Valentina Marino

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.

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.

Epidemiological role of dogs since the human leishmaniosis outbreak in Madrid

BackgroundCanine leishmaniosis (CanL) has been in the spotlight since the 2009 outbreak of human leishmaniosis in Madrid. In the framework of the Leishmaniosis Surveillance Programme set up in Madrid, this study examines Leishmania-specific seroprevalences in stray dogs for the outbreak area and rest of the Madrid region over the period spanning from the outbreak to the present (2009–2016). These data are of interest because stray dogs could be sentinels for disease surveillance in endemic areas. Since 2011, we have also been monitoring owned dogs in the outbreak area.MethodsOver the study period, Leishmania infantum seroprevalence was determined in 2,123 stray dogs from the outbreak and non-outbreak areas. A serological study was also performed for owned dogs in the outbreak area: high-risk dogs such as hunting or farm dogs (n = 1,722) and pets (n = 1372). All dogs were examined and blood was collected. The variables recorded for each animal were: breed, age, sex, and clinical history indicating if the animal was healthy or clinically suspected of having any disease, and if they showed a clinical picture compatible with CanL.ResultsSeroprevalences of L. infantum in stray dogs were similar in the two areas examined: 4.7% (20 out of 346) in the outbreak area and 5.4% (96 out of 1,777) in the remaining Madrid region (χ2 = 0.080, P = 0.777). A significant association was found between seroprevalence and age (z = -6.319; P < 0.001). Seroprevalence in owned dogs in the outbreak area was 2.1% in high-risk dogs (37 out of 1,722) and 1.2% in pets (17 out of 1,372) (χ2 = 3.561, P = 0.0591).ConclusionsBoth stray and owned dogs do not seem to play an important role in maintaining the transmission cycle of L. infantum in the Madrid outbreak area. The stable seroprevalence of infection observed in sentinel dogs suggests the good clinical management and prevention of CanL by local practitioners in owned dogs.

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.

First report of Leishmania infantum infection in the endangered orangutan ( Pongo pygmaeus pygmaeus ) in Madrid, Spain

BackgroundSome wild animals have been recognized as potential reservoirs of Leishmania infantum infection (e.g. carnivores, lagomorphs, rodents, etc.). Leishmania infantum was also identified infecting humans and lagomorphs (i.e. hares and rabbits) over the period of 2009–2016, with the latter acting as the main reservoirs involved in the human leishmaniosis outbreak in Madrid.ResultsTwo cases of clinical leishmaniosis are reported in orangutans (Pongo pygmaeus pygmaeus) housed at two different centres in Madrid. The first is the case of a 36-year-old male orangutan with severe weight loss and apathy. A complete blood count and biochemical profile revealed anaemia, neutropenia, hypoalbuminaemia and elevated transaminases. Hepato-splenomegaly was also observed. Four months later, due to worsening of clinical signs (mainly bilateral epistaxis), blood and bone marrow samples were collected. Amastigotes of L. infantum were detected in macrophages from a bone marrow aspirate and by specific polymerase chain reaction. The second case was a 34-year-old female orangutan with severe weight loss and apathy and no other apparent clinical signs. A complete blood count and biochemical profile revealed anaemia, pancytopenia and hypoalbuminaemia. Splenomegaly and pericardial effusion were also observed. As leishmaniosis was included in the differential diagnosis, both blood and bone marrow samples were collected. Leishmania infantum infection was confirmed by microscopy, molecular diagnosis and serology (immunofluorescence antibody test). Both animals were treated daily with oral miltefosine for 28 days; allopurinol was also given uninterruptedly in Case 2 for at least 6 months. During follow-up, though good clinical recovery was clear, a lack of parasitological cure was confirmed molecularly in both blood and bone marrow samples from the two orangutans. In both habitats, the presence of the sand fly vector identified as Phlebotomus perniciosus was confirmed.ConclusionsTo our knowledge, this is the first report of L. infantum infection in great apes and in the endangered species P. p. pygmaeus. We are presently looking for L. infantum in other non-human primates living in the same peri-urban areas. If detected, we will examine the impacts of this serious disease on these critically endangered species.

Detection and molecular characterization of Acanthamoeba spp. in stray cats from Madrid, Spain

Acanthamoeba spp. is a widespread protozoan that has been isolated from air, dust, soil, water and biological samples. An opportunistic pathogen of humans and animals, it may cause ocular keratitis, encephalitis, and even multisystem disease. The frequency of Acanthamoeba in animals is unknown. The aim of present study was determine the presence of Acanthamoeba spp. in immunocompromised stray cats - animals possibly more likely to harbour the infection given their immunocompromised status and frequenting of contaminated environments. Of 307 cats examined, 55 were positive for feline immunodeficiency virus and/or feline leukaemia virus and therefore included in the study. Corneal scrapings were obtained to isolate Acanthamoeba spp. by culture and molecular detection by conventional and real time PCR. None of the samples examined directly by molecular methods were positive for Acanthamoeba spp. However, two (3.6%) cases of the cultured samples provided positive results, which were confirmed by subsequent molecular analysis. Sequencing assigned one isolate to genotype T4 and the other to T2. Since Acanthamoeba spp. may also infect animals and humans, the present findings may raise some public health and veterinary concerns.