Potential role of Hepatozoon canis in a fatal systemic disease in a puppy

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 discusse

Molecular survey of Cytauxzoon spp. and Hepatozoon spp. in felids using a novel real-time PCR approach

Tick-transmitted apicomplexans of the genera Cytauxzoon and Hepatozoon affect a wide range of felids worldwide, but little is known about them. Recently, several studies addressed the species circulating in Europe, their distribution, and their hosts. Molecular assays are the method of choice for their detection. Unfortunately, conventional PCRs already described are time- and cost-consuming and specific for either Hepatozoon or Cytauxzoon detection. This study was developed to evaluate (i) the occurrence of Cytauxzoon and Hepatozoon in felids using a fast and cost-saving real-time PCR capable of detecting both protozoa simultaneously, (ii) the distribution of Cytauxzoon and Hepatozoon species in north-eastern Italy, and (iii) the involvement of other susceptible felid hosts in the same area. An SYBR® Green-based real-time PCR with primers targeting the 18S-rRNA was validated and applied to 237 felid samples, i.e., whole blood from 206 domestic cats and 12 captive exotic felids, and tissues from 19 wildcats. Positive results were obtained by melting temperature curve analysis due to the specific melting peak (i.e., 81°C Cytauxzoon spp.; 78–78.5°C Hepatozoon spp.). Positive samples were subjected to conventional PCR, followed by sequencing for species identification. Phylogenetic analyses were performed to assess relatedness among European isolates. Data on domestic cats (age class, sex, origin, management, and lifestyle) were recorded, and statistical analyses were performed to identify potential risk factors. A total of 31 (15%) domestic cats were positive for Hepatozoon spp. (i.e., 12 for H. felis, 19 for H. silvestris), while six (2.9%) for C. europaeus. The prevalence of Hepatozoon felis was significantly (p < 0.05) higher in domestic cats, while H. silvestris was higher in strays and animals from the Eastern region (i.e., Friuli- Venezia Giulia). Cytauxzoon europaeus was detected only in stray cats from Friuli-Venezia Giulia (province of Trieste). Among captive felids, one tiger was infected with H. felis and another with H. silvestris; eight out of 19 (42%) wildcats were positive for Hepatozoon spp. (i.e., six with H. felis, two with H. silvestris) and four out of 19 (21%) for Cytauxzoon europaeus. Outdoor lifestyle and origin (i.e., Friuli-Venezia Giulia region) were the most relevant risk factors for H. silvestris and C. europeus infections. Conversely, H. felis was most frequently isolated from domestic cats, suggesting different modes of transmission

Effective methods of estimation of pathogen prevalence in pooled ticks

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

Hepatozoon spp. in stray cats from the metropolitan area of Rio de Janeiro, Brazil

In the last few years, the number of studies on feline hepatozoonosis has increased, but our knowledge on the actual species of Hepatozoon and/or different genotypes affecting felines is still incipient. At least three species, namely Hepatozoon felis, H. canis, and H. silvestris, have been isolated from domestic cats in various countries. Additionally, there are indications that other species and genotypes may affect felines in given geographic areas. This study was carried out to investigate the occurrence of Hepatozoon spp. in cats from Niterói, a municipality within the metropolitan area of Rio de Janeiro, Brazil. Individual blood samples were collected from 28 cats enrolled in a spaying/castration program. DNA was extracted from all samples and subjected to sequencing specific for Hepatozoon spp. DNA of H. felis was found in 21/28 cats (75%), and four genetic polymorphisms never described thus far were detected. This is the first report of H. felis in cats living in the State of Rio de Janeiro, and the present data confirm that H. felis is a species complex encompassing different genotypes circulating within cat populations. Further studies are warranted to investigate whether different genotypes have different biology or pathogenicity for felids

ARTHROPOD-BORNE PARASITIC DISEASES IN WILD AND DOMESTIC FELIDS IN NORTH-EASTERN ITALY

Cytauxzoonosis, hepatozoonosis and dirofilariosis are vector-borne diseases transmitted by arthropods (i.e., ticks and mosquitos). Felids could be susceptible to blood-feeding vectors and, consequently, to all of these diseases, nevertheless, the awareness of VBDs in these animals is still lacking. During the past decades, arthropod-borne diseases in felines have stimulated the interest of the Scientific Community and epidemiological studies on Cytauxzoon spp., Hepatozoon spp. and Dirofilaria immitis are increased in Europe. Nevertheless, the presence and distribution of Cytauxzoon spp. and Hepatozoon spp. in Italy are still scant and mostly limited to a few areas of Northern and Southern regions, respectively. Although canine Heartworm (HW) is endemic in North-eastern Italy, very little information has been recorded in felids. Cytauxzoon and Hepatozoon are vector-borne parasites, even if the involved arthropod vector/s are not still known. Recently, Hepatozoon DNA was detected only in some species of engorged ticks suggesting that those tick species could act as a vector. Nonetheless, the vectorial competence of ticks in Cytauxzoon spp. and Hepatozoon spp. transmission is not yet proved by scientific studies. Stained blood smears are a not-sensitive method for these hemoprotozoa diagnosis due to the low parasitaemia. Molecular analyses are strongly recommended since they present higher sensitivity (i.e., conventional PCR and real-time PCR). Dirofilaria immitis has an elusive nature leading to unpredictable effect on cat host. Indeed, the heartworm disease diagnosis is difficult. In addition, no single test is able to detect D. immitis in each of its stages in felid hosts and multiple diagnostic methods are necessary to confirm the infestation. The present research project aims to provide new data on the circulation of Cytauxzoon spp., Hepatozoon spp. and D. immitis and to update the current epidemiological scenario in North-eastern Italy (i.e. Veneto, Friuli Venezia Giulia and Trentino Alto Adige regions) i) improving the knowledge on the presence and distribution of the considered parasites in different felid species (i.e., domestic cats, wildcats, exotic captive felids); (ii) investigating on the possible role of ticks in the transmission of Hepatozoon spp. and Cytauxzoon spp., and (iii) developing diagnostic protocols to provide fast and sensitive screening procedures.Cytauxzoonosis, hepatozoonosis and dirofilariosis are vector-borne diseases transmitted by arthropods (i.e., ticks and mosquitos). Felids could be susceptible to blood-feeding vectors and, consequently, to all of these diseases, nevertheless, the awareness of VBDs in these animals is still lacking. During the past decades, arthropod-borne diseases in felines have stimulated the interest of the Scientific Community and epidemiological studies on Cytauxzoon spp., Hepatozoon spp. and Dirofilaria immitis are increased in Europe. Nevertheless, the presence and distribution of Cytauxzoon spp. and Hepatozoon spp. in Italy are still scant and mostly limited to a few areas of Northern and Southern regions, respectively. Although canine Heartworm (HW) is endemic in North-eastern Italy, very little information has been recorded in felids. Cytauxzoon and Hepatozoon are vector-borne parasites, even if the involved arthropod vector/s are not still known. Recently, Hepatozoon DNA was detected only in some species of engorged ticks suggesting that those tick species could act as a vector. Nonetheless, the vectorial competence of ticks in Cytauxzoon spp. and Hepatozoon spp. transmission is not yet proved by scientific studies. Stained blood smears are a not-sensitive method for these hemoprotozoa diagnosis due to the low parasitaemia. Molecular analyses are strongly recommended since they present higher sensitivity (i.e., conventional PCR and real-time PCR). Dirofilaria immitis has an elusive nature leading to unpredictable effect on cat host. Indeed, the heartworm disease diagnosis is difficult. In addition, no single test is able to detect D. immitis in each of its stages in felid hosts and multiple diagnostic methods are necessary to confirm the infestation. The present research project aims to provide new data on the circulation of Cytauxzoon spp., Hepatozoon spp. and D. immitis and to update the current epidemiological scenario in North-eastern Italy (i.e. Veneto, Friuli Venezia Giulia and Trentino Alto Adige regions) i) improving the knowledge on the presence and distribution of the considered parasites in different felid species (i.e., domestic cats, wildcats, exotic captive felids); (ii) investigating on the possible role of ticks in the transmission of Hepatozoon spp. and Cytauxzoon spp., and (iii) developing diagnostic protocols to provide fast and sensitive screening procedures

High Prevalence of Tick-Borne Zoonotic <i>Rickettsia slovaca</i> in Ticks from Wild Boars, Northeastern Italy

Tick-borne rickettsiae are emerging pathogens that are becoming widespread in Europe. Rickettsiae are endemic in Italy, but epidemiological data are currently scarce. This study aimed to improve our knowledge about rickettsial infections in tick and wild boar populations. Blood and ticks were collected from 102 wild boars in 2010 and 2018. Ticks were also collected from the vegetation in the area. All of the samples were examined using real-time PCR targeting the gltA gene to detect Rickettsia DNA. Positivity was confirmed by PCR amplifying the gltA and/or ompB genes. A total of 254 ticks and 89 blood samples were analyzed. Zoonotic rickettsiae were detected in the ticks but not in the blood samples. Rickettsia slovaca (R. slovaca) was the most prevalent in ticks and was found in 23.7% of Dermacentor marginatus (D. marginatus) and in 3.4% of Ixodes ricinus (I. ricinus). Other zoonotic species were identified, such as Rickettsia monacensis, which was detected in 12% of I. ricinus ticks, and Rickettsia helvetica which was found in 3.4% of questing I. ricinus ticks and in 1.1% of D. marginatus collected from wild boars. This study highlights a high prevalence of zoonotic rickettsiae, particularly that of R. slovaca, in northeastern Italy. As rickettsioses are underreported and underdiagnosed in human medicine, both clinicians and researchers should pay more attention to this topic

The Influence of Temperature on the Larval Development of Aelurostrongylus abstrusus in the Land Snail Cornu aspersum

The metastrongyloid Aelurostrongylus&nbsp;abstrusus has an indirect lifecycle involving gastropod intermediate hosts. The widespread snail Cornu&nbsp;aspersum is an efficient intermediate host of A. abstrusus. As the temperature may influence the developmental rate of metastrongyloids from first (L1) to the third infective larval stage (L3) inside molluscs, this study evaluated the effect of two controlled temperatures on the development of A. abstrusus in C. aspersum. Overall, 300 snails were infected with 500 L1 of A. abstrusus and kept at ∼25 °C. Fifteen days post infection (D15), the overall developmental rate to L3 (0.8%) was assessed in a subset of 20 snails. The remaining gastropods were divided in 2 groups, i.e., 180 still kept at ∼25 °C (G1) and 100 hibernated at ∼4 °C (G2). On D30, the larval development was evaluated in 20 snails from each group, while another batch of 80 snails was selected random from G1 and hibernated at ∼4 °C (G3). The larval developmental rate was determined digesting 20 snails from each of the three groups on D45, D60, and D75. The higher mean developmental rate was registered in G1 (3.8%) compared to G2 (1.9%) and G3 (2.3%), indicating that the development to L3 of A. abstrusus in C. aspersum is positively influenced by the increase of temperature