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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.

The objective of this study was to study the changes in salivary proteins that occur in the dog after the ejaculation process. Saliva samples from eight dogs before and after induced ejaculation were analyzed by proteomic using Tandem Mass Tag (TMT) labeling and LC-MS/MS analysis. A total of 33 salivary proteins showed significant changes after the ejaculation process. The up-regulated proteins that showed changes of higher magnitude were mucin-7 (MUC- 7), peroxiredoxin-4 (PRDX4) and galectin-3 (LEGALS3) whereas proteins such as alpha-1-acid glycoprotein (A1G1) and alpha-1B-glycoprotein (A1BG) were the most down-regulated. MUC-7 and PRDX4 expression in saliva after ejaculation could be associated with the protective “environment” created by the organism to exert pr 3o-fertility activities and antioxidants benefits in spermatozoa. Also LEGALS3 increment could be associated with an improvement of wellbeing and could contribute to a positive global effect in the body. Down-regulations of A1G1 and A1GB proteins found in saliva after ejaculation could be associated with a reduction in systemic inflammation. Overall it can be concluded that, changes in proteins in saliva that are produced after ejaculation can reflect a state of increase immune defenses, improvement of antioxidant status and low inflammation.

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

Interdisciplinary collaborations are increasingly gaining popularity, as are active in higher education and innovative learning strategies. However, relatively little research has been performed related to interdisciplinary learning methodologies in higher education. In the present work, a pilot activity between communication and veterinary students was performed, consisting in performance of mock interviews at a professional television studio. Besides some drawbacks such as low participation rates by veterinary students, the activity was associated with a number of benefits, including enhanced acquirement of communication skills, greater topic-related knowledge assimilation, and reinforced practical application of the theoretical concepts.

The increasingly acknowledged "One Word, One Health" (OH) concept studies the collaborative multi-disciplinary approaches for the assessment of human and animal health and the environment. This study provides information about a module of activities created to teach the OH concept to undergraduate veterinary students. The module consisted of three different activities: theoretical classes, teamwork for the preparation of different concepts and practical examples related to OH, and public presentations of the students of these practical cases. This module was evaluated by two questionnaires' consisting of online surveys, which were filled in before (questionnaire 1, Q1) and after (questionnaire 2, Q2) the module about OH. Before the course, 80% of students recognized to have a poor or very poor knowledge about OH, and a 71% failed to include the three main items of the OH concept (humans, animals and environment) in their answers. After the course, the general knowledge about OH was improved and most students evaluated the course positively, although the lack of time and the high number of students per group were pointed out as the main drawbacks. In conclusion, the module of activities described in this report contributed to the increase of OH knowledge by veterinary students and could be a resource for future advances to improve the teaching of the OH concept in the curricula at University level in Veterinary and other Degrees related with OH.

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.

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.

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.

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

[Background] Leishmaniosis is associated with Phlebotomus sand fly vector density, but our knowledge of the environmental framework that regulates highly overdispersed vector abundance distributions is limited. We used a standardized sampling procedure in the bioclimatically diverse Murcia Region in Spain and multilevel regression models for count data to estimate P. perniciosus abundance in relation to environmental and anthropic factors., [Methods] Twenty-five dog and sheep premises were sampled for sand flies using adhesive and light-attraction traps, from late May to early October 2015. Temperature, relative humidity and other animal- and premise-related data recorded on site and other environmental data were extracted from digital databases using a geographical information system. The relationship between sand fly abundance and explanatory variables was analysed using binomial regression models., [Results] The total number of sand flies captured, mostly with light-attraction traps, was 3,644 specimens, including 80% P. perniciosus, the main L. infantum vector in Spain. Abundance varied between and within zones and was positively associated with increasing altitude from 0 to 900 m above sea level, except from 500 to 700 m where it was low. Populations peaked in July and especially during a 3-day heat wave when relative humidity and wind speed plummeted. Regression models indicated that climate and not land use or soil characteristics have the greatest impact on this species density on a large geographical scale. In contrast, micro-environmental factors such as animal building characteristics and husbandry practices affect sand fly population size on a smaller scale., [Conclusions] A standardised sampling procedure and statistical analysis for highly overdispersed distributions allow reliable estimation of P. perniciosus abundance and identification of environmental drivers. While climatic variables have the greatest impact at macro-environmental scale, anthropic factors may be determinant at a micro-geographical scale. These finding may be used to elaborate predictive distribution maps useful for vector and pathogen control programs., This study was funded by the Spanish Ministry of Science and Innovation (Pr. Ref: AGL2013-46981-R) and the Instituto de Salud Carlos III within the Network of Tropical Diseases Research (RICET RD06/0021/1007). The work was carried out under VectorNet, a European network for sharing data on the geographic distribution of arthropod vectors, transmitting human and animal disease agents (contract OC/EFSA/AHAW/2013/02-FWC1) funded by the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (ECDC). Authors of this paper are presently members and receive support from COST Action TD1303 (European Network for Neglected Vectors and Vector-borne infections).

This study developed a real-time quantitative PCR (qPCR) assay to detect L. infantum kinetoplast DNA (kDNA) in canine saliva. The qPCR showed an efficiency of 93.8%, a coefficient of correlation of 0.996 and a detection limit of 0.5 fg/reaction (0.005 parasites), although it detected until 0.25 fg/reaction (0.0025 parasites). When samples from 12 dogs experimentally infected with L. infantum were collected, L. infantum kDNA was detected at 16-weeks post-infection (wpi) in 41.7% and 91.7% of saliva and bone marrow samples, respectively, and at 47-wpi in 75% of both samples. L. infantum kDNA can be detected by qPCR in canine saliva, with lower sensitivity in the early stages of infection and a lower parasite load estimation compared to bone marrow. However, saliva had similar sensitivities to bone marrow in the later stages of the infection and could be used to detect L. infantum kDNA being aware of its limitations., This work was funded by the Seneca Foundation-Regional Agency for Science and Technology of the Government of Murcia, Spain [19894/GERM/15]. ACB was supported by a research contract [21327/PDGI/19] funded by the Research Talent Program and its Employability from the Seneca Foundation-Regional Agency for Science and Technology of the Government of Murcia, Spain and the European Social Fund. DE was supported by the Spanish Ministry of Economy, Industry, and Competitiveness [grant number IJC2018-035105-I]. MCT, RNAL, and MCL were supported by the Spanish Ministry of Economy, Industry, and Competitiveness [grant numbers RTC-2016-50005-1, SAF2016-80998-R] and the ISCIII and FEDER [RD16/0027/0005].

In this study, the circadian rhythm of IgG2 and IgA specific antibodies in serum and saliva samples of 6 dogs experimentally infected with Leishmania infantum was assessed. Sampling was performed at 8.00, 12.00, 16.00, 20.00, and 00.00 h on two consecutive days. Anti-Leishmania antibody levels in serum were expressed without any correction, whereas in saliva were shown in different ways: without any correction, adjusted by protein concentration and corrected by the salivary flow rate. No significant differences in anti-Leishmania IgG2 antibody levels in serum and saliva samples with or without correction were found. Significant differences were found when anti-Leishmania IgA levels were corrected by the salivary flow rate. In addition, a greater intra-individual variation of antibody levels was observed in saliva than in serum. However, this variation did not modify the serological status of the dogs. Therefore, it could be concluded that there is no circadian rhythm in serum and saliva samples and sampling can be performed at any time of the day., We thank ADL-Bionatur for supplying the dogs for the study. ACB is supported by a pre-doctoral FPU fellowship from the University of Murcia,Spain.DE held a post-doctoral“Juandela Cierva Formación ”fellowship from“Ministerio de Economíay Competitividad”(MINECO),Spain. AE and MCL were supported by grants RTC-2016-50005-1 and SAF2016-81003-R from the “Programa Estatal I+D+I” from MINECO and by the Network of Tropical Diseases Research RICET(RD16/0027/0005)funded by ISCIII and FEDER.This project was supported by grant 19894/GERM/15 of the Seneca Foundation of Murcia Region (Groups of Excellence).

In the present study, a quantitative proteomic approach to study changes in saliva proteins associated with canine leishmaniosis (CanL) was performed. For this, canine salivary proteins were analysed and compared between dogs before (TO) and after (T1) experimental infection with Leishmania infantum by high-throughput label-based quantitative LC-MS/MS proteomic approach and bioinformatic analysis of the in silico inferred interactome protein network was created from the initial list of differential proteins. More than 2000 proteins were identified, and of the 90 differentially expressed proteins between TO and T1, 12 were down-regulated with log2 fold change lower than -0.5849, and 19 were up-regulated with log2 fold change greater than 0.5849. This study provides evidence of changes in salivary proteome that can occur in canine leishmaniosis and revealed biological pathways in saliva modulated in canine leishmaniosis with potential for further targeted research., LFM was granted with predoctoral contract 'FPU' of University of Murcia, Spain. AT has a post-doctoral fellowship "Juan de la Cierva Incorporacion" supported by the "Ministerio de Economia y Competitividad", Spain. MCT and MCL were supported by grants RTC 2016 -50005-1 from the "Programa Estatal I+D+I" (MINECO). This work was supported by a grant from the Program for Research Groups of Excellence of the Seneca Foundation, Murcia, Spain (grant 19894/GERM/15) and European Commission ERA chair FP7 grant (VetMedZg #621394).

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., The project was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) (grants Pr. Ref. AGL2013-46981-R, RTC-2016-50005-1, SAF2016-81003-R and SAF2016-80998-R), the Instituto de Salud Carlos III within the Network of Tropical Diseases Research RICET (RD16/0027/0016 and RD16/0027/0005) and (FEDER). The authors of this paper are members of and receive support from COST Action TD1303 (European Network for Neglected Vectors and Vector-Borne Infections), INFRAVEC 2 (Research Infrastructures for the Control of Vector-Borne Diseases, H2020, no. 731060) and VectorNet, a European network for sharing data on the geographic distribution of arthropod vectors transmitting human and animal disease agents (contract OC/EFSA/AHAW/2013/02-FWC1) funded by the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (ECDC). TS and PV were supported by Ministry of Education, Youth and Sport of the Czech Republic (MSMT) (project OPVVV 16-019/0000759) and Charles University (Research Centre UNCE 204072).

The quantification of anti-Leishmania antibodies in serum and saliva by a time-resolved immunofluorometric assay is useful for the diagnosis and treatment monitoring of dogs with clinical leishmaniasis. We compared the kinetics of anti-Leishmania IgG2 and IgA antibodies in serum and saliva from 11 Beagle dogs experimentally infected with Leishmania infantum. Most dogs showed detectable concentrations of anti-Leishmania IgG2 earlier in serum (between 3 and 4 months p.i.) than in saliva (between 4 and 6 months p.i.). Overall, a high correlation between concentrations of anti-Leishmania IgG2 in serum and saliva (r = 0.853; P < 0.0001) was observed. The quantification of anti-Leishmania IgA showed less diagnostic value than IgG2, since detectable amounts of IgA were not observed in the saliva of four dogs and in the serum of one dog. In addition, a very low correlation between anti-Leishmania IgA in serum and saliva (r = 0.289; P < 0.001) was observed. Our results indicate that the antibodies against L. infantum in saliva appear approximately 1 month later than in serum, and suggest that there is a threshold for the passing of immunoglobulins from serum to saliva in dogs. These facts should be taken into consideration for a proper interpretation of saliva assays for quantification of antibodies.

Leishmania infantum causes human and canine leishmaniosis. The parasite, transmitted by phlebotomine sand flies, infects species other than dogs and people, including wildlife, although their role as reservoirs of infection remains unknown for most species. Molecular typing of parasites to investigate genetic variability and evolutionary proximity can help understand transmission cycles and designing control strategies. We investigated Leishmania DNA variability in kinetoplast (kDNA) and internal transcribed spacer 2 (ITS2) sequences in asymptomatically infected wildlife (n = 58) and symptomatically and asymptomatically infected humans (n = 38) and dogs (n = 15) from south-east Spain, using single nucleotide polymorphisms (SNPs) and in silico restriction fragment length polymorphism (RFLP) analyses. All ITS2 sequences (n = 76) displayed a 99%-100% nucleotide identity with a L. infantum reference sequence, except one with a 98% identity to a reference Leishmania panamensis sequence, from an Ecuadorian patient. No heterogeneity was recorded in the 73 L. infantum ITS2 sequences except for one SNP in a human parasite sequence. In contrast, kDNA analysis of 44 L. infantum sequences revealed 11 SNP genotypes (nucleotide variability up to 4.3%) and four RFLP genotypes including B, F and newly described S and T genotypes. Genotype frequency was significantly greater in symptomatic compared to asymptomatic individuals. Both methods similarly grouped parasites as predominantly or exclusively found in humans, in dogs, in wildlife or in all three of them. Accordingly, the phylogenetic analysis of kDNA sequences revealed three main clusters, two as a paraphyletic human parasites clade and a third including dogs, people and wildlife parasites. Results suggest that Leishmania infantum genetics is complex even in small geographical areas and that, probably, several independent transmission cycles take place simultaneously including some connecting animals and humans. Investigating these transmission networks may be useful in understanding the transmission dynamics, infection risk and therefore in planning L. infantum control strategies.

The objective of this study was to identify changes in serum proteome in dogs that may occur after an experimental infection at subclinical and clinical stages of canine leishmaniosis (CanL). For this purpose, canine pre‐ and post‐infection with Leishmania infantum serum proteomes in the same dogs were analysed by a high‐throughput label‐based quantitative LC‐MS/MS proteomic approach. A total of 169 proteins were identified, and 74 of them including complement C8 alpha chain, adiponectin, transferrin, sphingomyelin phosphodiesterase acid‐like 3A and immunoglobulins showed different modulation between the different stages of CanL. These proteins could be considered as potential serum biomarkers of early diagnostic or disease progression in CanL. Additionally, biological pathways modulated during CanL such as blood coagulation or gonadotropin‐releasing hormone receptor were revealed, which could help to understand the pathological mechanisms of the disease., LFM was granted with predoctoral contract ‘FPU' of University of Murcia, Spain. MV was supported by Research Plan of University of Castilla‐ La Mancha, Spain. AT has a postdoctoral fellowshisp ‘Ramón y Cajal' supported by the ‘Ministerio de Economía y Competitividad', Spain. MCT and MCL were supported by grants SAF2016‐81003‐R, SAF2016‐80996‐R, and RTC‐2016‐50005‐1 from the ‘Programa Estatal I+D+I' (MINECO). This work was supported by a grant from the Program for Research Groups of Excellence of the Seneca Foundation, Murcia, Spain (grant 19894/GERM/15).

Background Urea and creatinine in saliva have been reported to be possible markers of chronic kidney disease (CKD) in humans. The aim of this study was to assess if urea and creatinine could be measured in canine saliva, and to evaluate their possible changes in situations of CKD. Results The spectrophotometric assays for urea and creatinine measurements in saliva of dogs showed intra- and inter-assay imprecision lower than 12% and coefficients of correlation close to 1 in linearity under dilution tests. Healthy dogs showed median salivary concentrations of urea of 39.6 mg/dL and creatinine of 0.30 mg/dL, whereas dogs with CKD showed median salivary urea of 270.1 mg/dL and creatinine of 1.86 mg/dL. Positive high correlations were found between saliva and serum activities of the two analytes (urea, r = 0.909; P

Background Dogs with canine leishmaniosis (CanL) due to Leishmania infantum can show a wide spectrum of clinical and clinicopathological findings at the time of diagnosis. The aim of this paper is to describe the possible application of acute phase proteins (APPs) for the characterization and management of this disease, based on previously published information on the utility of APPs in CanL and the experience of the authors in using APPs as analytes in the profiling of canine diseases. Main body Dogs diagnosed with L. infantum infection by serology, polymerase chain reaction, cytological or histopathological identification, can be divided into three groups based on their clinical condition at physical examination and their APPs concentrations: Group 1: dogs with no clinical signs on physical examination and APPs in reference range; Group 2: dogs with changes in APPs but no clinical signs on physical examination; Group 3: dogs with clinical signs and changes in APPs. This report describes the main characteristics of each group as well as its association with the clinical classification schemes of CanL. Conclusion APPs concentration can be a useful clinical tool to characterize and manage CanL.

Funding: Fundación Séneca 19894/GERM/15 Salivary alpha-amylase (sAA) is considered a biomarker of sympathetic activation in humans, but there is controversy regarding the existence of sAA in dogs. The hypothesis of this study was that sAA exists in dogs and it could change in situations of sympathetic stimulation. Therefore, the aims of this study were: 1) to demonstrate the presence of alpha-amylase in saliva of dogs by Western-Blot, 2) to validate an spectrophotometric method for the measurement of sAA activity and 3) to evaluate the possible changes in sAA activity after the induction of an ejaculation in dogs which is known to produce a sympathetic activation. Western-Blot demonstrated a band in dog saliva specimens between 60 kDa and 50 kDa, similar to purified sAA. The spectrophotometric assay validated showed an adequate inter- and intra-assay precision, and a high correlation coefficient (r = 0.999) in the linearity under dilution study. sAA median activity significantly increased just after ejaculation compared with just before the ejaculation (2.06-fold, P = 0.005). This study demonstrated the existence of alpha-amylase in saliva of dogs and that this enzyme can be measured by a spectrophotometric assay. In addition, results showed that sAA increase after a sympathetic activation and could be potentially used as non-invasive biomarker of sympathetic activity in this species.

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 CO

[Background] Leishmania infantum is a protozoan parasite transmitted by phlebotomine sand flies that causes life-threatening disease in humans and dogs. The dog is the primary reservoir of the parasite and early diagnosis of canine leishmaniosis is crucial at the clinical and epidemiological level. The currently available serological tests for CanL diagnostic show limitations therefore the aim of the present study was to investigate the diagnostic performance of an indirect antibody ELISA based on the Leishmania infantum recombinant antigen PFR1 in asymptomatically infected dogs. One hundred fifty-six dogs including Leishmania-free experimental Beagles and pet dogs from England, Scotland and Leishmania-endemic Murcia in Spain, were tested with the assay. The later were also tested with two commercial L. infantum crude antigen ELISAs (INgezim and Civtest, respectively) and a real-time kinetoplast PCR test., [Results] Anti-PFR1 antibodies were detected in the four groups of dogs, and the mean log-transformed optical density (OD) values were lowest in Beagles and in dogs from England and highest among dogs from Murcia (p, [Conclusions] The ability shown by the PFR1 ELISA to detect infected dogs that go undetected by the crude antigen ELISAs is clinically and epidemiologically useful and PFR1 could be considered a candidate for a multi-antigen-based immunoassay for early detection of L. infantum infected dogs., The development of the PFR1 antigen was supported by the INNPACTO grant IPT-2012-0697-010000 from the Ministerio de Economía, Industria y Competitividad (MINECO). The sampling and testing of dogs was supported by the grants Pr. Ref: AGL2013-46981-R, RTC-2016-50,005-1, and SAF2016-81003-R from the MINECO, the Instituto de Salud Carlos III within the Network of Tropical Diseases Research RICET (RD16/0027/0016 and RD16/0027/0005) and FEDER. Darien Ledesma was supported by a FPU predoctoral fellowship (FPU-AP2012-1662) from MINECO.

We describe an atypical case of duodenal leishmaniasis in a boxer dog presenting with chronic diarrhea and poor general condition. Antidiarrheic therapy was previously administered without success and inflammatory bowel disease localized to the small intestine was suspected, given the chronic clinical signs and by ruling out other known causes of gastrointestinal inflammation. Endoscopic biopsy of duodenum showed a moderate increase in lamina propria lymphocytes, plasma cells, and macrophages. Basophilic bodies were seen in the cytoplasm of numerous macrophages, suggestive of Leishmania spp, confirmed by immunostaining, and a diagnosis of granulomatous duodenitis associated to Leishmania infection was made. After 7 mo of therapy, a significant clinical improvement and weight gain were observed, and endoscopic histology showed no evidence of Leishmania. A progressive decline of anti-leishmanial antibody titer was also observed during follow-up. This report emphasizes the importance of atypical symptoms and the unusual location of visceral leishmaniasis, suggesting the need to consider leishmaniasis in the differential diagnosis of canine chronic enteritis, especially in endemic areas.

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

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.

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.

The validation for Iberian red deer of a commercially available Time-resolved fluoroimmunoassay (TR-FIA) designed for analysis of progesterone in human beings was carried out. Intra-assay coefficients of variation ranged from 3.6% to 7.4%, while inter-assay coefficients of variation varied from 5.2% to 15.5%. Accuracy, evaluated by comparing results yielded by TR-FIA with those obtained from a validated radioimmunoassay (RIA) in the measurement of 14 samples, provided a high regression coefficient (R2= 0.93). Different progesterone concentrations added to pool plasma showed percentages of recovery that ranged between 102.6% and 82.48%. The limit of detection was 0.102 nmol/L. The results obtained indicate that the present method is suitable for the measurement of progesterone in female Iberian red deer. J. Exp. Zool. 301A:472–476, 2004. © 2004 Wiley-Liss, Inc.

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

We propose a probabilistic extension of the matching pursuit adaptive signal processing algorithm introduced by Mallat and others. In adaptive signal processing, signals are expanded in terms of a large linearly dependent “dictionary” of functions rather than in terms of an orthonormal basis. Matching pursuit is a simple greedy algorithm for generating an expansion of a given signal. In probabilistic matching pursuit multiple random expansions are obtained as estimates for a given signal. The new algorithm is illustrated in the context of signal denoising. Although most of the random expansions generated by probabilistic matching pursuit are poorer estimates for the signal than those obtained by matching pursuit, our final estimate, obtained as an expected value computed by means of an ergodic average, can improve the result obtained by MP in some denoising situations. One of the major underlying ideas is a novel notion of coherence between a signal and the dictionary. Several simulated examples are presented.

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.

A six-week-old male puppy was presented with a distended abdomen, dypsnoea and cyanosis. Auscultation revealed a grade II/VI systolic murmur. Thoracic radiographs showed gross cardiomegaly. An electrocardiogram revealed a narrow-complex tachycardia, deep S waves in leads I, II, III and aVF, and negative P a waves in lead III. Two-dimensional echocardiography showed a high ventricular septal defect and marked dilation of the right-sided chambers. There was also an echolucent structure lateral to the left atrium at a site corresponding to the coronary sinus. Contrast echocardiography revealed right-to-left shunting through the septal defect. Necropsy confirmed the existence of a septal defect in the membranous part of the septum and a persistent left cranial vena cava with dilation of the coronary sinus. In addition, a small patent t ductus arteriosus and tricuspid dysplasia were present.

Summary 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 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.

An automated method using 2,2’-dithiodipyridine (2-PDS) as chromophore for determination of wholeblood 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 Eilman 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.

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.

The paper describes an algorithm to count and classify cells of different geometrical shapes on a given image. The algorithm assumes that it is known a priori the type of geometries to be recognized and it allows for many different geometrical shapes to appear in the same image with different sizes, locations and orientations. The algorithm combines classical tools, mainly the two dimensional Fourier transform, with newly developed tools for edge enhancements as well as the main technical contribution of the present paper, which consists in the definition of an over-complete set of spanning functions. These functions are constructed from geometrical templates of size comparable to the image cells; moreover, the resulting functions are scaled and rotated to assure the recognition of all image cells. We then describe an algorithm that decomposes the image in its most likely elements. The combination of ingredients used by the algorithm provides a cell recognition tool that is very robust, provides high resolution to discern among competing candidate cells and delivers practical computational efficiency.

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.

Objective—To evaluate changes in serum concentrations of acute-phase proteins in dogs with leishmaniosis during short-term therapy in accordance with 2 treatment protocols and determine whether concentrations of acute-phase proteins could be used to monitor the initial response of dogs to treatment. Animals—12 dogs naturally infected with Leishmania infantum. Procedure—Dogs were allocated into 2 groups. Dogs of group 1 were treated by use of meglumine antimonate (100 mg/kg, SC, q 24 h) administered concurrently with allopurinol (15 mg/kg, PO, q 12 h) for 20 days and then with allopurinol alone at the same dosage for the subsequent 30 days. Dogs of group 2 were treated by administration of allopurinol alone (15 mg/kg, PO, q 12 h) for 60 days). Blood samples were obtained before and during treatment for measurement of serum concentrations of acute-phase proteins and determination of CBC counts, serum biochemical analyses, and electropherograms. Results—All dogs evaluated in the study had increased concentrations of C-reactive protein, haptoglobin, and ceruloplasmin at the time of diagnosis of leishmaniosis. Mean concentration of serum amyloid A before treatment was also increased, but some of the dogs had concentrations of serum amyloid A that were within the reference range. Concentrations of C-reactive protein and ceruloplasmin decreased significantly in all dogs at the end of the study period. Conclusions and Clinical Relevance—Measurement of concentrations of selected acute-phase proteins, such as C-reactive protein or ceruloplasmin, could be used to evaluate the initial response of dogs with leishmaniosis to treatment. (Am J Vet Res 2003;64:102–1026)

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.

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.

Serial electrocardiograms were recorded from 70 Mastin Espanol 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.

Purpose: Small-molecule inhibitors have revolutionized treatment of certain genomically defined solid cancers. Despite breakthroughs in treating systemic disease, central nervous system (CNS) metastatic progression is common, and advancements in treating CNS malignancies remain sparse. By improving drug penetration across a variably permeable blood–brain barrier and diffusion across intratumoral compartments, more uniform delivery and distribution can be achieved to enhance efficacy. Experimental Design: Ultrasmall fluorescent core-shell silica nanoparticles, Cornell prime dots (C9 dots), were functionalized with αv integrin-binding (cRGD), or nontargeting (cRAD) peptides, and PET labels (124I, 89Zr) to investigate the utility of dual-modality cRGD-C9 dots for enhancing accumulation, distribution, and retention (ADR) in a genetically engineered mouse model of glioblastoma (mGBM). mGBMs were systemically treated with 124I-cRGD- or 124I-cRAD-C9 dots and sacrificed at 3 and 96 hours, with concurrent intravital injections of FITC-dextran for mapping blood–brain barrier breakdown and the nuclear stain Hoechst. We further assessed target inhibition and ADR following attachment of dasatinib, creating nanoparticle–drug conjugates (Das-NDCs). Imaging findings were confirmed with ex vivo autoradiography, fluorescence microscopy, and p-S6RP IHC. Results: Improvements in brain tumor delivery and penetration, as well as enhancement in the ADR, were observed following administration of integrin-targeted C9 dots, as compared with a nontargeted control. Furthermore, attachment of the small-molecule inhibitor, dasatinib, led to its successful drug delivery throughout mGBM, demonstrated by downstream pathway inhibition. Conclusions: These results demonstrate that highly engineered C9 dots are promising drug delivery vehicles capable of navigating the complex physiologic barriers observed in a clinically relevant brain tumor model.

SUMMARY 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 ocurred at the age of 6 months and in the second year of life.