Effectiveness of the sesquiterpene (-)-α-bisabolol in dogs with naturally acquired canine leishmaniosis: an exploratory clinical trial.

The use of natural products is a promising approach for treating visceral leishmaniosis. (-)-α-Bisabolol is a sesquiterpene that have been proved active in vivo on Leishmania infantum-infected mice without showing toxicity. A single-centre, parallel-group, randomized, exploratory study was designed to assess its efficacy in a canine leishmaniosis model involving naturally infected dogs. In this clinical trial, 12 dogs were allocated into two groups and were treated with either meglumine antimoniate (100 mg/kg) through subcutaneous route or (-)-α-bisabolol (30 mg/kg) through oral route for two treatment series of 30 days, separated by a 30-day interval. A 4-month follow-up period was established as well. Parasite loads in bone marrow, lymph node and blood were estimated through quantitative PCR. Antibody titres were determined through immunofluorescence antibody test and cytokine expression values were estimated through real-time reverse transcription-PCR. Treatment safety was assessed through the evaluation of weight, gastrointestinal alterations and hematological and biochemical parameters in blood. Analyses were performed before and after treatment, and after a 4-months follow-up period. Treatment with the sesquiterpene was effective at decreasing parasite loads and increasing gamma-interferon expression level. Dogs treated with (-)-α-bisabolol did not show any toxicity sign. These results were better than those obtained using the reference drug, meglumine antimoniate. The natural compound seemed to induce a Th1 immune response that led to parasitological and clinical improvement without showing any safety issue, suggesting a high potential for the treatment of canine and human visceral leishmaniosis.

Hair parasite load as a new biomarker for monitoring treatment response in canine leishmaniasis.

Canine leishmaniasis treatment focuses on the reduction of parasite load, the clinical improvement of the animal, and the avoidance of relapses, in a scenario where the definitive parasite clearance is not achievable. Therefore, monitoring is crucial during the treatment of this disease. Quantitative PCR has been shown as an ideal tool for the treatment monitoring when quantifying parasite load in target organs such as lymph node or bone marrow, tissues that are too invasive for regular evaluation. This study aims to prove the potential of hair parasite load in the treatment monitoring of canine leishmaniasis. Six dogs were treated with meglumine antimoniate and monitored up to four months after the end of the treatment. Parasite loads in bone marrow, blood, lymph node and hair were quantified by real-time quantitative PCR. Total IgG, IgG1, and IgG2 antibody titres were analysed by immunofluorescent assay and a clinical assessment was carried out. Treatment consisted of two 28-day courses of meglumine antimoniate (100mg/kg/day) separated by an one-month interval. Analyses were performed before (day 0), during (day 60) and after treatment (day 120), and at the end of a follow-up period (day 210, four months after the end of treatment). Hair parasite load turned out to be strongly correlated with bone marrow, lymph node and blood parasite loads and with the clinical score and the IgG1 antibody titre. The evolution of this biomarker reflects the evolution of the parasitological, immunological and clinical state of the dog, highlighting its potential as a non-invasive marker for the treatment monitoring in canine leishmaniasis.

Situación de la leishmaniosis en Granada (Sur de España) después de 25 años de estudio / Current status of leishmaniasis in Granada (Southern Spain) after 25 years of study

INTRODUCCIÓN: En España, Leishmania infantum es la única especie responsable de la leishmaniosis cutánea (LC) y visceral (LV) humanas, así como de la leishmaniosis canina (L Ca).Como vectores actúan dípteros nematóceros del género Phlebotomus (Diptera, Phlebotomidae), con las especies Phlebotomus perniciosus y P. ariasi actuando en condiciones simpátricas en un mismo foco. OBJETIVO: Pretendemos analizar los cambios acaecidos en la epidemiología de la leishmaniosis en la provincia de Granada durante los últimos 25 años, prestando atención a la incidencia de la enfermedad humana, la prevalencia de la leishmaniosis canina y la densidad de los vectores. METODOLOGÍA: se han recopilado los casos de leishmaniosis humana declarados entre 1984 y2005. En el caso del reservorio, se ha realizado un estudio epidemiológico de la leishmaniosis canina en 20 pueblos de la Alpujarra, que fueron estudiados previamente hace 22 años. Los cambios en las poblaciones de vectores se han determinado haciendo capturas con papeles adhesivos. RESULTADOS Y DISCUSIÓN: En el periodo 1984-2005, la media de la tasa de incidencia de la leishmaniosis humana en la provincia de Granada ha sido de 0,4 casos anuales/100.000 habitantes, observándose un ligero aumento del número de casos a lo largo de los años. Con respecto a la L Ca, hemos observado un incremento progresivo de la seroprevalencia en el piso bioclimático Mesomediterráneo. La densidad de P.perniciosus y P. ariasi ha quedado constante en el Termomediterráneo y ha caído en el Mesomediterráneo; sin embargo se ha incrementado el periodo de actividad de P. perniciosus(AU)

Introduction: The human and canine leishmaniosis are endemic in Granada province (South Spain)since the beginning of 20th century. Object: to study the variation of the rate of human leishmaniosis, the seroprevalence of canine leishmaniosis, and the density of vectors (phlebotomine sandflies).Methodology: we have compiled the reported cases of human leishmaniosis in the period 1984-2004,we have analysed, by IFAT, the dogs of 20 villages of the Alpujarra region, and the results were compared with those obtained 22 years ago. In the same way, the vectors populations were studied using sticky traps. Results and discussion: we have observed a net increase in the rate of human leishmaniosis, and in the seroprevalence of canine leishmaniosis. Respect the vector there is a increase in the activity period but not in the density(AU)

Diagnosis of infections with Leishmania infantum using PCR-ELISA.

On the basis of partial amplification of a cloned fragment of kDNA of Leishmania infantum which is specific for this species, we developed a PCR-ELISA technique which avoids the problems associated with classical diagnostic techniques. This technique was tested on 33 L. infantum strains from 19 different zymodemes, which were recognized equally. It was also used on human and canine clinical samples. PCR-ELISA has a higher sensitivity than the other techniques used (IFAT, parasite cultures, optical microscopy of stained samples) and permits detection of a minimum of 0.1 promastigotes or 1 fg of genomic DNA. PCR-ELISA can be used to diagnose human cutaneous leishmaniasis using material obtained by scraping the lesion margin, and human visceral leishmaniasis in HIV(+) individuals and canine leishmaniasis with peripheral blood samples. The presence of L. infantum in dogs with low antibody titres with IFAT technique (20 and 40) was demonstrated indicating that seroprevalence data from epidemiological studies underestimate the true rates of infection.

Changes in antibody titres against Leishmania infantum in naturally infected dogs in southern Spain.

Monthly serological follow-up of the antibody titres against Leishmania infantum in hunting dogs in rural southern Spain was carried out during the transmission period (from April to October in 1996). Titres > or = 160 were regarded as indicating disease, while lower positive tires were also recorded as 'seropositive'. Over this period, a significant (p < 0.05) increase in the percentage of dogs with a titre > or = 160 was observed; from 12.6% in April to 19.2% in October. The same pattern was observed with the seropositives (titre > or = 20) which increased from 36.3% in April to 55.6% in October. Of seronegatives in April, 60% were still negative in October whereas 22% had a titre of 20-40 and 18% had a titre > or = 80. Similarly, 68% of those with a titre > or = 80 in April still had a titre in this range in October, 25% of the titre had decreased to 20-40 and in 7% to a zero titre. The most noteworthy is that 25% of animals reached titres of > or = 80 and decreased to zero or 20 more than once. Parallel to this study, another survey was conducted on dogs from Granada city centre. The seroprevalences (titres > or = 160) at the start and the end of the transmission period were 1.7 and 3.7% respectively and the seropositively values were 12 and 11% respectively.

Leishmaniasis eco-epidemiology in the Alpujarra region (Granada Province, southern Spain).

An eco-epidemiological study was carried out in the region of the Alpujarras (southern Spain, Granada province). Nine villages were chosen at random in 5 bioclimatic zones. A study of leishmaniasis in the canine and human populations, was carried out concentrating especially upon schoolchildren. A total of 615 dogs were screened, which represents almost 100% of the canine census. Of the screened dogs, 33 showed an antibody titer > or = 1/160 when tested by IFA (seroprevalence of 5.3%). Age, sex, activity, and clinical symptoms of each dog were taken into account. Among the human population, infection from Leishmania was studied using the Leishmanin skin test (LST). Of 1286 people who were tested, 568 (44.16%) were positive. Most of the subjects were schoolchildren (878; practically 100% of the pupils), of whom 288 (32.8%) tested positive. A close relation was seen to exist between the percentage of positive LST's and age. Finally, a close relationship was also observed between canine seroprevalence and percentage of schoolchildren who tested positive in the LST, in the 5 bioclimatic zones under consideration.

Isoenzymatic characterization of the etiologic agent of canine leishmaniasis in the Granada region of southern Spain.

Sixty-four strains of Leishmania of canine origin, 61 visceral and three cutaneous, were isoenzymatically examined. These were collected from 23 sites in the Granada region in southern Spain. Starch gel was used in electrophoresis and a total of 15 enzymes were studied. All of the visceral strains and two of the cutaneous ones were identified as L. infantum zymodeme GR-1 (= MON-1). The third cutaneous strain was of a different zymodeme, belonging to the same complex but differing in the glucose-6-phosphate dehydrogenase (= 105) enzyme. This zymodeme has been named GR-16 and is equivalent to L. infantum MON-105; it has not been previously reported in dogs or any other animal reservoir.

Phlebotomus perniciosus Newstead, 1911, infection by various zymodemes of the Leishmania infantum complex in the Granada province (southern Spain).

This study presents the results of the isoenzymatic characterization of 21 strains of Leishmania of sandfly (P. perniciosus) origin from the Torvizcón area. It forms an integral part of a larger eco-epidemiological study of the Alpujarras (Granada province, Southern Spain). The strains analysed were shown to belong to the L. infantum complex based on the results of 15 enzymes. The electrophoretic profiles for the enzymes MDH, G6PD and NP1 have permitted the identification of four zymodemes: GR-1 (5 strains), GR-2 (2 strains), GR-3 (13 strains) and GR-7 (1 strain); only one of these zymodemes, GR-1, was found in the Torvizcón area in the vertebrate host (man and dog). This is the first time zymodeme GR-7 has been described.

On Phlebotomus perniciosus Newstead, 1911 (Diptera, Phlebotomidae) in the Province of Almeria in southeastern Spain.

Several studies of Phlebotomine sandflies in the province of Almería (Southeastern Spain) showed Phlebotomus perniciosus to be the most abundant and wide-ranging number of the genus Phlebotomus. Samples collected during an entire year showed a population curve characterized by two peaks. Morphological examinations of males revealed marked variability to the peneane valves, and the existence od specimens with intermediate features between P. perniciosus and P. longicuspis. Dissections of females detected 24 specimens out of 522 (4.59%) infected with Leishmania sp.