Rocío Checa

Current situation of Leishmania infantum infection in shelter dogs in northern Spain

BackgroundCanine leishmaniosis (CanL) caused by Leishmania infantum is a widespread endemic disease in the Mediterranean basin, though, so far, the north of Spain has been considered a non-endemic area. The aim of the present study was to determine the prevalence of specific antibodies to L. infantum among stray dogs living in shelters in this area, and to evaluate the clinical status (both clinical signs and clinico-pathological abnormalities) of seropositive dogs. Besides L. infantum infection, the epidemiological role of variables like sex, breed and age was also assessed.MethodsOver the year 2011 a cross-sectional study was conducted on a total of 418 stray dogs. A preliminary entomological survey was carried out using CDC-light traps. The chi-squared test was used to examine relationships between L. infantum seroprevalence and the remaining variables.ResultsThe overall seroprevalence of L. infantum infection detected was 3% in the Cantabrian coast. In Orense the seroprevalence was 35.6%. In this latter region, the presence of sand fly, Phlebotomus perniciosus was also detected.In general, seropositivity for L. infantum was related to size (large breed dogs versus small) and age, with a significantly higher seroprevalence recorded in younger (0-3 years) and older dogs (> 7 years) than adult dogs. Clinical signs of CanL were observed in 41.3% of the seropositive dogs. The seropositivity for L. infantum infection associated with the presence of clinical signs and/or abnormal laboratory findings shows a prevalence of 4.5%.ConclusionOur data provide new insight into the prevalence of CanL across northern Spain. The situation observed in Orense seems to be worsening compared to the few reports available, with figures being similar to those cited for known endemic areas of Spain. Besides, the presence of P. perniciosus in Orense points out to a risk of the spread of this zoonotic disease in this geographical area. These findings identify a need for an active search for the sand fly vectors of L. infantum across the entire northern spanish region including the rest of Galicia, Asturias, Cantabria and the Basque Country.

Current status of L. infantum infection in stray cats in the Madrid region (Spain): implications for the recent outbreak of human leishmaniosis?

BackgroundSince 2009, the incidence of human leishmaniosis in the SW of the Madrid region has been unusually high. Although dogs are the main reservoir for this disease, a role played by dogs in this outbreak has been ruled out and investigators are now considering other hosts (eg. cats, rabbits, hares) as possible alternative reservoirs.This study was designed to examine the Leishmania infantum status of stray cats in Madrid to assess its possible implications in the human leishmaniosis outbreak.Methods346 captured stray cats were tested for antibodies against L. infantum by the indirect fluorescent antibody technique (IFAT) and nested-PCR methods were used to detect Leishmania DNA in blood samples of cats testing seropositive for L. infantum and/or retroviruses infection. Cats were also tested for Toxoplasma gondii using the direct agglutination test (DAT) and feline leukemia virus (FeLV) antigen and feline immunodeficiency virus (FIV) antibodies (PetChek* FIV/FeLV). The presence of intestinal parasites was determined using a routine coprological method.ResultsThe seroprevalence of L. infantum infection (cut off ≥ 1/100) was 3.2% (11/346). However, it was not possible to amplify Leishmania DNA in any of the blood samples. Seropositivity was not associated with sex, age, capture site, clinical status, retrovirus infection or T. gondii seropositivity. Of the 11 cats seropositive for L. infantum, 3 also tested positive for FIV, none for FeLV and 6 for T. gondii. It should be mentioned that the prevalence of FeLV p27 antigen was 4% and of FIV antibody was 9.2%. Although the seroprevalence of T. gondii was quite high at 53.5%, no T. gondii oocysts were found in any of the faeces samples analysed (n = 287). In contrast, intestinal parasites were detected in 76 (26.5%) samples, Toxocara cati being the most prevalent.ConclusionsOur results suggest a stable L. infantum infection situation among the stray cats of the Madrid area; the disease is uncommon and no clinical cases have been reported to date. The detection of other zoonotic parasites such as T. gondii and T. cati in stray cats indicates a need to adopt strict control measures in this population.

Theileria annae (syn. Babesia microti-like) infection in dogs in NW Spain detected using direct and indirect diagnostic techniques: clinical report of 75 cases

BackgroundIn north-western Spain, piroplamosis caused by Theileria annae is now recognized as a serious problem because veterinarians, despite being aware of the clinical signs of piroplasmosis, lack the necessary information on its epidemiology or specific diagnostic tools for its management. This, along with the fact that T. annae infection is also refractory to current piroplamosis treatments, prompted this study designed to assess the clinical presentation and diagnosis of this largely unknown parasitic disease in dogs.MethodsOne hundred and twenty dogs in NW Spain suspected clinically of having piroplasmosis were examined and piroplasm species detected by light microscopy (LM) observation of Giemsa-stained blood smears, immunofluorescent antibody test (IFAT), and PCR plus sequencing.ResultsSeventy five of the sick dogs were confirmed to be infected with T. annae by PCR (designated “true infection cases”). Intraerythrocytic ring-shaped bodies morphologically compatible with small piroplasms were observed by LM in 59 (57 true infections) of the 120 blood samples. Anti-Babesia antibodies were detected by IFAT in 59 of the 120 sera (55 of which were “true infections”). Using PCR as the reference method, moderate agreement was observed between positive LM vs PCR and IFAT vs PCR results (kappa values: 0.6680 and 0.6017, respectively). Microscopy examination and IFAT were moderately sensitive in detecting the pathogen (76% and 73.3%, respectively). In the 75 cases of “true infection”, the most common clinical signs observed were pale mucous membranes, anorexia and apathy. Blood cell counts consistently revealed severe regenerative anaemia and thrombocytopenia in dogs with piroplasmosis due to T. annae. Young dogs (≤3 year) (p = 0.0001) were more susceptible to the disease.ConclusionMicroscopy showed moderate diagnostic sensitivity for acute T. annae infection while IFAT-determined antibody titres were low (1/64 to 1/128). The infecting species should be therefore confirmed by molecular tests. Our results suggest that the disease affects dogs in regions of Spain bordering the endemic Galicia area where this piroplasm has not been previously reported (Asturias, northern Spain). Further epidemiological surveys based on serological and molecular methods are required to establish the current geographical range of T. annae infection.

LEISHMANIA INFANTUM INFECTION IN BENNETT'S WALLABIES (MACROPUS RUFOGRISEUS RUFOGRISEUS) IN A SPANISH WILDLIFE PARK

Abstract Although dogs are the main reservoir for human Leishmania infantum infection, the disease has also been reported in other domestic and wild mammals. In 2011, a fatal case of naturally acquired leishmaniosis was described for the first time in a Bennetts wallaby (Macropus rufogriseus rufogriseus) kept in a wildlife park in Madrid (Spain). This study was designed to assess the infection status of twelve Bennetts wallabies in the same park one year after this incident. Phlebotomus perniciosus, the main vector of L. infantum in Spain, was screened for using sticky and Centers for Disease Control miniature light traps. L. infantum infection was confirmed by molecular diagnosis in four animals, but only one wallaby returned a positive serology result. The presence of the sand fly vector was also confirmed in this habitat. These results suggest that the first case of L. infantum in a wallaby in this park was not an isolated incident and stress the need for further work to determine the role of this parasite in the morbidity and mortality of these macropods. Madrid was recently the scene of an outbreak of human cutaneous and visceral leishmaniosis. Epidemiological studies have so far revealed the widespread presence of L. infantum infection in animals other than the dog. Our ongoing work suggests a risk of L. infantum infection not only among captive animals in Madrid, but also among threatened species or even species that are already extinct in the wild.

Flea species infesting dogs in Spain: updated spatial and seasonal distribution patterns

This entomological survey examines the spatial and seasonal distribution patterns of flea species infesting dogs in Spain. Bioclimatic zones covering broad climate and vegetation ranges were surveyed according to size. In a cross‐sectional spatial survey carried out from late May 2013 to mid‐July 2015, 1084 dogs from 42 different locations were examined. A total of 3032 fleas were collected and identified as belonging to the following species: Ctenocephalides felis (Siphonaptera: Pulicidae) (81.7%, 2476 fleas); Ctenocephalides canis (11.4%, 347 fleas); Pulex irritans (Siphonaptera: Pulicidae) (6.9%, 208 fleas), and Echidnophaga gallinacea (Siphonaptera: Pulicidae) (0.03%, one flea). Variables observed to have effects on flea abundance were animal weight, sex, length of hair and habitat. In the seasonal survey conducted from June 2014 to June 2015, 1014 fleas were collected from 239 dogs at 30 veterinary practices across Spain. Peaks in C. felis abundance were observed in early summer and late autumn, whereas high numbers of P. irritans and C. canis were recorded in autumn. Numbers of fleas detected in winter were low overall. Based on these findings, the present study updates the spatial and seasonal distributions of flea species in Spain and assesses the impacts of host and habitat variables on flea infestation.

DNA sequence analysis suggests that cytb-nd1 PCR-RFLP may not be applicable to sandfly species identification throughout the Mediterranean region

Molecular methods are increasingly used for both species identification of sandflies and assessment of their population structure. In general, they are based on DNA sequence analysis of targets previously amplified by PCR. However, this approach requires access to DNA sequence facilities, and in some circumstances, it is time-consuming. Though DNA sequencing provides the most reliable information, other downstream PCR applications are explored to assist in species identification. Thus, it has been recently proposed that the amplification of a DNA region encompassing partially both the cytochrome-B (cytb) and the NADH dehydrogenase 1 (nd1) genes followed by RFLP analysis with the restriction enzyme Ase I allows the rapid identification of the most prevalent species of phlebotomine sandflies in the Mediterranean region. In order to confirm the suitability of this method, we collected, processed, and molecularly analyzed a total of 155 sandflies belonging to four species including Phlebotomus ariasi, P. papatasi, P. perniciosus, and Sergentomyia minuta from different regions in Spain. This data set was completed with DNA sequences available at the GenBank for species prevalent in the Mediterranean basin and the Middle East. Additionally, DNA sequences from 13 different phlebotomine species (P. ariasi, P. balcanicus, P. caucasicus, P. chabaudi, P. chadlii, P. longicuspis, P. neglectus, P. papatasi, P. perfiliewi, P. perniciosus, P. riouxi, P. sergenti, and S. minuta), from 19 countries, were added to the data set. Overall, our molecular data revealed that this PCR-RFLP method does not provide a unique and specific profile for each phlebotomine species tested. Intraspecific variability and similar RFLP patterns were frequently observed among the species tested. Our data suggest that this method may not be applicable throughout the Mediterranean region as previously proposed. Other molecular approaches like DNA barcoding or phylogenetic analyses would allow a more precise molecular species identification.

Unresponsiveness of Experimental Canine Leishmaniosis to a New Amphotericin B Formulation

This study was designed to evaluate the efficacy and safety of a novel free polyaggregated amphotericin B (FPA) formulation used to treat experimental canine leishmaniosis (CanL) caused by Leishmania infantum. Eight healthy beagles were intravenously challenged with promastigotes per mL of L. infantum. One year after infection, they received an intravenous dose of FPA (5 mg/kg) every 2 weeks three times. Dogs were assessed monthly for clinical signs, serology, and parasite detection during a follow-up period of 6 months. Transient adverse effects (i.e., hypotension, diarrhea, bodyweight loss, fever, and asthenia) were observed within 24–48 hours after treatment in 4 animals. In three dogs mean clinical signs scores were reduced. Antibody titers measured by immunofluorescence antibody test (IFAT) had significantly diminished at the end of the study, although according to bone marrow smears and cultures a high percentage of dogs tested positive for the parasite at 6 months posttreatment (PT6). Real-time quantitative PCR (rtQ-PCR) on blood, bone marrow, and urine samples revealed the presence of parasitic DNA in all animals at PT6, although blood loads of the parasite were reduced. These findings indicate that FPA at the dosing regimen used did not achieve clinical or parasitological cure in dogs experimentally infected with L. infantum.

Babesia microti-like piroplasm (syn. Babesia vulpes) infection in red foxes (Vulpes vulpes) in NW Spain (Galicia) and its relationship with Ixodes hexagonus

Piroplasmosis is caused by several species of protozoa such as the Babesia microti-like piroplasm (Bml), an emerging blood protozoan also known as Theileria annae or Babesia vulpes. Infection by Bml was first reported in dogs in Spain where it is endemic today. Recently, a high prevalence of Bml has been increasingly detected in red foxes (Vulpes vulpes) in European countries. The objective of this study was to determine infection levels of this parasite in foxes from Galicia, NW Spain, and ticks species infestation in these carnivores, where they are so far unknown. Samples of blood, spleen and ticks (if present) were taken from 237 hunted red foxes in the Galicia region. Blood smears were prepared for direct parasite observation, and spleen and tick samples were examined by nested PCR. Prevalences of Bml infection in Galician red foxes were estimated at 72% (171/237) by PCR and 38.23% (26/68) by direct observation. Among 837 ticks collected, the main tick identified was Ixodes hexagonus (present in 82.4% of the foxes) followed by Ixodes ricinus (12.3%), Dermacentor reticulatus (12.3%) and Rhipicephalus sanguineus sensu lato (3.5%). From 34 foxes testing positive for Bml, 616 ticks were collected: positive Bml PCR results were obtained in 55.6% (227/408) of ticks collected from 9 foxes, while the 208 ticks from the remaining 25 infected foxes returned negative PCR results. Given that canine piroplasmosis is endemic in this area, our observations point to the red fox as the main reservoir for Bml infection and the high proportion of I. hexagonus among ticks collected from red foxes suggests its likely role as vectors of B. microti-like piroplasm in this region. Further studies are needed for a better understanding of the link between the wild and domestic life cycles of this piroplasm.

Implications of zoonotic and vector-borne parasites to free-roaming cats in central Spain

Cats are definitive hosts and reservoirs for several parasites, some of which are responsible for serious zoonotic diseases. We conducted a case-control study of data from a trap-neuter-return (TNR) programme (years 2014-2017) designed to examine the prevalence of zoonotic parasites in free-roaming cats living in urban areas of central Spain. In the animal population tested (n = 263), we detected a 29.2% prevalence of endoparasites, including high rates of cestodes (12.9%) and Toxocara cati (11.7%). While faecal samples showed no Toxoplasma gondii oocysts, the seroprevalence of T. gondii infection was 24.2%. Antibodies to Leishmania infantum were detected in 4.8% of the animals, though all skin and blood samples analyzed were PCR negative for this parasite. Ectoparasites (ticks and fleas) were found in 4.6% of the cat population, and 10.6% of the cats were detected with Otodectes cynotis. Finally, 6.3% and 7.9% cats tested positive for feline leukaemia virus and feline immunodeficiency virus, respectively. Our study provides useful information for animal-welfare and public-health, as the parasites detected can affect native wild animals through predation, competition and disease transmission. Our detection of zoonotic parasites such as L. infantum, T. gondii, T. cati, Giardia duodenalis and several ectoparasites prompts an urgent need for health control measures in stray cats.

Detection of Thelazia callipaeda in Phortica variegata and spread of canine thelaziosis to new areas in Spain

BackgroundThe fruit fly Phortica variegata (Drosophilidae: Steganinae) feeds on the ocular secretions of animals and humans, and has been described as an intermediate host of the eye worm Thelazia callipaeda (Spirurida: Thelaziidae) in Italy. Despite the increased detection of T. callipaeda in many European countries, information about its vector role in natural conditions is still limited. In the Iberian Peninsula, thelaziosis caused by T. callipaeda has been reported in dogs, cats, red foxes, wild rabbits and humans.MethodsIn the last seven years, we have detected increased numbers of cases of canine thelaziosis at three locations in mainland Spain: Site 1, La Vera region (Cáceres Province, central-western Spain; 51 cases); Site 2, El Escorial municipality (Madrid Community, central Spain; 23 cases); and Site 3, Miraflores de la Sierra municipality (Madrid Community, central Spain; 41 cases). Site 1 is considered endemic for T. callipaeda while the other two sites have been recently recognised as risk zones for T. callipaeda infection.ResultsFrom June 2016 to September 2017, 2162 flies were collected and morphologically identified as Phortica spp. (Site 1, n = 395; Site 2, n = 1544; and Site 3, n = 223). Upon dissection, third-stage T. callipaeda larvae were found in two out of 155 flies examined from Site 1, and both these larvae tested molecularly positive for the eye worm. Of the 395 flies collected from Site 1, 371 were molecularly processed for arthropod species identification and T. callipaeda detection. All 371 flies were identified as P. variegata and 28 (7.5%; 95% CI: 4.8–10%) tested positive for T. callipaeda DNA haplotype 1.ConclusionsOur findings indicate that T. callipaeda circulates among dogs and P. variegata in Spain, where zoonotic cases have been also reported. The co-existence of canine thelaziosis and Phortica spp. in geographical areas previously considered free of the eye worm indicates a risk of infection for both animals and humans living in this region.