Luis J. Bernal

Urinary C reactive protein levels in dogs with leishmaniasis at different stages of renal damage

The objectives of the study were to validate a time-resolved immunofluorometric assay for C reactive protein (CRP) quantification in urine of dogs and to investigate the influence that the presence of proteinuria and azotemia could have on serum and urinary CRP (uCRP) values in dogs with leishmaniasis. Samples obtained from dogs naturally infected with Leishmania infantum were classified into four groups on the basis of the results of urinary protein/creatinine ratio and serum creatinine (sCr). In addition, 7 dogs were monitored at initial diagnosis and after a follow up visit. The assay showed good analytical performance based on precision, accuracy and limit of detection results. Results of the study suggested that CRP is present in urine of dogs with leishmaniasis and renal damage since uCRP/creatinine ratio was significantly increased in dogs with proteinuria, being the highest values observed in dogs with proteinuria and elevated sCr, and that the measurement of uCRP could be a tool to detect and evaluate the possible kidney damage associated with this disease.

Canine C-reactive protein measurements in cerebrospinal fluid by a time-resolved immunofluorimetric assay

In the current study, the quantification of C-reactive protein (CRP) in cerebrospinal fluid (CSF) of dogs using an adapted time-resolved immunofluorimetric assay (TR-IFMA) was investigated, as well as whether the assay could be used to detect the range of CRP concentrations found in different clinical situations. Intra- and interassay coefficients of variation were below 15% in all cases. The TR-IFMA measured the CRP values in a proportional and linear manner (r = 0.99); also CRP concentrations measured in CSF and in serum were significantly correlated (r = 0.80, P = 0.003). The limit of detection of the method was 7.1 × 10−6 mg/l. The assay was able to detect differences in CRP concentrations in CSF of dogs with inflammatory disorders compared with dogs with spinal cord compression or idiopathic epilepsy. In conclusion, TR-IFMA constitutes a very sensitive, precise, and accurate method for the measurement of CRP concentrations in CSF.

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 dogs residence was georeferenced and environmental data potentially associated with the dogs risk of L. infantum infection was obtained. A mixed logistic regression model was then developed to analyze the relationship between the dogs 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.

ACUTE PHASE PROTEIN RESPONSE IN THE CAPYBARA (HYDROCHOERUS HYDROCHAERIS)

We evaluated the acute phase protein response in capybaras (Hydrochoerus hydrochaeris). Three animal groups were used: 1) healthy animals (n=30), 2) a group in which experimental inflammation with turpentine was induced (n=6), and 3) a group affected with sarcoptic scabies (n=14) in which 10 animals were treated with ivermectin. Haptoglobin (Hp), acid-soluble glycoprotein (ASG) and albumin were analyzed in all animals. In those treated with turpentine, Hp reached its maximum value at 2 wk with a 2.7-fold increase, whereas ASG increased 1.75-fold and albumin decreased 0.87-fold 1 wk after the induction of inflammation. Capybaras affected with sarcoptic scabies presented increases in Hp and ASG of 4.98- and 3.18-fold, respectively, and a 0.87-fold decrease in albumin, compared with healthy animals. Haptoglobin and ASG can be considered as moderate, positive acute phase proteins in capybaras because they showed less than 10-fold increases after an inflammatory process and reached their peak concentrations 1 wk after the induction of inflammation. Conversely, albumin can be considered a negative acute phase protein in capybaras because it showed a reduction in concentration after inflammatory stimulus.

Quantification of anti-Leishmania antibodies in saliva of dogs

Detection of serum anti-Leishmania antibodies by quantitative or qualitative techniques has been the most used method to diagnose Canine Leishmaniosis (CanL). Nevertheless, saliva may represent an alternative to blood because it is easy to collect, painless and non-invasive in comparison with serum. In this study, two time-resolved immunofluorometric assays (TR-IFMAs) for quantification of anti-Leishmania IgG2 and IgA antibodies in saliva were developed and validated and their ability to distinguish Leishmania-seronegative from seropositive dogs was evaluated. The analytical study was performed by evaluation of assay precision, sensitivity and accuracy. In addition, serum from 48 dogs (21 Leishmania-seropositive and 27 Leishmania-seronegative) were analyzed by TR-IFMAs. The assays were precise, with an intra- and inter-assay coefficients of variation lower than 11%, and showed high level of accuracy, as determined by linearity under dilution (R2=0.99) and recovery tests (>88.60%). Anti-Leishmania IgG2 antibodies in saliva were significantly higher in the seropositive group compared with the seronegative (p<0.0001), whereas no significant differences for anti-Leishmania IgA antibodies between both groups were observed. Furthermore, TR-IFMA for quantification of anti-Leishmania IgG2 antibodies in saliva showed higher differences between seropositive and seronegative dogs than the commercial assay used in serum. In conclusion, TR-IFMAs developed may be used to quantify anti-Leishmania IgG2 and IgA antibodies in canine saliva with an adequate precision, analytical sensitivity and accuracy. Quantification of anti-Leishmania IgG2 antibodies in saliva could be potentially used to evaluate the humoral response in CanL. However, IgA in saliva seemed not to have diagnostic value for this disease. For future studies, it would be desirable to evaluate the ability of the IgG2 assay to detect dogs with subclinical disease or with low antibody titers in serum and also to study the antibodies behaviour in saliva during the treatment of CanL.

New wide dynamic range assays for quantification of anti-Leishmania IgG2 and IgA antibodies in canine serum

The aims of this study were (1) to develop and validate time resolved-immunofluorometric assays for the detection of anti-Leishmania IgG2 and IgA antibodies in canine serum and (2) to evaluate the ability of these assays to quantify different amounts of anti-Leishmania antibodies in Leishmania-seronegative and seropositive dogs, determined by a commercial ELISA assay, and between different clinical stages according to LeishVet guidelines. The analytical validation showed that the assays had a good precision with intra- and inter-assay coefficients of variation lower than 10%. In addition, the assays allowed the quantification of very low concentration of antibodies as well as demonstrated a high level of accuracy, as determined by linearity under dilution (R2=0.99) and recovery tests (>85%). Moreover, no cross-reactions with Ehrlichia canis, Canine Parvovirus Type 2, Anaplasma phagocytophilum, Babesia canis, Dirofilaria immitis and pyometra were found. The assays were able to detect higher values of anti-Leishmania IgG2 and IgA antibodies in seropositive dogs compared with seronegative dogs (p<0.0001), although an overlap between groups existed in the case of IgA. In addition, significantly higher values for both antibodies were detected in LeishVet groups II (p<0.05) and III (p<0.01) when compared with LeishVet group I. From our study, it could be concluded that the immunofluorometric assays developed would be suitable for determination of anti-Leishmania IgG2 and IgA antibodies in serum samples with an adequate precision, analytical sensitivity and accuracy. In addition, these assays showed a wider difference in the concentration of both IgG2 and IgA antibodies between seronegative and seropositive dogs and between different clinical stages of CanL than a current commercial ELISA kit. Further studies would be recommended to evaluate the diagnostic sensitivity and specificity of these new assays as well as their application in monitoring CanL.

Changes in the concentration of anti-Leishmania antibodies in saliva of dogs with clinical leishmaniosis after short-term treatment

The aim of this study was to evaluate the possible changes in the concentration of anti-Leishmania antibodies in saliva samples from dogs with clinical leishmaniosis after short-term treatment. Twenty dogs with clinical signs and laboratory abnormalities compatible with canine leishmaniosis (CanL) were diagnosed and treated with a standard antimonial plus allopurinol therapy. The concentration of anti-Leishmania IgG2 and IgA antibodies in saliva was measured at the time of diagnosis (day 0) and after treatment (day 30) by time-resolved immunofluorometric assays (TR-IFMAs) and results were compared with those of serum. In addition, correlations between antibody concentrations in saliva and serum, clinical scores and selected laboratory analytes were calculated. TR-IFMA results were expressed as Units of Fluorometry for Leishmania (UFL). Most dogs that adequately responded to treatment (n = 17) showed a reduction of anti-Leishmania antibodies in saliva [median IgG2: from 678.0 (day 0) to 201.1 UFL (day 30), p < 0.0001; median IgA: from 91.3 (day 0) to 60.2 UFL (day 30), p < 0.01] in accordance with clinical improvement (p < 0.0001). However, two of these dogs showed an increase of anti-Leishmania antibodies in saliva. Among dogs that did not improve after one month of treatment (n = 3), two showed a reduction in serum and saliva antibodies. In these two dogs, clinical recovery was achieved after one additional month of treatment with allopurinol. The other dog that did not respond to treatment showed increases in the concentration of anti-Leishmania antibodies, both in saliva and serum, and did not adequately respond to an additional month of treatment with allopurinol. From this pilot study, it could be concluded that, despite the low number of dogs used, the measurement of anti-Leishmania IgG2 and IgA antibodies in saliva could have a potential use for treatment monitoring of CanL, provided that a sufficient amount of specific antibodies is present at diagnosis. This is because, especially in the case of IgG2, there is a high correlation between the saliva and serum concentrations, and the reduction of antibodies is generally in accordance with the clinical improvement. Further long-term studies with a larger population should be undertaken to confirm this potential.

Relationship between serum anti-Leishmania antibody levels and acute phase proteins in dogs with canine leishmaniosis

This study examined the relationship between two serologic assays which quantify anti-Leishmania antibodies (a commercial enzyme-linked immunosorbent assay (ELISA) and a time-resolved immunofluorometric assay (TR-IFMA)) and selected acute phase proteins (APPs) and analytes related to protein concentration. Data were obtained from 205 canine serum samples from different veterinary clinics located in an area in which canine leishmaniosis (CanL) is endemic. The samples were submitted to the Interdisciplinary Laboratory of Clinical Analysis (Interlab-UMU), University of Murcia, Spain, for analysis. The biochemical analytes evaluated were serum ferritin, C-reactive protein (CRP), haptoglobin, paraoxonase-1 (PON-1) and albumin as APPs and total proteins and globulins as indicative analytes of protein concentration. Samples were submitted for the initial diagnosis of CanL, or to monitor the response to treatment in patients with CanL. The evaluation of the biochemical analytes did not show differences between Leishmania-seronegative and Leishmania-seropositive dogs. However, dogs with high antibody titers showed more pronounced clinicopathological abnormalities. Both serological assays had correlations of different significance with the biochemical analytes, showing higher significant correlations with total proteins and globulins than with the rest of the analytes. When the samples submitted for diagnosis and treatment monitoring were analyzed separately, serological assays showed lower correlation in samples for treatment monitoring (r = 0.531, p <  0.0001) than in samples for diagnosis (r = 0.769, p <  0.0001). In addition, higher correlations were found between TR-IFMA and analytes such as serum ferritin and CRP in the treatment monitoring group than with the ELISA. These results may help to clarify the relationship between anti-Leishmania antibody levels and selected biochemical analytes related to inflammation and protein concentration in CanL.

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: Phlebotomus perniciosus in rural areas

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.