Density assessment and reporting for Phlebotomus perniciosus and other sand fly species in periurban residential estates in Spain.

Green periurban residential areas in Mediterranean countries have flourished in the last decades and become foci for leishmaniasis. To remedy the absence of information on vector ecology in these environments, we examined phlebotomine sand fly distribution in 29 sites in Murcia City over a 3-year period, including the plots of 20 detached houses and nine non-urbanized sites nearby. We collected 5,066 specimens from five species using "sticky" interception and light attraction traps. The relative frequency of the main Leishmania infantum vector Phlebotomus perniciosus in these traps was 32% and 63%, respectively. Sand fly density was widely variable spatially and temporally and greatest in non-urbanized sites, particularly in caves and abandoned buildings close to domestic animal holdings. Phlebotomus perniciosus density in house plots was positively correlated with those in non-urbanized sites, greatest in larger properties with extensive vegetation and non-permanently lived, but not associated to dog presence or a history of canine leishmaniasis. Within house plots, sand fly density was highest in traps closest to walls. Furthermore, the study provides a guideline for insect density assessment and reporting and is envisioned as a building block towards the development of a pan-European database for robust investigation of environmental determinants of sand fly distribution.

A spatial ecology study in a high-diversity host community to understand blood-feeding behaviour in Phlebotomus sandfly vectors of Leishmania.

Molecular studies indicate that Phlebotomine sandflies (Diptera: Psychodidae) blood feed on many vertebrate species, of which only a few are proven parasite reservoirs. Investigating sandfly vector feeding preferences is therefore important and requires taking into account the availability and accessibility of host species. In terms of the latter, it is necessary to consider the metabolic cost to the insect of reaching the host and moving on to a suitable breeding site. The present study used statistical modelling to compare the feeding patterns of Phlebotomus perniciosus (n = 150), Phlebotomus papatasi (n = 35) and Phlebotomus ariasi (n = 7) on each of an average of 30 host species in a wildlife park in Murcia, Spain. Sandfly feeding movement costs were estimated as a function of the distance and altitude gradients saved by the insect, assuming that they displayed 'site fidelity'. Most (87%) engorged females were caught <100 m from the host on which they had fed. Although the percentage of bloodmeals was highest on fallow deer (Dama dama) (30%) and red deer (Cervus elaphus) (26%), the predicted feeding probability after considering movement cost was highest for red deer and common eland (Taurotragus oryx), and positively associated with host census. These results suggest that, under similar circumstances, sandflies prefer to feed on some host species more than on others.

Longitudinal monitoring of anti-saliva antibodies as markers of repellent efficacy against Phlebotomus perniciosus and Phlebotomus papatasi in dogs.

A 2-year longitudinal study of enzyme-linked immunosorbent assay (ELISA) antibodies against Phlebotomus perniciosus and Phlebotomus papatasi (Diptera: Psychodidae) sandfly saliva was performed in 32 Beagle dogs treated preventively with an imidacloprid-permethrin topical insecticide in an endemic area in Spain. Dogs were grouped into three sandfly exposure groups according to the time of inclusion in the study. Assays analysed immunoglobulin G (IgG) against salivary gland homogenates (SGH) of both species and recombinant P. papatasi rSP32 and P. perniciosus rSP03B proteins in serum. The dogs were participating in a Leishmania infantum (Kinetoplastida: Trypanosomatidae) vaccine trial and were experimentally infected with the parasite in the second year. No dog acquired natural L. infantum infections during the first year, but most developed anti-saliva antibodies, and median log-transformed optical densities (LODs) were seasonal, mimicking those of local sandflies. This indicates that the repellent efficacy of the insecticide used is below 100%. Multi-level modelling of LODs revealed variability among dogs, autocorrelation and differences according to the salivary antigen and the dog's age. However, dog seroprevalence, estimated using pre-exposure LODs as cut-offs, was relatively low. This, and the fact that dogs did not become naturally infected with L. infantum, would support the efficacy and usefulness of this imidacloprid-permethrin topical insecticide in canine leishmaniasis control.

Epidemiological and genetic studies suggest a common Leishmania infantum transmission cycle in wildlife, dogs and humans associated to vector abundance in Southeast Spain.

Leishmania infantum infection was investigated in 202 wild carnivores, rodents and lagomorphs in Southeast Spain using a real-time PCR (rtPCR) in skin and organ samples, mostly spleen. Lesions compatible with leishmaniosis were not observed in any of the animals. Prevalence defined as the percentage of rtPCR-positive animals was 32% overall, and 45% in foxes (n = 69), 30% in rabbits (n = 80) and stone martens (n = 10), 19% in wood mice (n = 16), 0% in black rats (n = 10) and ranged between 0% and 100% in other minoritarian species including badgers, wild cats, wolves, raccoons, genets and hares. Most infected rabbits were rtPCR-positive in skin and not in spleen samples and the opposite was the case for foxes (p < 0.05). L. infantum prevalence was lowest in spring following months of non-exposure to phlebotomine sand fly vectors, and spatially matched recently estimated Phlebotomus perniciosus vector abundance and the prevalence of subclinical infection in dogs and humans. Prevalence increased with altitude and was greater in drier and less windy South and West compared to the coastal Southeast of the study area (p < 0.05). Genetic diversity of L. infantum from foxes, investigated by PCR-restriction fragment length polymorphisms of kinetoplast DNA, revealed B genotype in all animals, which is frequent in people and dogs in the Iberian Peninsula and Morocco. The study provides further evidence that subclinical L. infantum infection is widespread in wildlife with prevalence depending on environmental factors and that parasite tissue tropism may vary according to host species. Moreover, it suggests that sylvatic and domestic transmission cycles are closely interconnected.

Investigations of Phlebotomus perniciosus sand flies in rural Spain reveal strongly aggregated and gender-specific spatial distributions and advocate use of light-attraction traps.

The spatial and temporal distribution of Phlebotomus perniciosus (Diptera: Psychodidae) (Newstead, 1911), the sand fly vector of pathogens of public and animal health importance, was investigated in a high sand fly density rural area in Spain using light-attraction and sticky-interception traps. Traps were placed inside animal buildings and outside at increasing distance from animals. A total of 8506 sand flies were collected, 87% with light traps. Species frequency differed between trap types. The abundance of P. perniciosus decreased exponentially with increasing distance to animals and, while females were most common in the animal enclosure, males predominated in adjoining storage places. Increasing CO2 concentration had an additional positive effect on female abundance only. Both male and female density increased with rising temperature, and there was some indication that females were more active than males at higher relative humidity. The study confirms that P. perniciosus aggregates around animal premises, although male and female distributions differ and should be analysed separately to account for biological and behavioural differences. This provides further evidence that light traps offer an accurate estimation of the relative spatial and temporal abundance of P. perniciosus, conferring an added value for the study of this species and the risk of pathogen transmission.

A cross-sectional study of the impact of regular use of insecticides in dogs on Canine Leishmaniosis seroprevalence in southeast Spain.

The relationship between Canine Leishmaniosis (CanL) seroprevalence and regular use of topical insecticides was investigated in 800 pet dogs with no visible signs of CanL in Murcia, southeast Spain in 2011. Dogs were clients to 17 veterinary practices and were analyzed for Leishmania infantum antibodies in blood plasma using two commercial ELISAs (Ingezim, Ingenasa®, Spain; Leishcan, Hipra®, Spain). Owners were interviewed to gather data on dog related variables. They included date of birth, home address and frequency, duration and timing of insecticide treatments used to prevent ectoparasite infestations. The dog's residence was georeferenced and environmental data potentially associated with the dog's risk of L. infantum infection was obtained. A mixed logistic regression model was then developed to analyze the relationship between the dog's serological status and insecticidal treatment adjusted for demographic and environmental variables. Overall, CanL seroprevalence (95% confidence limits) was 18% (16-21%) including 11% in dogs not using insecticide treatments (n=60) and 19% in those using them (n=740) (p>0.05). At least 16 different insecticide products were used and 73%, 26% and 1% of dogs received 1, 2 and 3 products a year. The most frequent commercial brands used and the only ones in the market claiming anti-sandfly activity, were Scalibor collars (deltametrin 40mg/g; MSD®), Advantix pipettes (permethrin 500mg/ml and imidacloprid 100mg/ml; Bayer®) and Exspot spot-on pipettes (permethrin 715mg/ml; MSD®). Seroprevalence was 9%, 16%, 20%, 22% and 25% for dogs with Scalibor collars plus Advantix pipettes, Scalibor collars plus ExSpot pipettes, Advantix pipettes alone, Scalibor collars alone and Exspot pipettes alone, respectively. The multivariable model confirmed a significant reduction in the risk of Leishmania spp. seropositivity in dogs using the Scalibor and Advantix combination compared to those using either product alone and provided evidence of greatly increased risk of CanL in rural areas situated at 300-500m altitude and average March-July temperatures of 18.6-19°C. The study highlights the difficulty in controlling CanL infection by means of insecticide use alone and that it could be improved by using the Scalibor and Advantix combination and identifying and targeting specific geographical areas.

Spatial distribution of human asymptomatic Leishmania infantum infection in southeast Spain: a study of environmental, demographic and social risk factors.

Recent PCR studies indicate that asymptomatic L. infantum infection is common in people in southern Europe. Understanding its spatial distribution is a requisite to evaluate the public health implications and to design disease control schemes. We investigated infection in blood samples from 657 donors in southeast Spain using PCR and antibody ELISA. They came from 19 blood centers and were interviewed about their residence, occupation, dog ownership and Leishmaniosis awareness. The percentage of PCR and ELISA positives were 8% (49/618) and 2% (13/657). Donor's residences were spatially clustered around blood donning centers and PCR prevalence was 18% in rural municipalities with 20-1330 inhabitants, 12% in those with 1467-5088 inhabitants and 3% in larger communities, and was associated with dog ownership (p<0.05). Further analysis of data from rural donors indicated that PCR status was strongly related to the climate, altitude and soil type in the donor's residence area and not to other demographic or sociologic variables. Mixed logistic regression analysis predicted PCR prevalence to be greatest in the 200-300m altitude range with a mean spring-summer (time of highest vector activity) temperature of 18.4-19.0°C. A temperature and altitude risk map was generated that will provide the basis for elaborating evidence-based vector surveillance studies.

The normal electrocardiogram of the unanaesthetized competition "Spanish Pouler" pigeon (Columba livia gutturosa).

This investigation was carried out on 64 healthy adult competition pigeons (Spanish Poulers) to determine reference values of electrocardiographic parameters in standard lead II; a comparative study between males and females was also performed to find changes in the electrocardiographic patterns related to sex. P wave was positive and monophasic; PR segment depression was present in 64% of records; the QRS pattern obtained was in most cases of the rS type, followed by the QS configuration. ST slurring (absence of ST) was observed in 47% of records and the mean electrical axis was in all cases negative. Statistically significant differences related to sex were found for P-wave, R-wave and T-wave amplitudes, showing the males have greater amplitudes than females, associated with the increase of cardiac tissue mass. No sex-related differences were found in heart rate and mean electrical axis.

The normal electrocardiogram of the unanesthetized peregrine falcon (Falco peregrinus brookei).

The mean duration and amplitudes of the lead II electrocardiogram were determined in the peregrine falcon (Falco peregrinus brookei) using 10 birds ranging in age from 1 to 5 yr. Electrocardiograms were performed on unanesthetized falcons in order to avoid the anesthesia effect on the electrocardiogram, by a method which seems to induce a tonic immobility-like reaction. All the falcons had a normal sinus rhythm, with a mean heart rate of 268 beats per minute. Mean durations of PR, ST, QT, and RR intervals were higher (but not statistically significant) in females than in males, except for the ST segment, with similar values in both sexes. P-wave deflections were positive in I, II, III, aVL, and aVF and negative in aVR. The normal patterns of wave forms of the QRS complexes in all leads were of QS and rS types, except for aVR and aVL, which presented an R configuration. The mean electrical axis was negative, with an average of -99.9 degrees. T-wave deflections were positive in I, II, III, and aVF leads II and negative in aVR and aVL. The data collected in this study may serve as a guide for electrocardiographic monitoring of peregrine falcons.

Cortisol and free thyroxine determination by time-resolved fluorometry in canine serum.

Validation for canine serum of 2 commercially available time-resolved fluoroimmunoassays (TR-FIAs) designed for analysis of cortisol and free thyroxine (fT4) in human serum was carried out. Included was the study of interference by hemolysis, lipemia, and bilirubinemia. With the dissociation enhancement lanthanide fluoroimmunoassay kits, the intra-assay coefficient of variation (CV) ranged from 6.4% to 8.7% for cortisol and from 5.3% to 9.8% for fT4; the interassay CVs ranged from 5.8% to 10.8% and from 3.9% to 14.1%, respectively. Accuracy was evaluated by comparing cortisol and fT4 results obtained with TR-FIA and those obtained with a validated enzyme-linked immunosorbent assay (ELISA) and an equilibrium dialysis (ED) assay, respectively. The regression equations obtained were y = 0.57x + 1.18 (r2 = 0.90) for cortisol and y = 0.87x + 0.82 (r2 = 0.93) for fT4. The limits of detection for cortisol and fT4 were 4.84 nmol/L and 2.68 pmol/L, respectively. The results of adrenocorticotropin-stimulation and dexamethasone-suppression tests were similar to those published previously; likewise, serial dilution of a canine serum sample with a high cortisol content demonstrated that the TR-FIA was immunologically specific. Serial dilution of a serum sample with a high fT4 concentration showed a methodologic bias, a dependence on serum binding capacity, which indicates that the results obtained with this method should be interpreted with caution. Finally, hemolysis and lipemia significantly interfered with cortisol and fT4 measurements, whereas bilirubinemia did not affect the results.

Arrhythmogenic right ventricular dysplasia/cardiomyopathy in a Siberian husky.

A seven-month-old male Siberian husky was presented with a recent history of anorexia, hindlimb weakness and syncope. Physical examination revealed severe tachycardia, tachypnoea and dyspnoea. Mucous membranes were pale and femoral pulses were weak. An electrocardiogram showed sustained ventricular tachycardia with a left bundle branch block configuration. Thoracic radiographs revealed slight right ventricular enlargement and two-dimensional echocardiography revealed mild right ventricular dilation at the cardiac apex and some hyperechogenic areas on the right side of the interventricular septum. Administration of intravenous lignocaine converted the ventricular tachycardia to sinus rhythm. The maintenance antiarrhythmic therapy consisted of oral procainamide and propranolol. Three weeks later the dog died suddenly. On postmortem examination, the right ventricular free wall was very thin at the apex, infundibulum and caudal aspect of the right ventricular parietal wall, similar to the 'triangle of dysplasia' of human patients. Histopathological examination revealed replacement of several areas of right ventricular free wall myocardium with connective tissue and fat. The right atrium and left ventricle were less severely affected by the same lesions. The clinical and pathological findings are similar to those reported in young people with arrhythmogenic right ventricular dysplasia/cardiomyopathy.

Use of whole blood for spectrophotometric determination of cholinesterase activity in dogs.

Whole blood has been compared with erythrocytes and plasma for spectrophotometric cholinesterase determination in the dog. Cholinesterase activity was characterized using two substrates: acetylthiocholine and butyrylthiocholine. Acetylcholinesterase was the only form of cholinesterase present on erythrocytes and hydrolysed only acetylthiocholine. Butyrylcholinesterase (pseudocholinesterase) was predominant in plasma, hydrolysing mainly butyrylthiocholine. Based on these results, a method based on the use of two substrates (acetylthiocholine for monitoring acetylcholinesterase and butyrylthiocholine for determining butyrylcholinesterase) in the same whole blood sample is recommended for canine cholinesterase analysis. This way of monitoring both enzymes can be easily automated, yielding good within (CVs < 5%) and between-run (CVs < 7%) precision.

Clinical and pathological findings of severe subvalvular aortic stenosis and mitral dysplasia in a rottweiler puppy.

Subvalvular aortic stenosis (SAS) and mitral dysplasia were diagnosed in an asymptomatic eight-week-old rottweiler. Clinical and pathological findings were compatible with a fixed and dynamic obstruction of the left ventricular outflow tract. Gross and microscopic pathological findings were consistent with the most severe form of SAS, described previously in Newfoundland dogs over six months of age. These observations demonstrate that very young asymptomatic puppies may suffer a severe complex form of SAS.

Automated spectrophotometric method using 2,2'-dithiodipyridine acid for determination of cholinesterase in whole blood.

An automated method using 2,2'-dithiodipyridine (2-PDS) as chromophore for determination of whole-blood cholinesterase activity was developed. Assay procedures, optimal concentrations of chromophore and substrate, detection limit, precision, backgrounds, and sensitivity of the method were compared with those of an earlier automated method based on the Ellman method and using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) as chromophore. The new method has the advantages of automation (resulting in increase throughput rate and decrease in amount of reagents used) and good precision and sensitivity. Sample dilutions also are reduced in the new method because hemoglobin interference is less.

Electrocardiographic values in Spanish-bred horses of different ages.

The duration of electrocardiograph wave forms and intervals were determined in 179 Spanish-bred (Andalusian) horses aged from 1 month to 17 years. The values were compared with those of other breeds, and the relationship between electrocardiographic data and age was examined. High correlation coefficients were found between PR, ST and QT intervals and the age of the horses, and an inverted relation between heart rate and age was found. A multiple range analysis was made and the results suggest that significant changes in duration values and heart rate occurred at the age of 6 months and in the second year of life.

Electrocardiographic changes in the growing Mastín Español.

Serial electrocardiograms were recorded from 70 Mastín Español dogs in right lateral recumbency, aged between one day and three years, in order to observe the changes in the waveform and intervals, QRS morphology, cardiac rhythm and heart rate caused by their growth. Age and bodyweight caused a gradual increase in the duration and amplitude of the P wave, duration of the PR, QT and RR intervals, amplitude of the R wave and duration of the QRS complex and ST segment. Q wave was observed in nearly all the recordings with different amplitudes. The S wave was only significant in one-day-old animals and QRS morphology showed significant important changes from qrS and rS to qR and qRs morphologies during the first two weeks of life. The T wave increased its amplitude until the age of two months and changed its polarity (from negative to positive) from five months of age onwards. The heart rate decreased until the age of seven months, reaching stabilised values of 110 +/- 7.3 beats per minute. Sinus tachycardia was commonplace in animals under one month old and respiratory sinus arrhythmia was found from six months of age. Sex only influenced the duration and amplitude of the T wave. Males had higher mean values than females.