Since tick-borne diseases (TBDs) incidence, both in human and animal populations, is increasing worldwide, there is the need to assess the presence, distribution and prevalence of tick-borne pathogens. Reliable estimates on tick-borne pathogens (TBPs) prevalence represent the public health foundation to create risk maps and take effective prevention and control actions against TBDs. Tick surveillance consists of collecting and testing (usually in pools) thousands of specimens. Construction and analysis of tick pools represent a challenge due to the complexity of tick-borne pathogens and tick-borne diseases ecology. The aim of this study is to provide a practical guideline on appropriate pooling strategies and statistical analysis of infection prevalence through: (i) reporting the different pooling strategies and statistical methodologies commonly used to calculate pathogen prevalence in tick populations and (ii) practical comparison between statistical methods utilising a real dataset of infection prevalence in ticks collected in Northern Italy. Reporting detailed information on tick pool composition and size is as important as the correct TBPs prevalence estimation. Among the prevalence indexes, we suggest using maximum-likelihood estimates of pooled prevalence instead of minimum infection rate or pool positivity rate given the merits of the method and availability of software.

Knowledge on the presence of Cytauxzoon sp. and Hepatozoon spp. in Italy is scant and mostly limited to a few areas of Northern and Southern regions, respectively. The present study updated the current epidemiological scenario by investigating the occurrence of these protozoa in domestic cats from three broad regions of North-Eastern Italy. Blood samples from cats at risk of vector-borne diseases were processed by PCR to detect Cytauxzoon and Hepatozoon DNA. Blood smears were observed for haemoparasite inclusions. The influence of cat individual data (e.g., provenance, management, indoor/outdoor lifestyle) on the prevalence of haemoprotozoan infections was statistically evaluated. Among 158 cats, Cytauxzoon and Hepatozoon DNA were detected in 6 (3.8%) and 26 (16.5%) animals, respectively. No Hepatozoon gamonts were detected in blood smears, whereas all Cytauxzoon PCR-positive samples were microscopically positive, though with low levels of parasitaemia. Two species of Hepatozoon were identified, Hepatozoon felis (n = 10) and Hepatozoon silvestris (n = 16). Hepatozoon silvestris prevalence values were significantly (p < 0.05) higher in the region Friuli Venezia Giulia and in stray cats. Cytauxzoon sp. was detected in 6/39 (15.4%) stray cats from Friuli Venezia Giulia (Trieste province). These data add new information on the occurrence of these neglected protozoa in domestic cats’ populations.

Dogs are known to host several tick-borne pathogens with zoonotic potential; however, scant information is available on the epidemiology of these pathogens in low-income tropical countries and in particular in sub-Saharan Africa. With the aim of investigating a wide range of tick-borne pathogens (i.e., Rickettsia spp., Anaplasma spp., Erhlichia spp., Borrelia spp., Hepatozoon spp. and Babesia spp.), 273 blood samples were collected from dogs in selected districts of Ethiopia and analyzed by real-time and/or end-point PCR. The results of the study showed that Hepatozoon canis was the most prevalent pathogen (53.8%), followed by Anaplasma phagocythophilum (7.0%), Babesia canis rossi (3.3%), Ehrlichia canis (2.6%) and Anaplasma platys (2.2%). Furthermore, five samples tested positive for Borrelia spp., identified as Borrelia afzelii (n = 3) and Borrelia burgdorferi (n = 2), and two samples for Rickettsia spp., identified as Rickettsia conorii (n = 1) and Rickettsia monacensis (n = 1). The finding of Anaplasma phagocythophilum and different species of the genera Borrelia and Rickettsia with zoonotic potential was unexpected and alarming, and calls for further investigation on the roles of dogs and on the tick, species acting as vector in this specific context. Other pathogens (Hepatozoon canis, Babaesia canis rossi, Anaplasma platys, Ehrlichia canis) are already known to have an important impact on the dogs’ health but have minor zoonotic potential as they were rarely or never reported in humans. Dogs from rural areas were found to be at higher risk for different pathogens, probably due to the presence of other wild canids in the same environment. The findings of the present study contribute to a better knowledge of the epidemiology of tick-borne pathogens, which is relevant to human and animal health.

Ticks and fleas are blood-sucking ectoparasites that cause irritation and anaemia to their hosts and act as vectors of pathogens (vector-borne pathogens, VBPs) of relevance for animal and human health. In the present study, tick and flea species in dogs and cats from Cyprus were recorded and VBPs were detected in the collected specimens. Ectoparasites were collected from 220 animals (161 dogs and 59 cats), and a questionnaire including demographic, clinical, and other information was filled out for each animal. The ectoparasites were morphologically identified and the detection of VBPs was performed by PCR-coupled sequencing. Rhipicephalus sanguineus sensu lato was found on 108 dogs and 13 cats, and Ixodes gibbosus on 2 dogs. Ctenocephalides felis was the predominant flea species (on 62 dogs and 45 cats), while one dog and one cat were infested by Ctenocephalides canis and Echidnophaga gallinacea, respectively. The VBPs in ticks were Anaplasma platys, Rickettsia massiliae, Rickettsia conorii, Rickettsia felis, Hepatozoon felis and Hepatozoon canis, while Rickettsia felis, Rickettsia sp., Bartonella koehlerae, Bartonella clarridgeiae, and Bartonella henselae were recorded in fleas. Statistical analysis (chi-square test and multiple univariate generalized linear model) showed that animals up to 6 months of age were less likely to be infested with ticks than older animals, but more likely to be infested with fleas. Ticks were more prevalent in sheltered than in owned animals, while the odds ratio of flea presence was higher in owned animals than those living in shelters. The present study is the first investigation on the occurrence of ticks and fleas in dogs and cats from Cyprus, showing the presence of different VBPs in these important ectoparasites. The results point out the importance of systematic ectoparasite control in dogs and cats.

Hepatozoon spp. is the causative agent of a vector-borne parasitic disease in many animal species. In felids, Hepatozoon felis, Hepatozoon canis and Hepatozoon silvestris have been molecularly isolated. Hepatozoonosis usually causes asymptomatic infections in domestic cats, but clinical cases have recently been reported in Europe. We describe the first Italian case of hepatozoonosis in a cat with an unusual presentation. An 11-year-old neutered European shorthair cat was urgently hospitalized for intestinal intussusception. Hematology, biochemistry, FIV-FeLV tests, blood smears and molecular investigation targeting the 18S rRNA gene of Hepatozoon spp. were performed on blood samples; in addition, histological and molecular investigations were performed to analyze surgical samples to identify Hepatozoon infection. Hepatozoon gamonts were detected in granulocytes in the blood smear, and Hepatozoon spp. DNA was confirmed by PCR on blood. The intussusception was caused by a sessile endoluminal nodule that was surgically removed. Histologically, many elements referring to parasitic tissue forms were identified in the intestinal cells, and then the specimens were molecularly confirmed to harbor H. silvestris. This is the first description of symptomatic hepatozoonosis in a domestic cat in Italy. Hepatozoon silvestris has been described in wild felids, which are usually resilient to the infection, whereas the domestic cat seems to be more susceptible. Indeed, H. silvestris in cats usually presents tropism for skeletal muscle and myocardium with subsequent clinical manifestations. This is the first description of a domestic cat with H. silvestris localized in the intestinal epithelium and associated with intussusception. Graphical abstract

Canine hepatozoonosis caused by Hepatozoon canis is an emerging disease in Europe. Clinical pictures vary from subclinical to life-threatening and non-specific clinical signs are predominantly reported. A 2-month-old female puppy originating from Southern Italy was adopted and moved to Northern Italy. Then, the dog was brought to a local veterinary practice for gastrointestinal signs, migrating lameness and pruritic dermatitis, and then tested positive for Hepatozoon spp. gamonts at the blood smear. After treatment with imidocarb dipropionate and doxycycline, the dog showed an initial clinical improvement. However, gastrointestinal signs recurred, and diffuse superficial pyoderma appeared on the thoracolumbar region, along with fever, lethargy, and weight loss. Eight months from the first onset of clinical signs, the dog was referred to a veterinary clinic and subjected to complete blood count, urine and fecal analysis, along with abdominal ultrasonography, whole-body CT and gastroduodenal endoscopy. Skin biopsies and blood samples were subjected to a PCR-coupled sequencing protocol, which scored both positive for H. canis. Alterations were consistent with a pre-existing cholangiohepatitis and multiple acquired extrahepatic shunts secondary to portal hypertension. The dog was euthanatized due to a clinical worsening two months later. The potential role of H. canis in the systemic disease observed, clinic-pathological findings and epizootiological implications are discussed.