Rosa Gálvez

Emerging trends in the seroprevalence of canine leishmaniosis in the Madrid region (central Spain)

This report describes a cross-sectional serological survey of the epidemiology of canine leishmaniasis (CanL) performed in 2006 and 2007 in the Madrid region (central Spain) where the disease is endemic. The work presented here is one of the several studies conducted in different Spanish regions under the Integrated Project of the European Commission entitled Emerging Diseases in a changing European eNvironment (EDEN). The aim of this project is to identify and catalogue European ecosystems and environmental conditions that determine the spatial and temporal distributions and dynamics of several pathogenic agents including Leishmania infantum (EDEN-LEI). The study area (Madrid Autonomous Region) was selected on the grounds of its wide altitude range. This area was surveyed from NE to SW across its mountain range (Sistema Central) and plateau area. One thousand and seventy-six dogs from 32 villages were examined for clinical signs of CanL, and serum samples were obtained to determine several haematological and biochemical variables. Leishmaniasis-specific antibodies were identified using an indirect immunofluorescence antibody test (IFAT). 87 of the 1076 dogs were seropositive for the protozoan (IFAT: cut-off>or=1/80) indicating a seroprevalence of 8.1% (0-16.1% depending on the village). On the basis of a physical examination and the biochemical/haematological status of each dog, 32 of the 87 infected dogs were described as clinically healthy (37%). Seroprevalence showed a peak in young dogs (1-2 years) and a second larger peak among the older dogs (7-8 years). Factors correlated with a higher infection risk were age (OR=1.15 [95% CI: 1.07-1.22]), weight (OR=1.10 [95% CI: 1.04-1.16]), and living outdoors as opposed to in a home (OR=3.38 [95% CI: 1.42-8.05]). According to data from studies performed in 1992 in the same area, the seroprevalence of CanL has increased 1.54-fold [95% CI: 1.04-2.29]. Given that this increasing trend cannot be attributed to differences in the sociodemographic characteristics of the dog populations, it is proposed that environmental changes could have had an impact on vector and reservoir densities and their geographical distributions. Further studies designed to explain this trend should attempt to correlate sand fly densities and CanL seroprevalences with climate, land use and human changes.

Predicting the distribution of canine leishmaniasis in western Europe based on environmental variables.

The domestic dog is the reservoir host of Leishmania infantum, the causative agent of zoonotic visceral leishmaniasis endemic in Mediterranean Europe. Targeted control requires predictive risk maps of canine leishmaniasis (CanL), which are now explored. We databased 2187 published and unpublished surveys of CanL in southern Europe. A total of 947 western surveys met inclusion criteria for analysis, including serological identification of infection (504, 369 dogs tested 1971-2006). Seroprevalence was 23 2% overall (median 10%). Logistic regression models within a GIS framework identified the main environmental predictors of CanL seroprevalence in Portugal, Spain, France and Italy, or in France alone. A 10-fold cross-validation approach determined model capacity to predict point-values of seroprevalence and the correct seroprevalence class (<5%, 5-20%, >20%). Both the four-country and France-only models performed reasonably well for predicting correctly the <5% and >20% seroprevalence classes (AUC >0 70). However, the France-only model performed much better for France than the four-country model. The four-country model adequately predicted regions of CanL emergence in northern Italy (<5% seroprevalence). Both models poorly predicted intermediate point seroprevalences (5-20%) within regional foci, because surveys were biased towards known rural foci and Mediterranean bioclimates. Our recommendations for standardizing surveys would permit higher-resolution risk mapping.

Mapping the Current Distribution and Predicted Spread of the Leishmaniosis Sand Fly Vector in the Madrid Region (Spain) Based on Environmental Variables and Expected Climate Change

Leishmaniosis caused by Leishmania infantum is a widespread zoonotic disease that is endemic in the Mediterranean basin. Based on prior point abundance data for the two sand fly vectors of leishmaniosis in the Madrid region (Phlebotomus perniciosus and Phlebotomus ariasi), models were constructed to predict the spatial distribution patterns of these vectors. The models were obtained by negative binomial regression of several environmental variables and then used to map vector distributions. To validate the maps, we used serological prevalence data of Leishmania infection in dogs and incidence data were obtained through questionnaires completed by veterinarians in the region. Seropositive dogs and veterinary clinics registering a higher incidence of canine leishmaniosis appeared closer to our modeled vector foci. In the face of climate change, we simulated the future distributions of the sand flies for each third of the 21st century and predicted their spread in the region.

Infectivity to Phlebotomus perniciosus of dogs naturally parasitized with Leishmania infantum after different treatments

BackgroundIn Europe most dogs with clinical leishmaniosis are treated with leishmanicides, typically antimonials combined with allopurinol and good clinical recovery is observed in a high number of these dogs. Through xenodiagnosis, the capacity of a treated animal to infect the vector of the disease under treatment is assessed as a measure of the chemotherapeutic efficacy of the drug used. The objective of the present study was to evaluate through direct xenodiagnosis the infectivity to Phlebotomus perniciosus of dogs naturally parasitized with Leishmania infantum after treatment, and to follow the clinical and parasite course of disease. Thirty two dogs with clinical leishmaniosis were assigned to one of three treatment groups: meglumine antimoniate plus allopurinol (Group A), meglumine antimoniate (Group B) or allopurinol (Group C). During the study, the dogs were examined before treatment (Day 0) and bimonthly thereafter until Day 180 (six months post-treatment onset).ResultsThe three groups were scored over time according to the effects of treatment on clinical signs and clinical-pathological variables. Significant differences in clinical scores were observed between Group A and the other two groups, indicating the combined treatment was the most effective. After treatment, bone marrow cultures were positive for the parasite in 30.8% of dogs in some of the check ups (3 or 25% in Group A, 1 or 11.1% in Group B, and 4 or 80% in Group C). Our xenodiagnosis experiments revealed that 15.4% of treated dogs were still able to infect sand flies at some point after treatment (2 dogs or 16.6% in Group A, 2 or 22.2% in Group B and none in Group C). Only 7.7% of the entire study population could infect sand flies as from the second month post-treatment onset.ConclusionThe three treatment regimens tested significantly reduced the infectivity of dogs towards sand flies, thus diminishing the epidemiological risks of treated dogs both for human beings and other healthy dogs. Despite its low cure rate, the use of allopurinol after a course of leishmanicide treatment is proposed to keep dogs non-infectious during the disease transmission season (4-6 months in southern Europe).

Genetic structure of Phlebotomus (Larroussius) ariasi populations, the vector of Leishmania infantum in the western Mediterranean: Epidemiological implications☆

In recent years there has been growing interest in analyzing the geographical variations between populations of different Phlebotomus spp. by comparing the sequences of various genes. However, little is known about the genetic structure of Phlebotomus ariasi. In this study, we were able to sequence a fragment of the mitochondrial Cyt b gene in 133 sandflies morphologically identified as P. ariasi and proceeding from a wide geographical range covering 35 locations in 11 different regions from five countries. The intra-specific diversity of P. ariasi is high, with 45 haplotypes differing from each other by one to 26 bases and they are distributed in two mitochondrial lineages, one limited geographically to Algeria and the other widely dispersed across Mediterranean countries. The Algerian lineage is characterized by having 13 fixed polymorphisms and is made up of one sole haplotype. The European/Moroccan P. ariasi lineage is characterized by being made up of a great diversity of haplotypes (44) which display some geographical structuring. This could be one of the multiple factors involved in the epidemiological heterogeneity of the foci of leishmaniasis. Phlebotomus chadlii is the sister group of European/Moroccan P. ariasi. The separation of the Algerian haplotype, H45, from the rest of the specimens, European/Moroccan P. ariasi and P. chadlii, is well supported by the bootstrap analysis.

Evaluation of a spray of permethrin and pyriproxyfen for the protection of dogs against Phlebotomus perniciosus

Dogs are the main domestic reservoir of Leishmania infantum in the Old World (Leishmania chagasi in the New World) a parasite responsible for many cases of human visceral leishmaniasis in both endemic and non-endemic regions. One strategy for the control of leishmaniasis would be to prevent dogs from being bitten by sandflies, the vector of leishmaniasis. This study was designed to assess the efficacy of spraying a combination of permethrin and pyriproxyfen on to dogs artificially exposed to sandflies. Two groups of four male dogs, one of them treated and the other left untreated as controls, were exposed for one hour to 100 female sandflies seven days before the treatment, on the day of treatment and seven, 14, 21, and 28 days later. After each exposure, sandflies were collected, counted and scored. The prevention of sandfly bite was calculated by measuring the number of fed sandflies (dead and alive) after treatment. In this experimental assay, the repellent effect of the treatment against sandfly bites after 21 days was 71·4 per cent, but the insecticidal effect was only 7·2 per cent.

Efficacy of 65% permethrin applied to dogs as a spot-on against Phlebotomus perniciosus

Leishmania infantum is a protozoan parasite causing leishmaniosis, a visceral disease transmitted by the bites of sand flies. As the main reservoir of the parasite, dogs are the principal targets of control measures against this disease, which affects both humans and dogs. Several studies have revealed the usefulness of topical insecticide treatment (collars, spot-ons and sprays) in reducing the incidence and prevalence of L. infantum. The present study was designed to test the efficacy of 65% permethrin applied to dogs as a spot-on against the sand fly vector Phlebotomus perniciosus. The duration of the desired effects was also estimated to help design an optimal treatment regimen. Twelve dogs assigned to treatment (n=6) and control (n=6) groups were exposed to sand flies once a week over a seven-week period. Repellent and insecticidal efficacies were estimated and compared amongst the groups. Our findings indicate satisfactory repellent, or anti-feeding, effects lasting 3 weeks and short-term insecticidal effects lasting 2 weeks after initial application. Accordingly, we recommend the use of this product every 2-3 weeks during the active phlebotomine sand fly period to protect dogs against the bites of P. perniciosus.

Questionnaire-based survey on the clinical management of canine leishmaniosis in the Madrid region (central Spain)

This paper describes a questionnaire designed to gain information on how veterinarians clinically manage canine leishmaniosis (CanL) in the Madrid region (central Spain). The present survey is one of the several similar questionnaire-based surveys conducted within the framework of the project EDEN (Emerging Diseases in a changing European eNvironment). The questionnaire sought to obtain data regarding the main clinical manifestations observed, the diagnostic methods used and the preventive measures recommended. Its Spanish version was sent by post to veterinary practitioners within the study area in two lots, one sent out in December 2006 and the other in March 2007. Only 174 of the 760 questionnaires sent were completed and returned (reply rate of 23%). Among the completed questionnaires, clinics differed widely in terms of features such as the habitats of the dogs (urban, peri-urban or rural) and patient volumes. Clinics attending dogs from peri-urban/rural habitats reported more suspected (p<0.001), confirmed (p=0.001) and newly diagnosed (p=0.001) cases/year than clinics providing service to a city clientele alone. According to the veterinary practitioners, skin lesions, lymphadenomegaly and weight loss were commonly observed, although these signs are not specific to CanL. Signs described to be of high diagnostic value were epistaxis and kidney disease. All the veterinarians polled reported that a suspicion of Leishmania infantum infection was confirmed by at least a serological method; the immunofluorescence antibody test (IFAT) being the technique most used. To prevent the disease, most vets recommended topical synthetic pyrethroids applied as impregnated collars or spot-ons. It is observed that despite considerable progress is being made in clinical management and controlling the disease, in Madrid Region its incidence continues to increase.

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.

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.