José Manuel Cristóvão

Leishmaniasis in Portugal: enzyme polymorphism of Leishmania infantum based on the identification of 213 strains

This study reports isoenzyme polymorphism of Leishmania strains isolated in different regions of Portugal between 1982 and 2005. A total of 213 strains were obtained from cases of visceral and cutaneous leishmaniasis isolated from immunocompetent patients (adults and children) and immunocompromised adults, as well as from dogs and sandflies. Four zymodemes were identified: MON‐1, MON‐24, MON‐29 and MON‐80. Zymodeme MON‐1 was identified in 96.7% of the strains, predominating in both immunocompetent and immunocompromised human patients, and it was the only zymodeme isolated from dogs. Isoenzyme diversity in HIV‐infected patients was higher than in the immunocompetent group, in which all the strains from visceral leishmaniasis were MON‐1. The domestic dog was confirmed as the reservoir host of zoonotic leishmaniasis in Portugal and Phlebotomus perniciosus and Phlebotomus ariasi as vectors. The overall low enzyme polymorphism observed in the Portuguese foci contrasts with the neighbouring foci in Spain.

Feline Leishmania infection in a canine leishmaniasis endemic region, Portugal.

Canine leishmaniasis (CanL) caused by Leishmania infantum is a serious zoonotic public health and veterinary problem in the Mediterranean basin. Leishmania infection in domestic cats (Felis catus domesticus) has been reported in several countries where this zoonosis is endemic, such as Portugal, Spain, Italy, France, Greece, Israel, Palestine and Brazil. The aim of this study was to contribute to the knowledge of the role played by cats in Leishmania epidemiology, in an endemic focus of zoonotic leishmaniasis, the Lisbon metropolitan area, Portugal. L. infantum DNA was detected in peripheral blood of 28 out of 138 cats (20.3%). The result of PCR in blood of cats was not closely associated with the level of specific circulating antibodies in their sera. Positive serology was observed only in one cat out of 76. In the same geographic region and time period the indirect immunofluorescent test revealed 20.4% (31/152) of dogs with antibodies and PCR detected Leismania DNA on 34.9% (53/152) animals. Despite the fact that specific antibodies have been validated for diagnosis of CanL, their detection does not seem to be sensitive enough to predict Leishmania infection in cats. On the other hand, the presence of parasite DNA in cats peripheral blood during the transmission season and out of the season suggests that these animals living in endemic areas are frequently exposed or infected with the parasite. Although dogs have been universally regarded as the major domestic/peridomestic reservoir hosts, the present data allow us to hypothesize that cats can act as an alternative reservoir host of L. infantum, rather than an accidental host. However, in order to evaluate the existence of a transmission cycle with cats sustaining and spreading zoonotic leishmaniasis is necessary to prove that these animals can transmit the parasite to the vector in nature.

Experimental canine leishmaniasis: Clinical, parasitological and serological follow-up

Canine leishmaniasis (CanL) caused by Leishmania infantum is transmitted by the bite of phlebotomine sand flies and affects millions of dogs in Europe, Asia, North Africa and South America. Canis familiaris is the major host for these parasites, and the main reservoir for human visceral infection. The development of effective molecules for therapy and immunoprophylaxis, would be an important tool in the control of this zoonosis. The aim of this study was to characterize an experimental CanL model in order to determine the best challenge model and which parameters are the most reliable to evaluate the efficacy of new drugs or vaccine candidates against L. infantum infection. The intravenous challenge with purified amastigotes used in this study allowed the development of infection in all animals inoculated (as confirmed by the detection of parasite in the different tissues and organs collected 6 months after inoculation). Molecular and serologic techniques were efficient methods for the follow-up. Lymph node and bone marrow aspirates were suitable clinical samples to detect the presence of Leishmania parasites. Despite ELISA was highly sensitive in detecting specific anti-Leishmania antibodies the use of two tests can improve the sensitivity and specificity of serological diagnosis.

Entomological and ecological studies in a new potential zoonotic leishmaniasis focus in Torres Novas municipality, Central Region, Portugal.

In Portugal human and canine leishmaniasis are caused by Leishmania infantum, and Phlebotomus perniciosus and P. ariasi are the proven vectors. Three main foci were identified in eightys decade: Trás-os-Montes and Alto Douro region, Lisbon region and Algarve region, but according to OnLeish observatory data, canine leishmaniasis cases have been reported from several other regions, for which sand fly species and their infection rates are unknown. This study is the first phlebotomine survey in Torres Novas municipality, Santarém District, Portugal. The main objectives were to identify the phlebotomine species, their bioecological aspects, Leishmania infection rate and the risk factors for the presence of phlebotomine species in the municipality. From June to November, 2010, 275 biotopes were surveyed with CDC light-traps. Captures covered the 17 parishes of the municipality and included domestic, peridomestic and sylvatic biotopes. Specimens were identified morphologically and females were used for molecular detection of Leishmania and bloodmeal identification. Simple and multiple logistic regression analysis were used to identify risk factors for phlebotomine presence. Nonparametric tests were used to compare densities of independent groups. A total of 1262 sand flies were captured and identified, and four species detected: P. perniciosus (73.69%), P. ariasi (8.16%), P. sergenti (6.58%) and Sergentomyia minuta (11.57%). In 71.4% localities at least one L. infantum proven vector species was present. Risk factors were identified as: high average temperatures and low relative humidities, sheltered locations and absence of strong wind, presence of pine trees as dominant vegetation, peridomestic biotopes, particularly sheep pens or proximity of sheep, poultry and house martin nests. L. infantum infection rate was 4% for P. ariasi and 0.48% for the total of Larroussius females. P. perniciosus females exhibited an opportunistic behavior, feeding in a wide variety of vertebrate hosts. The high abundance and distribution of proven vector species, together with a canine leishmaniasis seroprevalence of 7.93% in the District, and the capture of a gravid infective sand fly female, suggests that Torres Novas municipality is a potential zoonotic leishmaniasis focus in the country.

Molecular detection of Leishmania DNA and identification of blood meals in wild caught phlebotomine sand flies (Diptera: Psychodidae) from southern Portugal

BackgroundZoonotic visceral leishmaniasis caused by Leishmania infantum which is transmitted by phlebotomine sand flies (Diptera, Psychodidae) is endemic in the Mediterranean basin. The main objectives of this study were to (i) detect Leishmania DNA and (ii) identify blood meal sources in wild caught female sand flies in the zoonotic leishmaniasis region of Algarve, Portugal/Southwestern Europe.MethodsPhlebotomine sand flies were collected using CDC miniature light traps and sticky papers. Sand flies were identified morphologically and tested for Leishmania sp. by PCR using ITS-1 as the target sequence. The source of blood meal of the engorged females was determined using the cyt-b sequence.ResultsOut of the 4,971 (2,584 males and 2,387 females) collected sand flies, Leishmania DNA was detected by PCR in three females (0.13%), specifically in two specimens identified on the basis of morphological features as Sergentomyia minuta and one as Phlebotomus perniciosus. Haematic preferences, as defined by the analysis of cyt-b DNA amplified from the blood-meals detected in the engorged female specimens, showed that P. perniciosus fed on a wide range of domestic animals while human and lizard DNA was detected in engorged S. minuta.ConclusionsThe anthropophilic behavior of S. minuta together with the detection of Leishmania DNA highlights the need to determine the role played by this species in the transmission of Leishmania parasites to humans. In addition, on-going surveillance on Leishmania vectors is crucial as the increased migration and travelling flow elevate the risk of introduction and spread of infections by Leishmania species which are non-endemic.

Bacterial and protozoal agents of canine vector-borne diseases in the blood of domestic and stray dogs from southern Portugal.

BackgroundThe so-called canine vector-borne diseases (CVBD) are caused by a wide range of pathogens transmitted by arthropods. In addition to their veterinary importance, many of these canine vector-borne pathogens can also affect the human population due to their zoonotic potential, a situation that requires a One Health approach. As the prevalence of vector-borne pathogens in cats from southern Portugal has been recently evaluated, the aim of the present study was to assess if the same agents were present in dogs living in the same area, and to assess positivity-associated risk factors.MethodsOne thousand and ten dogs (521 domestic and 489 stray) from veterinary medical centres and animal shelters in southern Portugal were enrolled. Anaplasma spp./Ehrlichia spp., Bartonella spp., Borrelia burgdorferi sensu lato, Babesia spp., Hepatozoon spp. and Leishmania infantum infections were evaluated by polymerase chain reaction (PCR) assays in blood samples.ResultsSixty-eight (6.7%) dogs were PCR-positive to at least one of the tested CVBD agent species, genera or complex, including one dog found positive to two different genera. Nineteen (1.9%) dogs were positive to Anaplasma spp./Ehrlichia spp., eight (0.8%) to B. burgdorferi s.l., 31 (3.1%) to Hepatozoon spp. and 11 (1.1%) to L. infantum. Anaplasma platys, Ehrlichia canis, B. burgdorferis.l. and Hepatozoon canis were identified by DNA sequencing, including one animal confirmed with both A. platys and H. canis. Furthermore, Wolbachia spp. was amplified in blood from four dogs. None of the tested dogs was positive by PCR for Bartonella spp. or Babesia spp.ConclusionsThe molecular identification of CVBD agents in southern Portugal, some of them with zoonotic concern, reinforces the importance to alert the veterinary community, owners and public health authorities to prevent the risk of transmission of vector-borne pathogens among dogs and to other vertebrate hosts including humans. The prevalence of the selected pathogens was lower than that previously found in cats from the same region, probably because veterinarians and owners are more aware of them in the canine population and control measures are used more often.

Leishmania infection and host-blood feeding preferences of phlebotomine sandflies and canine leishmaniasis in an endemic European area, the Algarve Region in Portugal

The Algarve Region (AR) in southern Portugal, which is an international tourist destination, has been considered an endemic region of zoonotic leishmaniasis caused by Leishmania infantum since the 1980s. In the present study, phlebotomine and canine surveys were conducted to identify sandfly blood meal sources and to update the occurrence of Leishmania infection in vectors and dogs. Four sandfly species were captured: Phlebotomus perniciosus, Phlebotomus ariasi, Phlebotomus sergenti and Sergentomyia minuta. In one P. perniciosus female, L. infantum DNA was detected. Blood meal tests showed that this species had no host preferences and was an opportunistic feeder. An overall canine leishmaniasis (CanL) seroprevalence of 16.06% was found; the seroprevalence was 3.88% in dogs housed in kennels and 40.63% in dogs that attended veterinary clinics. The simultaneous occurrence of dogs and P. perniciosus infected with L. infantum in the AR indicates that the region continues to be an endemic area for CanL. Our results reinforce the need for the systematic spatial distribution of phlebotomine populations and their Leishmania infection rates and the need to simultaneously perform pathogen monitoring in both invertebrate and vertebrate hosts to investigate the transmission, distribution and spreading of Leishmania infection.

In vitro and in vivo behaviour of sympatric Leishmania (V.) braziliensis, L. (V.) peruviana and their hybrids

Leishmania (Viannia) braziliensis is the main cause of highly disfiguring mucocutaneous leishmaniasis (MCL) in South America. The related species L. (V.) peruviana has only been identified in simple cutaneous lesions (CL). Hybrids between L. braziliensis and L. peruviana have been reported although genetic exchange in Leishmania is considered to be rare. Here we compared growth in vitro, adaptive capacity under thermal and oxidative stress and behaviour in a hamster model, of L. braziliensis, L. peruviana, and their putative hybrids. At 24°C, the optimal temperature for in vitro growth, L. braziliensis had the highest growth rate. In in vitro studies hybrid clones presented heterogeneous phenotypes, from slower growth rates, similar to L. peruviana, to higher growth rates, as observed in L. braziliensis. Hamsters infected with hybrid strains, presented the highest parasite densities and aggressive relapses at a later stage of infection. Hybrids generally presented higher plasticity and phenotypic diversity than the putative parental species, with potential eco-epidemiological implications, including an impact on the success of disease control.

Neutralization-based seroprevalence of Toscana virus and sandfly fever Sicilian virus in dogs and cats from Portugal

Sandfly-borne phleboviruses are endemic in the Mediterranean basin. However, levels of exposure of human and animal populations are inadequately researched. Toscana virus (TOSV) is present in Portugal where it causes human infection and disease; in contrast there are few data for sandfly fever Sicilian virus (SFSV) which has neither been isolated nor detected by molecular tests and for which there are only limited serological data. The sera collected from 1160 dogs and 189 cats in southern Portugal were tested for the presence of neutralizing antibodies against TOSV and SFSV, two viruses recognized as distinct serocomplexes in the Mediterranean region. Our data showed (i) seropositivity to TOSV and SFSV in dogs at a rate of 6.8 and 50.8 %, respectively, and (ii) that 3.7 % of cats were seropositive for TOSV. TOSV findings are in line with previous results obtained with less stringent serological assays. Our results for SFSV in dogs clearly indicate that the virus is circulating widely and that humans may be exposed to infection via the dogs. Although the presence of SFSV was suggested by haemagglutination inhibition in 4/1690 human sera in 1974, this is the first time, as far as we know, that SFSV has been shown to circulate so widely in dogs in Portugal. Future studies should be directed at isolating strains of SFSV in Portugal from dogs, humans and sandflies collected in high prevalence regions. As dogs appear to be good sentinels for SFSV, their role as a possible reservoir in the natural cycle should also be considered.

Exploring the utility of phylogenetic analysis of cytochrome oxidase gene subunit I as a complementary tool to classical taxonomical identification of phlebotomine sand fly species (Diptera, Psychodidae) from southern Europe.

Phlebotomine sand flies (Diptera, Psychodidae) are known to be vectors of several pathogens such as Leishmania and Phlebovirus genera. The identification of phlebotomine sand fly species is currently based on morphological characters, and requires considerable taxonomic expertise and skilfulness, but may be complemented by DNA-based analyses for (i) accurate species identification and (ii) for estimating sand fly diversity. The aim of this study was to evaluate the utility of mitochondrial cytochrome oxidase gene subunit I (cox1) sequence analysis as a complementary tool to classical taxonomical for the identification of the most prevalent phlebotomine sand fly species from southern Europe (i.e. Phlebotomus ariasi, P. perniciosus, P. sergenti and Sergentomyia minuta). Phylogenetic analyses of cox1 sequences allowed conclusive assignment of most of the sand flies into individual species, and revealed the genetic heterogeneity that characterizes some of the identified genetic clusters. Nevertheless, it showed some limitations, as it failed to (i) allocate correctly all of all species of a given subgenus to a single lineage, or (ii) conclusively identify sequences amplified from individuals classified morphologically as P. ariasi. A more extensive analysis of cox1 sequences together with morphometric characterization of specimens from different geographic areas/regions might be useful for the correct assessment of the phylogenetic relationship within the P. ariasi/P. chadlii cluster and/or help to ascertain the usefulness of cox1 for molecular taxonomy of sand flies.