Changes in salivary analytes in canine parvovirus : A high-resolution quantitative proteomic study

The present study evaluated the changes in salivary proteome in parvoviral enteritis (PVE) in dogs through a high-throughput quantitative proteomic analysis. Saliva samples from healthy dogs and dogs with severe parvovirosis that survived or perished due to the disease were analysed and compared by Tandem Mass Tags (TMT) analysis. Proteomic analysis quantified 1516 peptides, and 287 (corresponding to 190 proteins) showed significantly different abundances between studied groups. Ten proteins were observed to change significantly between dogs that survived or perished due to PVE. Bioinformatics' analysis revealed that saliva reflects the involvement of different pathways in PVE such as catalytic activity and binding, and indicates antimicrobial humoral response as a pathway with a major role in the development of the disease. These results indicate that saliva proteins reflect physiopathological changes that occur in PVE and could be a potential source of biomarkers for this disease

Advances in Canine Leishmaniosis with emphasis on the use of saliva samples

Esta tesis como compendio de publicaciones fue concebida para profundizar en el estudio del desarrollo de ensayos que permitieran el uso de muestras de saliva para diagnosticar leishmaniosis en perros. Los objetivos fueron: (1) desarrollar y validar un ensayo ultrasensible para la cuantificación de anticuerpos anti-Leishmania que permitiera su medición en suero y saliva de perro; (2) evaluar la utilidad del nuevo ensayo con muestras de suero y saliva para monitorizar el tratamiento frente a leishmaniosis canina (LCan); (3) evaluar la relación entre los niveles de anticuerpos anti-Leishmania en suero de perro medidos con el nuevo ensayo y por un ELISA comercial y la concentración de proteínas de fase aguda; (4) evaluar, mediante el nuevo ensayo, la cinética de los niveles de IgG2 e IgA anti-Leishmania en suero y saliva de perros infectados experimentalmente con L. infantum durante un año de seguimiento; (5) estudiar el posible ritmo circadiano en los niveles de IgG2 e IgA anti-Leishmania en suero y saliva de perros infectados experimentalmente; (6) y desarrollar y validar una PCR a tiempo real para la cuantificación de ADN de Leishmania spp. en saliva canina. La precisión, la exactitud y la sensibilidad analítica de cada ensayo fueron evaluadas mediante los coeficientes de variación intra- e inter-ensayo, la linealidad bajo dilución, el porcentaje de recuperación y el estudio de correlación, y los límites de detección y cuantificación. Para ello, se recogieron muestras de suero y saliva de perros seronegativos y seropositivos a Leishmania spp. Para evaluar la utilidad de los TR-IFMAs en suero en la monitorización del tratamiento se obtuvieron muestras de suero de 16 perros seropositivos a Leishmania spp. en el momento del diagnóstico y tras 30 y 180 días de tratamiento. Del mismo modo, para evaluar la utilidad de los TR-IFMAs en la monitorización del tratamiento con muestras de saliva, se recogieron muestras de suero y saliva de 20 perros con signos clínicos compatibles con LCan en el momento del diagnóstico y después de 30 días de tratamiento. También se usaron 205 muestras de suero para evaluar la correlación entre TR-IFMA y un ELISA comercial y la concentración de proteínas de fase aguda en el momento del diagnóstico de LCan y durante la monitorización del tratamiento. Para estudiar la cinética de los niveles de anticuerpos anti-Leishmania se tomaron mensualmente durante 1 año muestras de suero y saliva de 11 perros infectados experimentalmente con Leishmania infantum y se analizaron por TR-IFMA. Para evaluar el posible ritmo circadiano en los niveles de anticuerpos anti-Leishmania, muestras de suero y saliva de 6 perros infectados experimentalmente que presentaban signos clínicos compatibles con LCan se recogieron a intervalos de 4 horas durante un periodo de 16 horas en dos días consecutivos. Finalmente, para el desarrollo y validación de una PCR a tiempo real para la detección de ADN de Leishmania spp. en saliva de perro se recogieron salivas de 16 perros infectados experimentalmente pre-infección y 16 semanas post-infección. Las conclusiones de esta tesis doctoral son: 1. Los ensayos inmunofluorométricos desarrollados en esta tesis doctoral permiten la cuantificación de anticuerpos IgG2 e IgA anti-Leishmania en suero y saliva de perro, siendo los niveles más elevados en perros seropositivos. Se observó solapamiento en los niveles de IgA anti-Leishmania entre seropositivos y seronegativos. 2. Los ensayos desarrollados detectan disminuciones en los niveles de IgG2 e IgA anti-Leishmania en suero y saliva de perros después del tratamiento, asociado con mejoría clínica. 3. El ensayo desarrollado para detectar IgG2 anti-Leishmania mostró mayor correlación con los resultados de CRP y ferritina en perros tratados que el ELISA. 4. Los niveles de IgG2 e IgA anti-Leishmania en suero y saliva de perros aumentan por encima del punto de corte del ensayo TR-IFMA tras la infección experimental. Además, el TR-IFMA desarrollado para la detección de IgG2 anti-Leishmania ofrece mejor valor diagnóstico que el desarrollado para IgA anti-Leishmania. 5. No se ha observado ritmo circadiano en los niveles de IgG2 e IgA anti-Leishmania tanto en suero como en saliva de perros con LCan en un periodo de 16 horas en dos días consecutivos. Además, se han observado mayores variaciones en los niveles de estos anticuerpos en saliva que en suero. 6. La PCR a tiempo real desarrollada permite la cuantificación de ADN del cinetoplasto de Leishmania spp. en saliva de perros infectados experimentalmente con L. infantum. No obstante, hay que tener en cuenta que la sensibilidad del ensayo en saliva es menor que en médula ósea.This PhD thesis as a compendium of publications was conceived to go in-depth on the study of the development of assays that could allow the use of saliva samples to diagnose leishmaniosis in dogs. The objectives were: (1) to develop and validate an ultrasensitive assay for anti-Leishmania antibodies quantification that could allow their measurement in serum and saliva of dogs; (2) to evaluate the usefulness of the assay for anti-Leishmania antibodies measurement in both serum and saliva samples as a tool for monitoring the treatment of canine leishmaniosis (CanL); (3) to assess the possible relationship between serum anti-Leishmania antibody levels measured by the new assay and by a commercially-available ELISA kit and the concentration of acute phase proteins; (4) to evaluate and compare the kinetics of anti-Leishmania IgG2 and IgA by the new assay in serum and saliva from experimentally infected dogs with L. infantum during one-year follow-up; (5) to assess the possible circadian rhythm of anti-Leishmania IgG2 and IgA levels in serum and saliva from experimentally infected dogs; and (6) to develop and validate a qPCR for quantification of Leishmania spp. DNA in canine saliva. Precision, accuracy and analytical sensitivity were evaluated by the coefficients of variation intra- and inter-assay, linearity under dilution, percentage of recovery and correlation study, and limits of detection and limits of quantification to validate the new assays. For that, serum and saliva samples from Leishmania-seronegative and Leishmania-seropositive dogs were collected. To evaluate the usefulness of the TR-IFMAs for treatment monitoring, serum samples were obtained from 16 Leishmania-seropositive dogs at the time of diagnosis and after 30 and 180 days of treatment. As well as, to evaluate the utility of the TR-IFMAs for treatment monitoring using saliva samples, serum and saliva samples were collected from 20 dogs with clinical signs compatible with CanL at the time of diagnosis and after 30 days of treatment. Also, 205 serum samples were evaluated to study the correlation between the TR-IFMA, a commercial ELISA and the concentration of acute phase proteins in dogs at the time of diagnosis and during treatment monitoring. To study the kinetics of anti-Leishmania antibody levels, 11 dogs were infected with Leishmania infantum. After, serum and saliva samples were monthly collected during 1-year and analyzed by TR-IFMAs. To assess the possible circadian rhythm in the levels of anti-Leishmania antibodies serum and saliva of 6 dogs experimentally infected presenting clinical signs of CanL were collected during a 16-h period at 4-h intervals on two consecutive days. Finally, to develop and validate a qPCR for the detection of Leishmania spp. DNA in saliva of dogs, saliva samples from 16 dogs experimentally infected were collected pre-infection and at 16-weeks post-infection. The conclusions of this PhD thesis are: 1. The immunofluorometric assays developed in this PhD thesis allow the quantification of anti-Leishmania IgG2 and IgA in canine serum and saliva samples in a reliable way. An overlap between seropositive and seronegative dogs was observed for IgA. 2. The assays developed detect decreases in anti-Leishmania IgG2 and IgA levels in serum and saliva from dogs with clinical leishmaniosis after treatment, which is associated with clinical improvement. 3. The assay developed for anti-Leishmania IgG2 is more correlated with CRP and ferritin concentrations than the ELISA results in treated dogs. 4. Levels of anti-Leishmania IgG2 and IgA in canine serum and saliva increase after experimental infection, surpassing the cut-off approximately one month earlier in serum than in saliva. Anti-Leishmania IgG2 shows better diagnostic value than IgA. 5. No circadian rhythm is observed for anti-Leishmania IgG2 and IgA levels in both serum and saliva samples of dogs with CanL during a 16 h-period. Additionally, higher intra-individual variations in the specific antibody levels are observed in saliva than in serum. 6. The qPCR assay developed allows the quantification of Leishmania kDNA in saliva samples of experimentally infected dogs with L. infantum. However, the sensitivity of this assay in the saliva is lower than in bone marrow

Evaluation of the circadian rhythm of anti-Leishmania IgG2 and IgA antibodies in serum and saliva of dogs with clinical leishmaniosis

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)

One-year follow-up of anti-Leishmania antibody concentrations in serum and saliva from experimentally infected dogs

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

A systematic review and meta-analysis of the validation of serological methods for detecting anti-Toxoplasma gondii antibodies in humans and animals

Toxoplasma gondii is a paradigmatic zoonotic parasite from the One Health perspective, since it is broadly distributed and virtually infects all warm-blooded species. A wide variety of serological techniques have been developed to detect T. gondii infection in humans and animals. Our aim was to describe and compare the main characteristics of these serological tests and validation processes and to critically analyze whether these tests meet the standards required to ensure an accurate serological diagnosis. The current systematic review and meta-analysis included 134 studies that were published from 2013 to 2023. QUADAS 2 tool was used to evaluate the quality of the included studies. A total of 52 variables related to the characteristics of the techniques and analytical and diagnostic validation parameters were studied. A wider panel of tests was developed for humans, including techniques exclusively developed for humans that involve costly equipment and the measurement of different Ig isotypes that are considered biomarkers of congenital toxoplasmosis. Studies conducted in humans frequently employed commercial techniques as reference tests, measured different immunoglobulin isotypes with a predominance for IgG (>50%) and discriminated between acute and chronic infections. In animals, the most commonly used reference techniques were in-house tests, which almost exclusively detected IgG. Common limitations identified in a large number of studies were some misunderstandings of the terms “gold standard” and “reference test” and the absence of information about the negative and positive control sera used or the exact cutoff employed, which were independent of the quality of the study. There is a lack of analytical validation, with few evaluations of cross-reactivity with other pathogens. Diagnostic odds ratio values showed that indirect ELISA based on native or chimeric antigens performed better than other tests. The reproducibility of serological test results in both humans and animals is not guaranteed due to a lack of relevant information and analytical validation. Thus, several key issues should be considered in the future, including interlaboratory ring trials.Universidad de MurciaEuropean CommissionFundación Séneca-Agencia de Ciencia y Tecnología de la Región de MurciaDepto. de Sanidad AnimalFac. de VeterinariaTRUEpu

Detection of Leishmania infantum DNA by real-time PCR in saliva of dogs

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

Detection of Leishmania infantum DNA by real-time PCR in saliva of dogs

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]