Management, prevention and treatment of canine leishmaniosis in north-eastern Spain: an online questionnaire-based survey in the province of Girona with special emphasis on new preventive methods (CaniLeish vaccine and domperidone).

Knowledge of how canine leishmaniosis (CanL) is being managed clinically and its epidemiology is very important, since dogs are the main reservoir of human leishmaniosis. This study reports the results obtained through a questionnaire-based survey of veterinary practitioners in Girona province, a recognised, but non-documented endemic area in north-eastern Spain. The primary objective was to obtain data on the clinical management of CanL, focusing particularly on new preventive methods and therapeutic tools. The results show an extensive routine management of CanL cases and a widespread use of the CaniLeish (Virbac) vaccine and domperidone (Leisguard, Esteve). Adverse reactions were detected by a vast majority of the vaccine users (82 per cent), the most frequent being local reactions, apathy, fever and gastroenteritis. All the respondents had treated confirmed cases, and the therapeutic protocol most used was the combination of meglumine antimoniate (Glucantime, Merial) and allopurinol (Zyloric, GlaxoSmithKline).

Cold-water immersion decreases cerebral oxygenation but improves recovery after intermittent-sprint exercise in the heat.

This study examined the effects of post-exercise cooling on recovery of neuromuscular, physiological, and cerebral hemodynamic responses after intermittent-sprint exercise in the heat. Nine participants underwent three post-exercise recovery trials, including a control (CONT), mixed-method cooling (MIX), and cold-water immersion (10 °C; CWI). Voluntary force and activation were assessed simultaneously with cerebral oxygenation (near-infrared spectroscopy) pre- and post-exercise, post-intervention, and 1-h and 24-h post-exercise. Measures of heart rate, core temperature, skin temperature, muscle damage, and inflammation were also collected. Both cooling interventions reduced heart rate, core, and skin temperature post-intervention (P < 0.05). CWI hastened the recovery of voluntary force by 12.7 ± 11.7% (mean ± SD) and 16.3 ± 10.5% 1-h post-exercise compared to MIX and CONT, respectively (P < 0.01). Voluntary force remained elevated by 16.1 ± 20.5% 24-h post-exercise after CWI compared to CONT (P < 0.05). Central activation was increased post-intervention and 1-h post-exercise with CWI compared to CONT (P < 0.05), without differences between conditions 24-h post-exercise (P > 0.05). CWI reduced cerebral oxygenation compared to MIX and CONT post-intervention (P < 0.01). Furthermore, cooling interventions reduced cortisol 1-h post-exercise (P < 0.01), although only CWI blunted creatine kinase 24-h post-exercise compared to CONT (P < 0.05). Accordingly, improvements in neuromuscular recovery after post-exercise cooling appear to be disassociated with cerebral oxygenation, rather reflecting reductions in thermoregulatory demands to sustain force production.

Temporal trends in canine leishmaniosis in the Balearic Islands (Spain): a veterinary questionnaire. Prospective canine leishmaniosis survey and entomological studies conducted on the Island of Minorca, 20 years after first data were obtained.

Leishmaniosis is present in the Mediterranean region of Europe, where Leishmania infantum is responsible for the disease, dogs are the main reservoir, and sand flies of the Phlebotomus genus, subgenus Larroussius, are proven vectors. Some areas, including Minorca in the Balearic Islands, are considered free of the disease, despite the presence of vectors. However, in the context of the current expansion of canine leishmaniosis in parts of Europe, an epidemiological study using a veterinary questionnaire was carried out to establish the current situation of the disease in the Balearic Islands. While 50% of veterinarians thought that the incidence of canine leishmaniosis had not changed over time, 26.2% perceived an increasing trend, mainly those from Minorca, where most of the veterinarians polled (88.1%) considered the new diagnosed cases as autochthonous. A cross-sectional serological study performed in this island gave a seroprevalence rate of 24%. Seroprevalence among animals of local origin and with no history of movements to endemic areas was 31%. The presence of autochthonous canine leishmaniosis in Minorca was not correlated with an increase in vector density. The environmental and climatic factors that influenced the distribution and density of Phlebotomus perniciosus on the island and the possible causes of the apparent emergence of canine leishmaniosis in Minorca are discussed.

Distribution of phlebotomine sand flies (Diptera: Psychodidae) in Andorra: first finding of P. perniciosus and wide distribution of P. ariasi.

During July 2007 sand fly captures were carried out in Andorra using sticky castor oil traps set in sand fly resting places for four consecutive nights. The sampling stations were located between 800 and 2400 m above sea level. The specimens captured belong to two species of the genus Phlebotomus subgenus Larroussius, Phlebotomus ariasi and Phlebotomus perniciosus. The results shed new light on the wide geographical and altitudinal distribution of P. ariasi in Andorra, where it was located between 800 and 2200 m a.s.l. The study also identified P. perniciosus in Andorra for the first time, with captures below 1000 m a.s.l. The finding of these species, both proven vectors of human and canine leishmaniasis in the bordering areas of France and Spain, is considered in terms of a possible emergence of leishmaniasis in Andorra, as has occurred in other parts of Europe.

Is leishmaniasis widespread in Spain? First data on canine leishmaniasis in the province of Lleida, Catalonia, northeast Spain.

Canine leishmaniasis (CanL) is a widespread disease present in 42 countries. It is considered of epidemiological importance because of its role as a reservoir of human leishmaniasis. Knowledge of the real distribution of CanL and its emergence and/or re-emergence is of great importance in order to determine the extension of the disease. This work reports the detection of CanL in a farm dog located in a Pyrenean area of northwest Catalonia (Spain) where the disease was previously unknown. Since the dog had never left the region and sandfly vectors, Phlebotomus ariasi and P. perniciosus, were present in the farm the case is considered as autochthonous and is the first to be published in this region of Spain.

In vitro evaluation of the effectiveness and cytotoxicity of meglumine antimoniate microspheres produced by spray drying against Leishmania infantum.

The aim of the present study was to evaluate the in vitro activity and cytotoxicity of meglumine antimoniate microspheres produced by spray drying on Leishmania infantum and the effect of the excipients used in them. The parasite strain shows sensitivity to the meglumine antimoniate microspheres prepared. All the antimony IC50 values from encapsulated meglumine antimoniate (3.80 +/- 0.34 to 9.53 +/- 0.70 microg SbV/ml for promastigotes assay) are considerably lower compared to the mean value of IC50 in Glucantime solution (112 +/- 12.74 microg SbV/ml). Interesting IC50 values for the excipient chitosan (112.64 +/- 0.53 mg/ml for promastigotes and 100.81 +/- 26.45 mg/ml for amastigotes) were obtained (without cytotoxic activity), whereas the rest of the excipients did not show any activity. This new delivery system could offer a new pharmacological tool for the treatment of leishmaniosis that reduces the doses required, lowering toxic side effects because of meglumine antimoniate.

Off-side front foot drives in men's high performance cricket.

We investigated the techniques used by nine right-handed, international batsmen to perform front foot off-side drives in first class and international matches. All strokes were captured using two synchronised high-speed video cameras; nine were selected for kinematic analysis. These movement sequences were then manually digitised at a sampling frequency of 125 Hz using APAS motion analysis software. The results of this study indicated that the batsmen used movement patterns that enabled important aspects of stroke production, such as the front stride and the downswing of the bat, to be delayed so that additional information from ball flight could be assimilated. Front upper limb segments were constrained to work in a unitary fashion, with the peak horizontal end point speed of each segment occurring almost simultaneously just before impact. It has been suggested that these strategies serve to enhance stroke accuracy. Other aspects of their techniques included a distinctively looped bat path, a front foot placement that occurred only just before impact, and a front ankle that was positioned well inside the line of the ball at impact. Various technical parameters, such as the alignment of the trunk relative to ground and the continuous flow of the bat between the backswing and downswing, were similar to findings in previous batting research. Other characteristics of stroke production not previously addressed, including the path of the bat and the timing of the front stride, may challenge some long held beliefs evident in current coaching literature.

Evaluation of a latex agglutination test (KAtex) for detection of Leishmania antigen in urine of patients with HIV-Leishmania coinfection: value in diagnosis and post-treatment follow-up.

The usefulness of antigen detection in urine as an alternative tool for diagnosis of leishmaniasis and post-treatment follow-up in patients with Leishmania-HIV coinfection was evaluated with a latex agglutination test (KAtex; Kalon Biological, UK). Forty-nine HIV-infected patients with visceral leishmaniasis were included in the study. Antigen detection in urine (ADU) was positive in 42 of 49 (sensitivity, 85.7%) samples obtained during a primary episode. After treatment, a follow-up study in 23 patients was performed by simultaneous ADU and culture of peripheral blood mononuclear cells in 148 determinations. The two methods gave concordant results in 94 cases, 38 of which were positive and 56 negative. In five cases, ADU was negative and culture of peripheral blood mononuclear cells was positive: two of these cases corresponded to clinical relapses. In 49 cases, culture of peripheral blood mononuclear cells was negative and ADU was positive. In the absence of clinical symptoms, the detection of parasite antigens in 71 of 130 (54.6%) urine samples was not associated with clinical disease. The Kaplan-Meier estimates of the probability of relapse at 6, 12, 18, and 24 months were 16% (95%CI, 15-17%), 20% (95%CI, 18-22%), 31% (95%CI, 27-35%), and 71% (95%CI, 52-89%), respectively, in patients with a positive ADU result. In contrast, when ADU was negative, the probability of relapse was 5% at 6 months (95%CI, 2-8%) (only 2 of 11 patients who relapsed had a negative test). ADU by KAtex is appropriate for primary diagnosis of visceral leishmaniasis, for monitoring the efficacy of treatment, and for detection of subclinical infection.

Detection of Leishmania infantum cryptic infection in asymptomatic blood donors living in an endemic area (Eivissa, Balearic Islands, Spain) by different diagnostic methods.

The extent of cryptic leishmaniasis in blood donors from a Spanish endemic area, (Eivissa Island) was studied using various immunological and parasitological methods. Sera from 656 blood donors were analysed: 16 (2.4%) were positive by ELISA and 50 (7.6%) by Western blot. Peripheral blood mononuclear cells (PBMC) and buffy coat (BC) samples, were analyzed by culture and nested-PCR. DNA of L. infantum was amplified in 27 (22.1%) of 122 PBMC. Parasites were isolated in 3 (4.5%) of 67 BC cultures and the strains were identified as L. infantum zymodeme MON-28. No parasites were isolated in PBMC culture. After 12 months, a second blood sample was obtained from 18 blood donors who were positive by nested-PCR in the first extraction; nine of them remained positive. Delayed type hypersensitivity (DTH) tests on 15/67 donors (22.3%) were positive. Comparison of results obtained by ELISA, WB and DTH; ELISA, WB and nested-PCR and nested-PCR and BC culture showed a significant association (Pearson test, P < 0.05). L. infantum zyodeme MON-28 was identified in three strains isolated from asymptomatic donors, which suggests a low virulence capacity of these strains. The detection of Leishmania DNA in a high number of asymptomatic subjects supports the need to monitor it in blood donors endemic areas.

Detection of anti-Leishmania immunoglobulin G antibodies in urine specimens of dogs with leishmaniasis.

For years, anti-Leishmania immunoglobulin G (IgG) antibodies have been detected in the sera of dogs living in areas of leishmaniasis endemicity. They have also been found in the aqueous humor and cerebrospinal fluid. In contrast, a review of the literature failed to identify the detection of anti-Leishmania antibodies in urine samples from dogs with leishmaniasis. Ninety-five dog urine samples were examined for the presence of anti-Leishmania antibodies by using a protein A enzyme-linked immunosorbent assay (ELISA). Twenty additional urine samples were collected from healthy dogs as controls. An IgG2 ELISA was performed on 26 urine samples found positive by the protein A ELISA. Twenty-three urine samples found positive to anti-Leishmania antibodies were tested for the local production of anti-Leishmania antibodies in the urinary tract by means of the urine antibody coefficient. Ten urine samples (and the corresponding serum samples) were compared by Western blot (WB) analysis. Thirty-five out of the 95 urine samples were found positive, 57 were found negative, and 3 were found inconclusive for antibody detection by the protein A ELISA. A high correlation between protein A and IgG2 levels was found in positive urine samples. Anti-Leishmania antibodies were present in the urine of dogs that had leishmaniasis, urinary protein/creatinine (U P/C) ratios of greater than one, and normal urinary sediment. A statistically significant correlation was observed between the U P/C ratios and the levels of anti-Leishmania antibodies in positive urine samples. In general, WB analysis and the urine antibody coefficient suggested that the presence of anti-Leishmania antibodies in urine was the consequence of an impairment of filtration of the glomerular barrier. However, in some dogs, WB analysis could be interpreted as suggesting that the presence of anti-Leishmania antibodies was caused, to a lesser extent, by local antibody production in the urinary tract. Antibody detection in urine could be a noninvasive method for leishmaniasis diagnosis and prognosis in dogs with glomerulonephropathies.

A nested polymerase chain reaction for diagnosis and follow-up of human visceral leishmaniasis patients using blood samples.

A nested polymerase chain reaction (PCR) assay for the diagnosis of human visceral leishmaniasis (VL) due to Leishmania infantum infection was developed using primers selected from the parasite's genomic deoxyribonucleic acid (DNA). The assay, which is based on the use of leucocytes separated from blood samples by Ficoll-Paque centrifugation, was compared with culture in vitro. Blood samples were collected from 17 patients in Spain with a history of clinical VL, 15 of whom were also infected with human immunodeficiency virus (HIV) (13 samples during the VL episode and 31 samples during post-treatment monitoring) and one sample was collected from each of 28 patients with HIV infection and fever but no history of VL. The nested PCR using blood detected all the cases of parasitologically confirmed, clinically active VL, while culture detected 92%. The nested PCR detected Leishmania DNA in 18% of the HIV-infected patients with fever and no history of VL, none of whom gave a positive culture. Follow-up examination of the VL patients by nested PCR and culture demonstrated the persistence of L. infantum in blood for a long time after treatment.

Exact moment of a gastrocnemius muscle strain captured on video.

A left gastrocnemius strain was sustained by an elite cricket batsman while he was taking off to run. The exact moment of injury, captured by a camera in the middle stump, appears to correspond to the sudden appearance of a deficit in the gastrocnemius muscle, seen through the player's trousers. The strain occurred when the entire body weight was on the left foot with the centre of mass well in front of the leg. The injury probably occurred close to the time when the gastrocnemius complex was moving from an eccentric to an isometric phase.

Isoenzymatic identification of Leishmania isolates from repeated clinical human leishmaniasis episodes in Catalonia (Spain).

Forty human strains of Leishmania infantum isolated in 1985-99 from 17 patients with repeated cutaneous, mucosal or visceral leishmaniasis episodes in Catalonia (Spain) were examined by isoenzyme electrophoresis. Six zymodemes were revealed: MON-1, MON-24, MON-28, MON-29, MON-33 and MON-34. In 2 patients 2 different zymodemes were identified in consecutive episodes.

Nested PCR for diagnosis of canine leishmaniosis in peripheral blood, lymph node and bone marrow aspirates.

A nested polymerase chain reaction (PCR) assay was developed using primers selected from the genomic DNA of Leishmania infantum and applied to the diagnosis of leishmaniosis in peripheral blood in dogs. Blood of 39 dogs of different breeds, all sampled in Catalonia (Spain), were tested for leishmaniosis by enzyme linked immunosorbent assay (ELISA), western blotting (WB) and peripheral blood mononuclear cell (PBMC) culture and nested PCR. Twenty negative controls (healthy dogs less than 1-year-old that had not been exposed to a sandfly season) were also studied. Nineteen of the 39 dogs studied were positive by ELISA and/or WB, and 18 of these had a positive PBMC nested PCR. PBMC nested PCR was negative in all the remaining animals that were negative by serological examination, including the 20 negative controls. Parasitological examination and nested PCR of bone marrow and lymph node aspirate from the 19 dogs positive by serological examination, were also positive. These results indicate that PBMC nested PCR is a sensitive and specific tool to diagnose leishmaniosis in dogs. The use of PBMC has the advantage over bone marrow and lymph node aspirates in that it is a less invasive sample.

Long term improvement in the treatment of canine leishmaniosis using an antimony liposomal formulation.

Pharmacokinetic and clinical effectiveness of liposome-encapsulated N-methylglucamine antimoniate (LMA) was performed in dogs suffering from experimental leishmaniosis. LMA was compared with N-methylglucamine antimoniate (MGA), the same drug in its free form. Sb plasma concentrations for LMA were always higher than those for MGA. Mean residence time (MRT), half-life time (t(1/2)) and clearance (Cl) showed that Sb was eliminated slower after liposome administration. The high volume of distribution (Vd) obtained with LMA suggests that Sb could achieve therapeutic concentrations in parasite-infected tissues. Average plasma concentration at steady state (Css(ave)) shows that Sb body concentrations after LMA treatment (9.8 mg/kg Sb, each 24h) would be effective in Leishmania infantum canine infection. Comparing LMA with MGA in a 1-year follow-up we observed no relapses for LMA and total protein and gammaglobulin concentrations were within normal range, while for MGA both began to rise 3 months after treatment. Use of antimonial liposomal formulations may restore effectiveness to an existing drug and reduce toxicity.

Leishmania infantum-specific IgG, IgG1 and IgG2 antibody responses in healthy and ill dogs from endemic areas. Evolution in the course of infection and after treatment.

The expression of IgG, IgG1 and IgG2 specific antibodies for Leishmania infantum was studied in five groups of dogs in Catalonia (Spain): I, 99 asymptomatic dogs (infected and uninfected) from a highly endemic area for leishmaniosis; II, 139 untreated dogs with clinically patent leishmaniosis; III, 11 naturally infected asymptomatic dogs monitored for up to 5 years since they were found seropositive to Leishmania antigen and without treatment; IV, 25 naturally infected dogs with clinically patent leishmaniosis and treated with either meglumine antimoniate and allopurinol or allopurinol alone and V, six experimentally infected dogs, treated with meglumine antimoniate and controlled for 5 years. The levels (ELISA units) of IgG, IgG1 and IgG2 in asymptomatic dogs (group I) were very variable (24+/-33, 32+/-31 and 26+/-31, respectively), and, as expected, lower than in ill dogs (group II) (168+/-34, 84+/-71 and 172+/-31, respectively). In both groups, the correlation between IgG and IgG2 levels (r=0.95, P<0.001 in group I and r=0.63, P<0.001 in group II) was higher than between IgG and IgG1 levels (r=0.01, P>0.05 in group I and r=0.31, P<0.001 in group II). In group III, IgG and IgG2 expression increased during infection, while IgG1 expression remained the same. In dogs of group IV, IgG levels after 1 year of treatment decreased more in responsive (mean values, 163+/-42 before treatment (b.t.) and 100+/-36 after treatment (a.t.)) than in unresponsive dogs (158+/-29 b.t. and 124+/-51 a.t.), especially for IgG1 (94+/-89 b.t. and 20+/-21 a.t. in responsive dogs and 35+/-25 b.t. and 22+/-13 a.t. in unresponsive dogs) rather than for IgG2 (156+/-16 b.t. and 114+/-45 a.t. in responsive and 151+/-11 b.t. and 125+/-36 a.t. in unresponsive dogs). Similar results were observed in the evolution of experimentally infected animals after consecutive and specific treatments. Overall results show the great variation in Leishmania-specific IgG1 expression in asymptomatic and symptomatic dogs, their lack of correlation with that of IgG2 and chemotherapy is more effective in dogs with initially high expression of IgG1.