on tick, enabled proper diagnosis and therapy.

Usual or unusual presentations of Dirofilaria repens in two sibling dogs: a case report.

This study describes two different manifestations of Dirofilaria repens infection in sibling dogs with microfilaremia. Dog 1, asymptomatic, harbored a gravid female of D. repens on the parietal layer of tunica vaginalis of one testicle and showed a marked circulating eosinophilia (3.3·103/µL). Both testicles were normal in shape and size without any gross lesions. Dog 2 had a pyotraumatic dermatitis. The cases were confirmed by PCR and sequencing. The sequences obtained showed 100% identity with those of D. repens isolated from human scrotum in Croatia. The treatment with moxidectin 2.5% and imidacloprid 10%/kg was effective in eliminating microfilariae after just one application, as demonstrated by negative modified Knott's tests and PCR analyses of blood samples. This status was maintained during the post-treatment observation period. The classical localization of D. repens in dogs is in subcutaneous tissues, within nodules or free; however, it can also occur with some frequency in testicles, as described in humans. The infection can be associated with circulating eosinophilia or pyotraumatic dermatitis, as reported in this study. Thus, in endemic areas, it is advisable to carefully inspect the removed testicles at neutering since parasite localization can take place without any macroscopic changes. Moreover, in the case of circulating eosinophilia or pyotraumatic dermatitis, investigations should include modified Knott's test and PCR to ensure that D. repens is not the cause of these alterations. Rapid and sensitive tests for the early detection of infected animals would help to prevent or limit the spread of this zoonosis.

Rickettsia helvetica in Human-Parasitizing and Free-Living Ixodes ricinus from Urban and Wild Green Areas in the Metropolitan City of Rome, Italy.

Rickettsia helvetica is an emerging human pathogen, belonging to the spotted fever group (SFG) rickettsiae, associated with generally aneruptive fever, meningitis, and sudden death in chronic perimyocarditis. In this study, we describe the detection of R. helvetica in human-parasitizing and free-living Ixodes ricinus from the Metropolitan City of Rome. The pathogen was found in a tick acquired by a woman in an urban park. The circulation of R. helvetica was further confirmed by its detection in free-living ticks from a wild green area. These findings demonstrate that urban as well as wild green areas can represent a risk of infection to humans by R. helvetica, with potentially severe sequelae. To the best of our knowledge, this is the first report of R. helvetica in the Lazio region. Large-scale studies are needed to evaluate and quantify the presence of R. helvetica and other SFG rickettsiae in the urban and periurban context and to assess the risk to humans and animals related to their frequentation.

Leishmania infantum, Dirofilaria spp. and other endoparasite infections in kennel dogs in central Italy.

Prevalence and risk factors of Leishmania infantum, Dirofilaria spp. and other potentially zoonotic or canine-specific endoparasite infections were assessed in 639 kennel dogs from central Italy. To this end, individual blood and fecal samples were examined using parasitological, immunological and molecular techniques. The presence of compatible clinical pictures, as well as age and gender were considered as putative risks factors. To evaluate risk factors, multivariable analysis with logistic regression and univariable analysis with a Chi square test and a Fischer's exact test were performed. Overall, 52.6% of dogs (95% CI 48.6-56.5) were found positive, while 39.6% of dogs (95% CI 35.8-43.5) were infected by potentially zoonotic species. Leishmania infantum and Dirofilaria repens showed prevalences of 2.5% (95% CI 1.5-4.1) and 2.8% (95% CI 1.7-4.5), respectively. The prevalence of cardiorespiratory parasites was 7.8% (95% CI 5.9-10.3) and included the species Angiostrongylus vasorum, Eucoleus aerophilus, Eucoleus boehmi and D. immitis; the latter showed a prevalence of 0.2% (95% CI 0.001-1). Intestinal parasites were significantly prevalent (38.8%, 95% CI 35-42.7) and they consisted mainly of species of major zoonotic concern, including ancylostomatids, Toxocara canis, Giardia duodenalis, Dipylidium caninum, Taeniidae, Strongyloides stercoralis and Cryptosporidium parvum. Endoparasites were significantly prevalent in clinically suspected dogs. Leishmania infantum and cardiorespiratory nematodes were prevalent in older dogs, while intestinal parasites were prevalent in younger dogs. Results show high dog and public health risks in kennels in central Italy, and suggest the need for more effective control measures.

Comparison of indirect fluorescent antibody test and modified agglutination test for detecting Toxoplasma gondii immunoglobulin G antibodies in dog and cat.

The present study describes the comparison between a modified agglutination test (MAT) and the indirect fluorescent antibody test (IFAT) for the detection of Toxoplasma specific IgG antibodies in dog and cat sera. MAT showed an "almost perfect" agreement with IFAT in detecting positive and negative results in cat sera, where as only a "substantial" agreement was observed in dog sera due to false negative results. Differences relative to sample dilution were recorded and serological titres obtained by MAT and IFAT are not directly comparable in cat and dog sera.

New findings on anaplasmosis caused by infection with Anaplasma phagocytophilum.

Ixodes ricinus (I. ricinus) is one of the vectors of Anaplasma phagocytophilum (A. phagocytophilum) in Europe, in which rates of infection range from 1.9% to 34%. In 1998, human granulocytic ehrlichiosis-like (HGE-like) Ehrlichia DNA was detected in Italy, by PCR technique in one I. ricinus nymph out of 55 ticks that were examined. In 1996, 6.3% of 310 human sera in high-risk subjects from Italy were found positive for antibodies to Ehrlichia phagocytophila (E. phagocytophila). In the same year, the authors reported the first case of equine granulocytic ehrlichiosis. In 1997, only 2 out of 563 equine blood samples examined were found positive for antibodies to E. phagocytophila in the Latium region. In 1998, serological positivity was not observed in 14 symptomatic race horses. In 2002, a symptomatic horse living in Rome was found positive for Ehrlichia equi (E. equi) antibodies, as confirmed by PCR. E. equi was also demonstrated in horses by detection of specific antibodies from two asymptomatic ponies. We tested 128 sera from sheep in different flocks, and antibodies to E. phagocytophila were detected in 17 sera (13.3%) of these sheep. From 2000 to 2004, 147 dog sera were tested for antibodies against A. phagocytophilum, and 7 of these sera were positive (4.8%). These data confirm the presence of the infection in human, domestic animals, and pets in Italy. Studies are under way to correlate the distributions of the disease and tick vector, I. ricinus.

Equine ehrlichiosis in Italy.

The authors review equine granulocytic ehrlichiosis (EGE) in Italy from 1996 to 2002. In 1996, the first case of EGE has been observed in a horse affected with specific symptomatology (fever, lethargy, anorexia, limb edema, thrombocytopenia, and petechiae). In 1997, a seroepidemiological survey was performed in the province of Rome on 563 animals using IFAT. The authors describe the last case, which occurred on 2002 in a 15-year-old male, bay, half-breed, tick-infested horse. Clinical features included fever, lethargy, limb edema, icterus, leukocytopenia, and thrombocytopenia. Laboratory tests were positive for ELISA and IFAT and several morulae were seen in the cytoplasm of neutrophils in buffy coat smears. The authors think that in Italy the prevalence of EGE is underestimated because of the nonpathognomonic clinical symptoms, the diffusion of specific infected vector, and the nonroutine nature of specific laboratory tests.