- Email: [email protected]
Relationship between serum anti-Leishmania antibody levels and acute phase proteins in dogs with canine leishmaniosis
Veterinary Parasitology 260 (2018) 63–68
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Research paper
Relationship between serum anti-Leishmania antibody levels and acute phase proteins in dogs with canine leishmaniosis
T
⁎
Ana Cantos-Barredaa, Damián Escribanoa, , José J. Ceróna, Luis J. Bernala, Tommaso Furlanellob, Fernando Teclesa, Luis Pardo-Marína, Silvia Martínez-Subielaa a b
Interdisciplinary Laboratory of Clinical Analysis, Interlab-UMU, Campus of Excellence Mare Nostrum, University of Murcia, 30100 Espinardo, Murcia, Spain San Marco Veterinary Clinic, Veggiano, Padova, Italy
A R T I C LE I N FO
A B S T R A C T
Keywords: Canine leishmaniosis Correlation study TR-IFMA ELISA Acute phase proteins Anti-Leishmania antibodies
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 (TRIFMA)) 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.
1. Introduction Canine leishmaniosis (CanL) is an infectious disease in the Mediterranean area caused by Leishmania infantum. It is marked by its zoonotic potential and endemic character (Mettler et al., 2005; Baneth and Aroch, 2008). The spread of CanL depends on the distribution of the vectors and reservoir animals (Kato et al., 2005). Dogs play an important role in the transmission of the disease to humans and other mammals since they are the main reservoirs for sandfly infection (Ready, 2010). CanL ranges from subclinical disease to fatality depending on the host’s immune response (Barbosa et al., 2009). When the immune response is mainly mediated by Th2 lymphocytes, the antibody production is high, leading to the deposition of soluble immune-complexes in
⁎
organs and tissues (Solano-Gallego et al., 2001; Proverbio et al., 2014). This pathogenic reaction is associated with the presence of a great variety of clinical signs, the presence of skin lesions being the most common manifestation in dogs with clinical leishmaniosis. Other clinical signs that may be present are generalized lymphadenomegaly, weight loss, blepharitis, conjunctivitis, epistaxis or polyarthritis, among others (Solano-Gallego et al., 2011). The estimated seroprevalence of CanL in endemic areas is typically 10–30%, depending on where the dogs live and their exposure to infection, as well as on the sensitivity and specificity of the diagnostic tests (Maia and Campino, 2008). A variety of laboratory techniques to detect anti-Leishmania antibodies using quantitative serological techniques have been developed (Wolf et al., 2014). The most commonlyused techniques are the enzyme-linked immunosorbent assay (ELISA)
Corresponding author. E-mail address: [email protected] (D. Escribano).
https://doi.org/10.1016/j.vetpar.2018.08.010 Received 15 May 2018; Received in revised form 22 August 2018; Accepted 23 August 2018 0304-4017/ © 2018 Elsevier B.V. All rights reserved.
Veterinary Parasitology 260 (2018) 63–68
A. Cantos-Barreda et al.
total proteins and globulins; (3) in the case of samples submitted for initial diagnosis of CanL there should be no history of suffering a previous episode of CanL; (4) there should be no history of Leishmania vaccination; and (5) the dogs involved should be free from other diseases. Dogs were allocated into two groups: (1) animals whose samples were submitted to our laboratory for the initial serological diagnosis of CanL and (2) animals whose samples were submitted for treatment monitoring of CanL.
and the immunofluorescence antibody test (IFAT) (Solano-Gallego et al., 2011). Recently, a highly-sensitive time-resolved immunofluorometric assay (TR-IFMA) for the quantification of anti-Leishmania antibodies in canine serum has been developed and validated (CantosBarreda et al., 2017). This TR-IFMA was based on the recombinant antigen K39 (rK39), a repetitive and highly conserved protein among viscerotropic Leishmania spp. (Burns et al., 1993). In addition, this TRIFMA showed a wider difference in anti-Leishmania antibody levels between Leishmania-seropositive and Leishmania-seronegative dogs and between clinical stages, than did a commercially-available ELISA (Cantos-Barreda et al., 2017). Acute phase proteins (APPs) are proteins present in plasma whose concentrations are modified in response to tissue injury, infection or inflammation, and which allow the assessing of the innate immune system of the host. Their quantification provides relevant information in support of clinical diagnosis, prognosis and monitoring response to treatment (Murata et al., 2004; Eckersall and Bell, 2010). APPs can be classified as positive or negative if their levels increase or decrease, respectively, after inflammation. Serum ferritin, C-reactive protein (CRP) and haptoglobin are considered positive APPs, while albumin and paraoxonase-1 (PON-1) are considered negative APPs (Cerón et al., 2005; Martínez-Subiela et al., 2014). Previous studies have reported an acute phase response in dogs with CanL, characterized by variations of APPs with increases in CRP, serum ferritin and haptoglobin (MartínezSubiela et al., 2011; Cantos-Barreda et al., 2018a). Furthermore, the progression of the disease through the determination of the APPs at the time of diagnosis and following treatment, showed an improvement in the concentration of APPs as the clinical manifestations disappeared (Martínez-Subiela et al., 2016). To the authors’ knowledge, there are no studies that correlate antibody levels and APPs in dogs with CanL. The objective of this study was to assess the possible relationship between serum anti-Leishmania antibody levels measured by two different assays and selected biochemical analytes related to inflammation and protein concentration in dogs naturally infected with L. infantum. For this purpose, we evaluated: (1) the possible differences in the concentration of APPs and analytes related to proteins between Leishmania-seronegative and Leishmania-seropositive dogs; (2) the values of these analytes in the different quartiles of anti-Leishmania antibodies; and, finally, (3) the possible differences in the correlations between antibody levels and the biochemical analytes evaluated, depending on whether they are measured at the time of diagnosis or during treatment monitoring.
2.3. Sampling Blood samples were obtained at different clinics using venipuncture of the cephalic or jugular vein, and were collected in tubes containing a coagulation activator and a gel separator. Tubes were allowed to clot at room temperature and centrifuged at 3500 rpm for 5 min. The serum was separated and sent the same day to the Interdisciplinary Laboratory of Clinical Analysis (Interlab-UMU, University of Murcia, Spain). Biochemical analytes included in the study were analyzed on arrival. Samples were stored at -80 °C until analyzed for antibody titers. Such an analysis was undertaken in the lab once a week. 2.4. Anti-Leishmania antibodies In order to determine the level of anti-Leishmania antibodies in the serum samples two different quantitative methods, a commerciallyavailable ELISA test and a TR-IFMA, were employed. The ELISA was performed following manufacturer’s instructions using the recommended 1/20 dilution for serum samples and results were expressed as sample-to-positive (S/P) ratio calculated as optical density (OD) sample/OD low positive control. TR-IFMA was performed as previously described (Cantos-Barreda et al., 2017) and samples were diluted 1/4000 in an assay buffer. The results were expressed as Units of Fluorometry for Leishmania (UFL). 2.5. Biochemical analytes CRP concentration was measured using a human immunoturbidimetric test (CRP OSR 6147 Olympus Life and Material Science Europe GmbH, Lismeehan, O'Callaghan Mills, Co. Clare, Ireland) used previously in dogs (Muñoz-Prieto et al., 2017). Haptoglobin was determined using a hemoglobin-binding method (Tridelta Phase, Tridelta Development Ltd., Bray, Ireland) in a biochemistry autoanalyzer (Cobas Mira Plus, ABX Diagnostics, Montpellier, France). PON-1 activity was analyzed following a previously described method (Tvarijonaviciute et al., 2012). Ferritin concentrations were measured using an immunoturbidimetric assay with polyclonal anti-human ferritin antibodies (Tina-quant Ferritin, Boehringer Mannheim, Germany) previously validated for use in dogs (Martínez-Subiela et al., 2014). Serum total protein and albumin concentrations were measured using a human colorimetric assay (Total protein OSR 6132, Albumin OSR 6102, Olympus Life and Material Science Europe GmbH, Hamburg, Germany). Globulin concentrations were calculated by the difference between total proteins and albumin concentrations as previously described in dogs with CanL (Silvestrini et al., 2012). Determinations of CRP, total proteins and albumin concentrations were performed using an automated biochemistry analyzer (Olympus AU600; Olympus Diagnostica GmbH, Hamburg, Germany).
2. Materials and methods 2.1. Ethics statement This study was approved by the Local Ethical Committee of the University of Murcia under protocol no. 36/2014. All procedures were conducted in accordance with law RD 53/2013 regarding animal experimentation in Spain and with the European Directive 2010/63/EU concerning the protection of animals used for scientific purposes. 2.2. Animals and study design Samples from 205 dogs were included in the study. These samples were submitted to our laboratory from different veterinary clinics throughout Spain in order to measure anti-Leishmania antibody levels and APPs. A set of criteria were taken into consideration when it came to accepting the samples for this study: (1) they had to have undergone the quantification of anti-Leishmania antibodies by a commerciallyavailable ELISA (Leiscan® Leishmania ELISA Test, Esteve Veterinaria, Laboratorios Dr. Esteve SA, Barcelona, Spain) and the TR-IFMA between January 2016 and January 2018; (2) the serological measurement had to be accompanied by the analysis of a panel of inflammatory analytes including CRP, haptoglobin, serum ferritin, PON-1, albumin,
2.6. Statistical analysis All data were assessed for normality of distribution using the Kolmogorov-Smirnov normality test. Descriptive statistics for the different groups were determined using the non-parametric Kruskal-Wallis test. To compare the TR-IFMA and the ELISA results, and to compare the serological results and the biochemical analytes values, a 64
Veterinary Parasitology 260 (2018) 63–68
A. Cantos-Barreda et al.
correlation is described by Spearman’s rank correlation coefficient (rs). Median values were given with regard to the respective 25th–75th percentiles. Antibody levels determined by TR-IFMA and ELISA were parameterized as a dummy variable comparing each quartile with each other. The significance level of p < 0.05 was chosen. All analyses were conducted using the GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA). 3. Results 3.1. Animals A total of 205 dogs were included in this study. One hundred and sixty-one dogs (78.5%) were included in the group of samples for serological diagnosis of CanL because these cases were submitted for initial diagnosis. Forty-four dogs (21.5%) were included in the group of samples submitted for treatment monitoring because these dogs were previously diagnosed with CanL and were receiving anti-leishmanial treatment. Additionally, all animals were allocated by quartiles of antiLeishmania antibodies for each serological assay. The ages of the dogs included in the study ranged from 7 months to 14.5 years, with a mean ± SD of 6.8 ± 3.4 years. Among the recruited dogs, 100 were male and 99 were female. The sex data from 6 dogs were not specified in their signalment. Purebred dogs were predominant in this study, with a total of 125 dogs, mainly represented by the German shepherd (14), Labrador retriever (13), French bulldog (8) and Boxer (6) breeds. Seventy-one crossbred dogs were also included in the study. Breed data from 9 dogs were not found in their signalment.
Fig. 1. Concentration of the selected biochemical analytes regarding the timeresolved immunofluorometric assay (TR-IFMA) and the commercially-available ELISA test (Leiscan® Leishmania ELISA Test) classification as Leishmania-seronegative and Leishmania-seropositive. The box plot shows median (horizontal line within box), 25th and 75th percentiles (box) and minimum and maximum values (whiskers). TP: total proteins (g/dL); Glob: globulins (mg/dL); Alb: albumin (g/dL); CRP: C-reactive protein (μg/mL); SF: serum ferritin (μg/L); PON1: paraoxonase-1 (UI/mL); Hp: haptoglobin (g/L). NS: not significant.
what is shown in Fig. 1 for the Leishmania-seronegative and Leishmaniaseropositive groups, significant differences between quartiles were observed. Animals belonging to the quartile 4, with higher levels of antiLeishmania antibodies, showed more pronounced changes in the biochemical analytes, with significant increases in total proteins, CRP, serum ferritin, haptoglobin and globulins, and significant decreases in albumin and PON-1 compared with other quartiles. CRP, PON-1, serum ferritin and albumin showed greater significant differences between quartiles when TR-IFMA was used compared with the ELISA. The distribution of cases by quartile of anti-Leishmania antibodies in which there was an increase, decrease or normal value of the different biochemical analytes is shown in Table 2. The higher number of abnormal values of the biochemical analytes correspond to quartile 4 of antibodies.
3.2. Relationship between Leishmania-seronegative and Leishmaniaseropositive dogs and the selected biochemical analytes Data were classified regarding the serological status and the clinical test cut-offs into two different groups: Leishmania-seronegative and Leishmania-seropositive. The cut-off between a negative and a positive result was established at 22 UFL and at 0.9 S/P ratio for TR-IFMA and ELISA, respectively. Samples close to the threshold were included in the Leishmania-seropositive group in both assays. The anti-Leishmania antibody levels and concentrations of APPs and analytes related to protein concentration in the Leishmania-seronegative and the Leishmania-seropositive groups were evaluated. Significant differences (p < 0.0001) were observed in anti-Leishmania antibody levels in the seropositive groups with respect to the seronegative groups. The seropositive groups showed a median value of 235 IgG2 UFL (25th–75th percentiles: 77.5–1014) and a median value of 3.2 S/P ratio (1.9–4.1) while the seronegative groups showed a median value of 4.9 IgG2 UFL (4.8–8.9) and a median value of 0.5 S/P ratio (0.3–0.8). However, no differences were detected in the concentration of the selected biochemical analytes between seronegative and seropositive dogs (Fig. 1).
3.4. Relationship between serology at diagnosis or during treatment monitoring and the selected biochemical analytes Three correlation studies were carried out separately: one for all the samples included in the study, another for the samples belonging to the diagnostic group, and another for the samples belonging to the treatment monitoring group. The correlation study performed with the totality of data revealed that IgG2 UFL were significantly correlated with the S/P ratio (Spearman rank r = 0.768; p < 0.0001). Both serological assays showed positive correlations with total proteins, globulins and serum ferritin and negative correlations with albumin. However, it is interesting to note that these correlations were more significant with regard to TR-IFMA. In the correlation study with regard to the diagnostic group both serological assays showed a significant positive correlation (r = 0.769, p < 0.0001). The TR-IFMA was significantly correlated with serum ferritin (r = 0.338, p < 0.0001), total proteins (r = 0.458, p < 0.0001), albumin (r = -0.168, p < 0.05), globulins (r = 0.436, p < 0.0001) and haptoglobin (r = 0.161, p < 0.05). The ELISA was significantly correlated with serum ferritin (r = 0.303, p < 0.0001), total proteins (r = 0.502, p < 0.0001), albumin (r = -0.234, p < 0.01) and globulins (r = 0.504, p < 0.0001). For the treatment monitoring group, TR-IFMA and the ELISA showed a correlation of r = 0.531 (p < 0.0001). The TR-IFMA was correlated with CRP (r = 0.563, p < 0.0001), haptoglobin (r = 0.311, p < 0.05), PON-1 (r = -0.381, p < 0.05), serum ferritin (r = 0.519,
3.3. Relationship between the magnitude of serology values and the selected biochemical analytes Table 1 shows the median, 25th–75th percentiles and the statistical differences for anti-Leishmania antibodies and the biochemical analytes among the different quartiles of anti-Leishmania antibodies for each serological assay. Quartiles were broken at 6.47, 40.12 and 304.9 UFL for TR-IFMA, and 0.50, 1.68 and 3.58 S/P ratio for ELISA. 88 and 78 dogs were considered seronegative according to the cut-offs for TRIFMA and ELISA, respectively. Significant differences in anti-Leishmania antibody levels were observed between quartiles 1 and 2 (p < 0.05), 1 and 3 (p < 0.0001), 2 and 3 (p < 0.0001), 1 and 4 (p < 0.0001), 2 and 4 (p < 0.0001) and 3 and 4 (p < 0.0001) for TR-IFMA. For the ELISA, significant differences were found between quartiles 1 and 3 (p < 0.001), 1 and 4 (p < 0.0001) and 2 and 4 (p < 0.0001). Related to the concentration of the selected biochemical analytes, contrary to 65
Veterinary Parasitology 260 (2018) 63–68
241 (168 – 498) 7.2 (6.5 – 7.5) α 2.9 (2.4 – 3.1) 4.2 (3.6 – 5.0)
*p < 0.05 comparing quartile 1 and 2; α p < 0.05 comparing quartile 1 and 3; Ͼ p < 0.01 comparing quartile 1 and 3; Ͻ p < 0.001 comparing quartile 1 and 3; β p < 0.0001 comparing quartile 1 and 3; ѱ p < 0.05 comparing quartiles 1 and 4; γ p < 0.01 comparing quartile 1 and 4; δ p < 0.001 comparing quartile 1 and 4; ε p < 0.0001 comparing quartile 1 and 4; Ͽ p < 0.05 comparing quartile 2 and 3; † p < 0.0001 comparing quartile 2 and 3; ζ p < 0.05 comparing quartile 2 and 4; η p < 0.01 comparing quartile 2 and 4; θ p < 0.001 comparing quartile 2 and 4; ι p < 0.0001 comparing quartile 2 and 4; κ p < 0.05 comparing quartile 3 and 4; λ p < 0.01 comparing quartile 3 and 4; μ p < 0.001 comparing quartile 3 and 4; ν p < 0.0001 comparing quartile 3 and 4. a Units of Fluorometry for Leishmania. b Sample-to-positive ratio. c C-reactive protein. d Paraoxonase 1. e Interlab-UMU biochemical laboratory reference values.
60-190 5.4-7.2 2.5-3.6 2.6-3.8 631 (203 – 866) γ, ζ 7.9 (7.0 – 8.4) ε, ι 2.1 (1.8 – 2.8) γ, η, κ 5.5 (4.6 – 6.5) ε, ι, λ 261 (197 – 377) 6.6 (6.2 – 7.1) 2.7 (2.5 – 3.0) 3.7 (3.5 – 4.4) 224 (166 – 443) 6.5 (5.6 – 7.0) 2.8 (2.4 – 3.2) 3.5 (3.2 – 4.3) 222 (167 – 338) 6.6 (6.2 – 7.3) 2.8 (2.5 – 3.1) 3.7 (3.2 – 4.6) 261 (187 – 375) 6.5 (5.9 – 7.2) 2.7 (2.3 – 3.1) 3.8 (3.3 – 4.6) Serum ferritin (μg/L) Total proteins (g/dL) Albumin (g/dL) Globulins (g/dL)
25.5 (5.1 – 60.9) η, κ 4.3 (2.9 – 4.8) 2.6 (2.2 – 3.2) η, ν 631 (312 – 874) δ, ι, μ 7.9 (6.9 – 8.9) ε, ι, κ 2.2 (1.8 – 2.7) ѱ, θ, μ 5.7 (4.2 – 6.8) ε, ι, λ 5.0 (5.0 – 20.2) 4.1 (2.2 – 4.8) 3.4 (2.9 – 3.9) α
1463 (543 – 3541) ε, ι, ν 129 (72.0 – 198) β, †
13.3 (5.0 – 57.7) 3.9 (2.0 – 4.8) 3.0 (2.4 – 3.7) 5.3 (5.0 – 14.4) 3.6 (1.5 – 4.8) 3.2 (2.7 – 3.8) 13.4 (5.0 – 64.2) 3.9 (2.1 – 4.6) 3.0 (2.4 – 3.5) CRPc(μg/mL) Haptoglobin (g/L) PON-1d(UI/mL)
6.6 (5.0 – 28.6) 3.1 (1.8 – 4.7) 3.0 (2.6 – 3.5)
245 (177 – 587) 7.3 (6.5 – 7.7) Ͼ, Ͽ 2.7 (2.2 – 3.0) 4.3 (3.6 – 5.1) Ͼ
< 12 < 3 3-4.3 (5.0 – 20.2) (1.5 – 4.8) (2.7 – 3.9)
4.4 (3.9 – 5.4) ε, ι 20.3 (5.0 -56.0) κ 4.4 (3.8 – 4.8) ζ 2.8 (2.4 – 3.5) 2.7 Ͻ 5.5 3.7 3.3 0.9 (0.7 – 1.3) 12.1 (8.2 – 25.0) * 4.8 (4.8 – 4.8) Anti-Leishmania antibodies (UFLa; S/P ratiob)
0.3 (0.3 – 0.4)
1 3
4 2 1
2
3
(2.1 – 3.1)
4
Reference valuese Quartiles (ELISA) Quartiles (TR-IFMA) Analyte (units)
Table 1 Values of the different analytes (median and 25th–75th percentiles within parenthesis) of the population study stratified by quartiles of anti-Leishmania antibody levels measured using a time-resolved immunofluorometric assay (TR-IFMA) and a commercially-available ELISA test.
A. Cantos-Barreda et al.
p < 0.001), total proteins (r = 0.481, p < 0.01), albumin (r = -0.548, p < 0.0001) and globulins (r = 0.649, p < 0.0001). The ELISA was correlated with CRP (r = 0.321, p < 0.05), haptoglobin (r = 0.526, p < 0.001), total proteins (r = 0.582, p < 0.001), albumin (r = -0.465, p < 0.01) and globulins (r = 0.679, p < 0.0001). 4. Discussion This study evaluated the possible relationships between two serological assays and some inflammatory biomarkers in 205 canine serum samples. The intention was to assess this relationship in three ways: (1) considering only if the dogs are Leishmania-seronegative or Leishmaniaseropositive, (2) taking into account the different amounts of antiLeishmania antibodies, and (3) considering if this relationship could be influenced if it is studied at the time of diagnosis or during treatment. These results will help to clarify the association between serological and APPs results in the case of CanL. When samples were assigned in positive and negative based on serological assays, no significant differences in the selected biochemical analytes were found between the groups. This presumably reflects the fact that in many cases a positive serology would not imply the existence of an active inflammation, and that the animal would be considered to be a clinically healthy seropositive dog (Solano-Gallego et al., 2016). In the other hand, the presence of a negative serology but changes in APPs may reflect the occurrence of undetected other diseases or infections that may alter the concentration of the biochemical analytes (Cerón et al., 2005). When the relationship between serology and biochemical analytes was studied by quartiles of anti-Leishmania antibodies, it was observed that dogs belonging to the quartile 4 with the highest anti-Leishmania antibody levels showed the highest concentration of total proteins and globulins, the highest median values of positive APPs, and lowest median values of negative APPs in relation to the lower quartiles. Increases in the concentration of total proteins and globulins would be related to augmentation in anti-Leishmania antibody levels since, as immunoglobulins, they are a fraction of the total globulins produced by the individual (Shenton et al., 2015). It is worthy of note that increases in specific antibodies are usually associated with an active disease (Solano-Gallego et al., 2011) and, as a result, abnormalities in the concentration of APPs may be observed as previously reported (Martínez-Subiela et al., 2016; Cantos-Barreda et al., 2018a). In addition, the differences in the biochemical analytes were more significant when TR-IFMA was used compared to the ELISA, in agreement with previous reports (Cantos-Barreda et al., 2017). A higher correlation between serology and biochemical analytes for treatment monitoring was observed using TR-IFMA. A debate exists about the use of serology for treatment monitoring. Some authors suggest that measuring Leishmania-specific antibodies for treatment monitoring of CanL is useful (Solano-Gallego et al., 2016; Segarra et al., 2017) whereas others are of the opposite opinion (Ferrer et al., 1995; Rougier et al., 2012). Our results would suggest that the use of serology for the treatment monitoring of CanL could depend on the assay. For instance, moderate correlations between TR-IFMA and serum ferritin and CRP were found, suggesting that TR-IFMA could be of use for monitoring the treatment of CanL (Cantos-Barreda et al., 2018b). In any case, the correlations between the TR-IFMA and APPs were moderate, and therefore they could not substitute for each other. One limitation of the research reported in this paper is that there was not enough clinical data to evaluate the scores of dogs’ clinical signs. This might prove to be a limitation with regard to the interpretation of the results, as the degree of severity of the disease in relation to biochemical analysis could not be evaluated. 5. Conclusion In conclusion, in a set of serum samples that were evaluated for the 66
Veterinary Parasitology 260 (2018) 63–68
A. Cantos-Barreda et al.
Table 2 Distributions of APPs and analytes related to protein concentration values by quartiles of anti-Leishmania antibody levels measured using the time-resolved immunofluorometric assay (TR-IFMA) and the commercially-available ELISA test for all dogs included in the study (n = 205). Quartile (TR-IFMA) Analyte
CRPa (n = 205) Below Above Haptoglobin (n = 205) Below Above PON-1b (n = 205) Below In range Above Serum ferritin (n = 205) Below In range Above Total proteins (n = 205) Below In range Above Albumin (n = 205) Below In range Above Globulins (n = 205) Below In range Above a b c
Reference valuesc (units)
Quartile (ELISA)
1 No (%)
2 No (%)
3 No (%)
4 No (%)
1 No (%)
2 No (%)
3 No (%)
4 No (%)
23 (45.1) 28 (54.9)
39 (75.0) 13 (25.0)
34 (66.7) 17 (33.3)
19 (37.3) 32 (62.7)
25 (49.0) 26 (51.0)
34 (65.4) 18 (34.6)
34 (66.7) 17 (33.3)
22 (43.1) 29 (56.9)
22 (43.1) 29 (56.9)
21 (40.4) 31 (59.6)
19 (37.3) 32 (62.7)
13 (25.5) 38 (74.5)
20 (39.2) 31 (60.8)
25 (40.1) 27 (51.9)
21 (41.2) 30 (58.8)
9 (17.7) 42 (82.3)
26 (51.0) 24 (47.0) 1 (2.0)
21 (40.4) 29 (55.8) 2 (3.8)
15 (29.4) 30 (58.8) 6 (11.8)
37 (72.5) 11 (21.6) 3 (5.9)
25 (49.0) 23 (45.1) 3 (5.9)
26 (50.0) 24 (46.2) 2 (3.8)
20 (39.2) 28 (54.9) 3 (5.9)
28 (54.9) 19 (37.3) 4 (7.8)
0 (0.0) 13 (25.5) 38 (74.5)
0 (0.0) 17 (32.7) 35 (67.3)
0 (0.0) 15 (29.4) 36 (70.6)
0 (0.0) 7 (13.7) 44 (86.3)
0 (0.0) 16 (31.4) 35 (68.6)
0 (0.0) 12 (23.1) 40 (76.9)
0 (0.0) 14 (27.5) 37 (72.5)
0 (0.0) 10 (19.6) 41 (80.4)
8 (15.7) 30 (58.8) 13 (25.5)
1 (1.9) 36 (69.2) 15 (28.9)
1 (2.0) 26 (51.0) 24 (47.0)
0 (0.0) 16 (31.4) 35 (68.6)
6 (11.8) 33 (64.7) 12 (23.5)
3 (5.8) 37 (71.1) 12 (23.1)
1 (1.9) 23 (45.1) 27 (53.0)
0 (0.0) 15 (29.4) 36 (70.6)
19 (37.3) 32 (62.7) 0 (0.0)
12 (23.1) 39 (75.0) 1 (1.9)
13 (25.5) 38 (74.5) 0 (0.0)
32 (62.8) 18 (35.3) 1 (1.9)
15 (29.4) 35 (68.7) 1 (1.9)
15 (28.8) 37 (71.2) 0 (0.0)
15 (29.4) 36 (70.6) 0 (0.0)
31 (60.8) 19 (37.3) 1 (1.9)
1 (1.9) 27 (53.0) 23 (45.1)
2 (3.8) 28 (53.9) 22 (42.3)
0 (0.0) 18 (35.3) 33 (64.7)
1 (1.9) 6 (11.8) 44 (86.3)
1 (1.9) 33 (64.8) 17 (33.3)
1 (1.9) 26 (50.0) 25 (49.1)
1 (1.9) 15 (29.4) 35 (68.2)
1 (1.9) 5 (9.8) 45 (88.2)
< 12 (μg/mL)
< 3 (g/L)
3-4.3 (UI/mL)
60-190 (μg/L)
5.4-7.2 (g/dL)
2.5-3.6 (g/dL)
2.6-3.8 (g/dL)
C-reactive protein. Paraoxonase 1. Interlab-UMU biochemical laboratory reference values.
initial diagnosis and treatment monitoring of CanL, the APPs and the analytes related to protein concentration did not show significant changes depending on whether the serological assays were positive or negative. However, they showed changes indicating an inflammatory status when the titer was high, and also showed moderate correlations with the TR-IFMA in treatment monitoring. These results may help to clarify the relationship between the measurement of anti-Leishmania antibody levels and the concentration of APPs and analytes related to proteins in CanL.
Acad. Sci. U. S. A. 90, 775–779. Cantos-Barreda, A., Escribano, D., Bernal, L.J., Pardo-Marín, L., Cerón, J.J., MartínezSubiela, S., 2017. New wide dynamic range assays for quantification of antiLeishmania IgG2 and IgA antibodies in canine serum. Vet. Immunol. Immunopathol. 189, 11–16. Cantos-Barreda, A., Escribano, D., Cerón, J.J., Tecles, F., Bernal, L.J., Martínez-subiela, S., 2018a. Changes in the concentration of anti-Leishmania antibodies in saliva of dogs with clinical leishmaniosis after short-term treatment. Vet. Parasitol. 254, 135–141. Cantos-Barreda, A., Escribano, D., Martínez-Subiela, S., Pardo-Marín, L., Segarra, S., Cerón, J.J., 2018b. Changes in serum anti-Leishmania antibody concentrations measured by time-resolved immunofluorometric assays in dogs with leishmaniosis after treatment. Vet. Immunol. Immunopathol. 198, 65–69. Cerón, J.J., Eckersall, P.D., Martínez-Subiela, S., 2005. Acute phase proteins in dogs and cats: current knowledge and future perspectives. Vet. Clin. Pathol. 34, 85–99. Eckersall, P.D., Bell, R., 2010. Acute phase proteins: biomarkers of infection and inflammation in veterinary medicine. Vet. J. 185, 23–27. Ferrer, L., Aisa, M.J., Roura, X., Portús, M., 1995. Serological diagnosis and treatment of canine leishmaniasis. Vet. Rec. 136, 514–516. Kato, H., Uezato, H., Katakura, K., Calvopiña, M., Marco, J.D., Barroso, P.A., Gomez, E.A., Mimori, T., Korenaga, M., Iwata, H., Nonaka, S., Hashiguchi, Y., 2005. Detection and identification of Leishmania species within naturally infected sand flies in the andean areas of ecuador by a polymerase chain reaction. Am. J. Trop. Med. Hyg. 72, 87–93. Maia, C., Campino, L., 2008. Methods for diagnosis of canine leishmaniasis and immune response to infection. Vet. Parasitol. 158, 274–287. Martínez-Subiela, S., Strauss-Ayali, D., Cerón, J.J., Baneth, G., 2011. Acute phase protein response in experimental canine leishmaniosis. Vet. Parasitol. 180, 197–202. Martínez-Subiela, S., Cerón, J.J., Strauss-Ayali, D., García-Martinez, J.D., Tecles, F., Tvarijonaviciute, A., Caldin, M., Baneth, G., 2014. Serum ferritin and paraoxonase-1 in canine leishmaniosis. Comp. Immunol. Microbiol. Infect. Dis. 37, 23–29. Martínez-Subiela, S., Pardo-Marín, L., Tecles, F., Baneth, G., Cerón, J.J., 2016. Serum Creactive protein and ferritin concentrations in dogs undergoing leishmaniosis treatment. Res. Vet. Sci. 109, 17–20. Mettler, M., Grimm, F., Capelli, G., Camp, H., 2005. Evaluation of enzyme-linked immunosorbent assays, an immunofluorescent-antibody test, and two rapid tests and gel tests for serological diagnosis of symptomatic and asymptomatic Leishmania infections in dogs. J. Clin. Microbil. 43, 5515–5519. Muñoz-Prieto, A., Martínez-Subiela, S., Tvarijonaviciute, A., Cerón, J.J., 2017. Use of heterologous immunoassays for quantification of serum proteins: the case of canine C-reactive protein. PLoS One 12, 1–14. Murata, H., Shimada, N., Yoshioka, M., 2004. Current research on acute phase proteins in veterinary diagnosis: an overview. Vet. J. 168, 28–40. Proverbio, D., Spada, E., Giorgi De, G.B., Perego, R., Valena, E., 2014. Relationship
Acknowledgements Ana Cantos Barreda was awarded a pre-doctoral fellowship “FPU” by the University of Murcia, Spain. Damián Escribano held a postdoctoral fellowship “Juan de la Cierva” supported by the “Ministerio de Economía y Competitividad”, Spain. This study was funded by the Program for Research Groups of Excellence of the Seneca Foundation (grant 19894/GERM/15). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.vetpar.2018.08.010. References Baneth, G., Aroch, I., 2008. Canine leishmaniasis: a diagnostic and clinical challenge. Vet. J. 175, 14–15. Barbosa, A., Martins-Filho, O.A., Mayrink, W., Tafuri, W.L., Corre, R., 2009. Systemic and compartmentalized immune response in canine visceral leishmaniasis. Vet. Immunol. Immunopathol. 128, 87–95. Burns, J.M., Shreffler, W.G., Benson, D.R., Ghalib, H.W., Badaro, R., Reed, S.G., 1993. Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis. Proc. Natl.
67
Veterinary Parasitology 260 (2018) 63–68
A. Cantos-Barreda et al.
Solano-Gallego, L., Morell, P., Arboix, M., Alberola, J., Ferrer, L., 2001. Prevalence of Leishmania infantum infection in dogs living in an area of canine leishmaniasis endemicity using PCR on several tissues and serology. J. Clin. Microbiol. 39, 560–563. Solano-Gallego, L., Miró, G., Koutinas, A., Cardoso, L., Pennisi, M.G., Ferrer, L., Bourdeau, P., Oliva, G., Baneth, G., The LeishVet Group, 2011. LeishVet guidelines for the practical management of canine leishmaniosis. Parasit. Vectors 4, 86. Solano-Gallego, L., Di Filippo, L., Ordeix, L., Planellas, M., Roura, X., Altet, L., MartínezOrellana, P., Montserrat, S., 2016. Early reduction of Leishmania infantum-specific antibodies and blood parasitemia during treatment in dogs with moderate or severe disease. Parasit. Vectors 9, 235. Tvarijonaviciute, A., Tecles, F., Caldin, M., Tasca, S., Cerón, J., 2012. Validation of spectrophotometric assays for serum paraoxonase type-1 measurement in dogs. Am. J. Vet. Res. 73, 34–41. Wolf, D., Failing, K., Taubert, A., Pantchev, N., 2014. Serological diagnosis of canine leishmaniosis: comparison of three commercially available tests. Parasitol. Res. 113, 1997–2002.
between Leishmania IFAT titer and clinicopathological manifestations (clinical score) in dogs. Biomed Res. Int. 2014, 412808. Ready, P.D., 2010. Leishmaniasis emergence in Europe. Euro Surveill. 15, 19505. Rougier, S., Hasseine, L., Delaunay, P., Michel, G., Marty, P., 2012. One-year clinical and parasitological follow-up of dogs treated with marbofloxacin for canine leishmaniosis. Vet. Parasitol. 186, 245–253. Segarra, S., Miró, G., Montoya, A., Pardo-Marín, L., Boqué, N., Ferrer, L., Cerón, J., 2017. Randomized, allopurinol-controlled trial of the effects of dietary nucleotides and active hexose correlated compound in the treatment of canine leishmaniosis. Vet. Parasitol. 239, 50–56. Shenton, J.M., Henson, K.L., Rebelatto, M.C., 2015. Consequences and indicators of test article-related immune suppression in nonhuman primates. In: Bluemel, J., Korte, S., Schenck, E., Weinbauer, G. (Eds.), The Nonhuman Primate in the Nonclinical Drug Development and Safety Assessment. Academic Press, Amsterdam, pp. 215–234. Silvestrini, P., Planellas, M., Roura, X., Brien, P.J.O., Pastor, J., 2012. Serum cardiac troponin I concentrations in dogs with leishmaniasis: correlation with age and clinicopathologic abnormalities. Vet. Clin. Pathol. 4, 568–574.
68
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Research paper
Relationship between serum anti-Leishmania antibody levels and acute phase proteins in dogs with canine leishmaniosis
T
⁎
Ana Cantos-Barredaa, Damián Escribanoa, , José J. Ceróna, Luis J. Bernala, Tommaso Furlanellob, Fernando Teclesa, Luis Pardo-Marína, Silvia Martínez-Subielaa a b
Interdisciplinary Laboratory of Clinical Analysis, Interlab-UMU, Campus of Excellence Mare Nostrum, University of Murcia, 30100 Espinardo, Murcia, Spain San Marco Veterinary Clinic, Veggiano, Padova, Italy
A R T I C LE I N FO
A B S T R A C T
Keywords: Canine leishmaniosis Correlation study TR-IFMA ELISA Acute phase proteins Anti-Leishmania antibodies
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 (TRIFMA)) 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.
1. Introduction Canine leishmaniosis (CanL) is an infectious disease in the Mediterranean area caused by Leishmania infantum. It is marked by its zoonotic potential and endemic character (Mettler et al., 2005; Baneth and Aroch, 2008). The spread of CanL depends on the distribution of the vectors and reservoir animals (Kato et al., 2005). Dogs play an important role in the transmission of the disease to humans and other mammals since they are the main reservoirs for sandfly infection (Ready, 2010). CanL ranges from subclinical disease to fatality depending on the host’s immune response (Barbosa et al., 2009). When the immune response is mainly mediated by Th2 lymphocytes, the antibody production is high, leading to the deposition of soluble immune-complexes in
⁎
organs and tissues (Solano-Gallego et al., 2001; Proverbio et al., 2014). This pathogenic reaction is associated with the presence of a great variety of clinical signs, the presence of skin lesions being the most common manifestation in dogs with clinical leishmaniosis. Other clinical signs that may be present are generalized lymphadenomegaly, weight loss, blepharitis, conjunctivitis, epistaxis or polyarthritis, among others (Solano-Gallego et al., 2011). The estimated seroprevalence of CanL in endemic areas is typically 10–30%, depending on where the dogs live and their exposure to infection, as well as on the sensitivity and specificity of the diagnostic tests (Maia and Campino, 2008). A variety of laboratory techniques to detect anti-Leishmania antibodies using quantitative serological techniques have been developed (Wolf et al., 2014). The most commonlyused techniques are the enzyme-linked immunosorbent assay (ELISA)
Corresponding author. E-mail address: [email protected] (D. Escribano).
https://doi.org/10.1016/j.vetpar.2018.08.010 Received 15 May 2018; Received in revised form 22 August 2018; Accepted 23 August 2018 0304-4017/ © 2018 Elsevier B.V. All rights reserved.
Veterinary Parasitology 260 (2018) 63–68
A. Cantos-Barreda et al.
total proteins and globulins; (3) in the case of samples submitted for initial diagnosis of CanL there should be no history of suffering a previous episode of CanL; (4) there should be no history of Leishmania vaccination; and (5) the dogs involved should be free from other diseases. Dogs were allocated into two groups: (1) animals whose samples were submitted to our laboratory for the initial serological diagnosis of CanL and (2) animals whose samples were submitted for treatment monitoring of CanL.
and the immunofluorescence antibody test (IFAT) (Solano-Gallego et al., 2011). Recently, a highly-sensitive time-resolved immunofluorometric assay (TR-IFMA) for the quantification of anti-Leishmania antibodies in canine serum has been developed and validated (CantosBarreda et al., 2017). This TR-IFMA was based on the recombinant antigen K39 (rK39), a repetitive and highly conserved protein among viscerotropic Leishmania spp. (Burns et al., 1993). In addition, this TRIFMA showed a wider difference in anti-Leishmania antibody levels between Leishmania-seropositive and Leishmania-seronegative dogs and between clinical stages, than did a commercially-available ELISA (Cantos-Barreda et al., 2017). Acute phase proteins (APPs) are proteins present in plasma whose concentrations are modified in response to tissue injury, infection or inflammation, and which allow the assessing of the innate immune system of the host. Their quantification provides relevant information in support of clinical diagnosis, prognosis and monitoring response to treatment (Murata et al., 2004; Eckersall and Bell, 2010). APPs can be classified as positive or negative if their levels increase or decrease, respectively, after inflammation. Serum ferritin, C-reactive protein (CRP) and haptoglobin are considered positive APPs, while albumin and paraoxonase-1 (PON-1) are considered negative APPs (Cerón et al., 2005; Martínez-Subiela et al., 2014). Previous studies have reported an acute phase response in dogs with CanL, characterized by variations of APPs with increases in CRP, serum ferritin and haptoglobin (MartínezSubiela et al., 2011; Cantos-Barreda et al., 2018a). Furthermore, the progression of the disease through the determination of the APPs at the time of diagnosis and following treatment, showed an improvement in the concentration of APPs as the clinical manifestations disappeared (Martínez-Subiela et al., 2016). To the authors’ knowledge, there are no studies that correlate antibody levels and APPs in dogs with CanL. The objective of this study was to assess the possible relationship between serum anti-Leishmania antibody levels measured by two different assays and selected biochemical analytes related to inflammation and protein concentration in dogs naturally infected with L. infantum. For this purpose, we evaluated: (1) the possible differences in the concentration of APPs and analytes related to proteins between Leishmania-seronegative and Leishmania-seropositive dogs; (2) the values of these analytes in the different quartiles of anti-Leishmania antibodies; and, finally, (3) the possible differences in the correlations between antibody levels and the biochemical analytes evaluated, depending on whether they are measured at the time of diagnosis or during treatment monitoring.
2.3. Sampling Blood samples were obtained at different clinics using venipuncture of the cephalic or jugular vein, and were collected in tubes containing a coagulation activator and a gel separator. Tubes were allowed to clot at room temperature and centrifuged at 3500 rpm for 5 min. The serum was separated and sent the same day to the Interdisciplinary Laboratory of Clinical Analysis (Interlab-UMU, University of Murcia, Spain). Biochemical analytes included in the study were analyzed on arrival. Samples were stored at -80 °C until analyzed for antibody titers. Such an analysis was undertaken in the lab once a week. 2.4. Anti-Leishmania antibodies In order to determine the level of anti-Leishmania antibodies in the serum samples two different quantitative methods, a commerciallyavailable ELISA test and a TR-IFMA, were employed. The ELISA was performed following manufacturer’s instructions using the recommended 1/20 dilution for serum samples and results were expressed as sample-to-positive (S/P) ratio calculated as optical density (OD) sample/OD low positive control. TR-IFMA was performed as previously described (Cantos-Barreda et al., 2017) and samples were diluted 1/4000 in an assay buffer. The results were expressed as Units of Fluorometry for Leishmania (UFL). 2.5. Biochemical analytes CRP concentration was measured using a human immunoturbidimetric test (CRP OSR 6147 Olympus Life and Material Science Europe GmbH, Lismeehan, O'Callaghan Mills, Co. Clare, Ireland) used previously in dogs (Muñoz-Prieto et al., 2017). Haptoglobin was determined using a hemoglobin-binding method (Tridelta Phase, Tridelta Development Ltd., Bray, Ireland) in a biochemistry autoanalyzer (Cobas Mira Plus, ABX Diagnostics, Montpellier, France). PON-1 activity was analyzed following a previously described method (Tvarijonaviciute et al., 2012). Ferritin concentrations were measured using an immunoturbidimetric assay with polyclonal anti-human ferritin antibodies (Tina-quant Ferritin, Boehringer Mannheim, Germany) previously validated for use in dogs (Martínez-Subiela et al., 2014). Serum total protein and albumin concentrations were measured using a human colorimetric assay (Total protein OSR 6132, Albumin OSR 6102, Olympus Life and Material Science Europe GmbH, Hamburg, Germany). Globulin concentrations were calculated by the difference between total proteins and albumin concentrations as previously described in dogs with CanL (Silvestrini et al., 2012). Determinations of CRP, total proteins and albumin concentrations were performed using an automated biochemistry analyzer (Olympus AU600; Olympus Diagnostica GmbH, Hamburg, Germany).
2. Materials and methods 2.1. Ethics statement This study was approved by the Local Ethical Committee of the University of Murcia under protocol no. 36/2014. All procedures were conducted in accordance with law RD 53/2013 regarding animal experimentation in Spain and with the European Directive 2010/63/EU concerning the protection of animals used for scientific purposes. 2.2. Animals and study design Samples from 205 dogs were included in the study. These samples were submitted to our laboratory from different veterinary clinics throughout Spain in order to measure anti-Leishmania antibody levels and APPs. A set of criteria were taken into consideration when it came to accepting the samples for this study: (1) they had to have undergone the quantification of anti-Leishmania antibodies by a commerciallyavailable ELISA (Leiscan® Leishmania ELISA Test, Esteve Veterinaria, Laboratorios Dr. Esteve SA, Barcelona, Spain) and the TR-IFMA between January 2016 and January 2018; (2) the serological measurement had to be accompanied by the analysis of a panel of inflammatory analytes including CRP, haptoglobin, serum ferritin, PON-1, albumin,
2.6. Statistical analysis All data were assessed for normality of distribution using the Kolmogorov-Smirnov normality test. Descriptive statistics for the different groups were determined using the non-parametric Kruskal-Wallis test. To compare the TR-IFMA and the ELISA results, and to compare the serological results and the biochemical analytes values, a 64
Veterinary Parasitology 260 (2018) 63–68
A. Cantos-Barreda et al.
correlation is described by Spearman’s rank correlation coefficient (rs). Median values were given with regard to the respective 25th–75th percentiles. Antibody levels determined by TR-IFMA and ELISA were parameterized as a dummy variable comparing each quartile with each other. The significance level of p < 0.05 was chosen. All analyses were conducted using the GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA). 3. Results 3.1. Animals A total of 205 dogs were included in this study. One hundred and sixty-one dogs (78.5%) were included in the group of samples for serological diagnosis of CanL because these cases were submitted for initial diagnosis. Forty-four dogs (21.5%) were included in the group of samples submitted for treatment monitoring because these dogs were previously diagnosed with CanL and were receiving anti-leishmanial treatment. Additionally, all animals were allocated by quartiles of antiLeishmania antibodies for each serological assay. The ages of the dogs included in the study ranged from 7 months to 14.5 years, with a mean ± SD of 6.8 ± 3.4 years. Among the recruited dogs, 100 were male and 99 were female. The sex data from 6 dogs were not specified in their signalment. Purebred dogs were predominant in this study, with a total of 125 dogs, mainly represented by the German shepherd (14), Labrador retriever (13), French bulldog (8) and Boxer (6) breeds. Seventy-one crossbred dogs were also included in the study. Breed data from 9 dogs were not found in their signalment.
Fig. 1. Concentration of the selected biochemical analytes regarding the timeresolved immunofluorometric assay (TR-IFMA) and the commercially-available ELISA test (Leiscan® Leishmania ELISA Test) classification as Leishmania-seronegative and Leishmania-seropositive. The box plot shows median (horizontal line within box), 25th and 75th percentiles (box) and minimum and maximum values (whiskers). TP: total proteins (g/dL); Glob: globulins (mg/dL); Alb: albumin (g/dL); CRP: C-reactive protein (μg/mL); SF: serum ferritin (μg/L); PON1: paraoxonase-1 (UI/mL); Hp: haptoglobin (g/L). NS: not significant.
what is shown in Fig. 1 for the Leishmania-seronegative and Leishmaniaseropositive groups, significant differences between quartiles were observed. Animals belonging to the quartile 4, with higher levels of antiLeishmania antibodies, showed more pronounced changes in the biochemical analytes, with significant increases in total proteins, CRP, serum ferritin, haptoglobin and globulins, and significant decreases in albumin and PON-1 compared with other quartiles. CRP, PON-1, serum ferritin and albumin showed greater significant differences between quartiles when TR-IFMA was used compared with the ELISA. The distribution of cases by quartile of anti-Leishmania antibodies in which there was an increase, decrease or normal value of the different biochemical analytes is shown in Table 2. The higher number of abnormal values of the biochemical analytes correspond to quartile 4 of antibodies.
3.2. Relationship between Leishmania-seronegative and Leishmaniaseropositive dogs and the selected biochemical analytes Data were classified regarding the serological status and the clinical test cut-offs into two different groups: Leishmania-seronegative and Leishmania-seropositive. The cut-off between a negative and a positive result was established at 22 UFL and at 0.9 S/P ratio for TR-IFMA and ELISA, respectively. Samples close to the threshold were included in the Leishmania-seropositive group in both assays. The anti-Leishmania antibody levels and concentrations of APPs and analytes related to protein concentration in the Leishmania-seronegative and the Leishmania-seropositive groups were evaluated. Significant differences (p < 0.0001) were observed in anti-Leishmania antibody levels in the seropositive groups with respect to the seronegative groups. The seropositive groups showed a median value of 235 IgG2 UFL (25th–75th percentiles: 77.5–1014) and a median value of 3.2 S/P ratio (1.9–4.1) while the seronegative groups showed a median value of 4.9 IgG2 UFL (4.8–8.9) and a median value of 0.5 S/P ratio (0.3–0.8). However, no differences were detected in the concentration of the selected biochemical analytes between seronegative and seropositive dogs (Fig. 1).
3.4. Relationship between serology at diagnosis or during treatment monitoring and the selected biochemical analytes Three correlation studies were carried out separately: one for all the samples included in the study, another for the samples belonging to the diagnostic group, and another for the samples belonging to the treatment monitoring group. The correlation study performed with the totality of data revealed that IgG2 UFL were significantly correlated with the S/P ratio (Spearman rank r = 0.768; p < 0.0001). Both serological assays showed positive correlations with total proteins, globulins and serum ferritin and negative correlations with albumin. However, it is interesting to note that these correlations were more significant with regard to TR-IFMA. In the correlation study with regard to the diagnostic group both serological assays showed a significant positive correlation (r = 0.769, p < 0.0001). The TR-IFMA was significantly correlated with serum ferritin (r = 0.338, p < 0.0001), total proteins (r = 0.458, p < 0.0001), albumin (r = -0.168, p < 0.05), globulins (r = 0.436, p < 0.0001) and haptoglobin (r = 0.161, p < 0.05). The ELISA was significantly correlated with serum ferritin (r = 0.303, p < 0.0001), total proteins (r = 0.502, p < 0.0001), albumin (r = -0.234, p < 0.01) and globulins (r = 0.504, p < 0.0001). For the treatment monitoring group, TR-IFMA and the ELISA showed a correlation of r = 0.531 (p < 0.0001). The TR-IFMA was correlated with CRP (r = 0.563, p < 0.0001), haptoglobin (r = 0.311, p < 0.05), PON-1 (r = -0.381, p < 0.05), serum ferritin (r = 0.519,
3.3. Relationship between the magnitude of serology values and the selected biochemical analytes Table 1 shows the median, 25th–75th percentiles and the statistical differences for anti-Leishmania antibodies and the biochemical analytes among the different quartiles of anti-Leishmania antibodies for each serological assay. Quartiles were broken at 6.47, 40.12 and 304.9 UFL for TR-IFMA, and 0.50, 1.68 and 3.58 S/P ratio for ELISA. 88 and 78 dogs were considered seronegative according to the cut-offs for TRIFMA and ELISA, respectively. Significant differences in anti-Leishmania antibody levels were observed between quartiles 1 and 2 (p < 0.05), 1 and 3 (p < 0.0001), 2 and 3 (p < 0.0001), 1 and 4 (p < 0.0001), 2 and 4 (p < 0.0001) and 3 and 4 (p < 0.0001) for TR-IFMA. For the ELISA, significant differences were found between quartiles 1 and 3 (p < 0.001), 1 and 4 (p < 0.0001) and 2 and 4 (p < 0.0001). Related to the concentration of the selected biochemical analytes, contrary to 65
Veterinary Parasitology 260 (2018) 63–68
241 (168 – 498) 7.2 (6.5 – 7.5) α 2.9 (2.4 – 3.1) 4.2 (3.6 – 5.0)
*p < 0.05 comparing quartile 1 and 2; α p < 0.05 comparing quartile 1 and 3; Ͼ p < 0.01 comparing quartile 1 and 3; Ͻ p < 0.001 comparing quartile 1 and 3; β p < 0.0001 comparing quartile 1 and 3; ѱ p < 0.05 comparing quartiles 1 and 4; γ p < 0.01 comparing quartile 1 and 4; δ p < 0.001 comparing quartile 1 and 4; ε p < 0.0001 comparing quartile 1 and 4; Ͽ p < 0.05 comparing quartile 2 and 3; † p < 0.0001 comparing quartile 2 and 3; ζ p < 0.05 comparing quartile 2 and 4; η p < 0.01 comparing quartile 2 and 4; θ p < 0.001 comparing quartile 2 and 4; ι p < 0.0001 comparing quartile 2 and 4; κ p < 0.05 comparing quartile 3 and 4; λ p < 0.01 comparing quartile 3 and 4; μ p < 0.001 comparing quartile 3 and 4; ν p < 0.0001 comparing quartile 3 and 4. a Units of Fluorometry for Leishmania. b Sample-to-positive ratio. c C-reactive protein. d Paraoxonase 1. e Interlab-UMU biochemical laboratory reference values.
60-190 5.4-7.2 2.5-3.6 2.6-3.8 631 (203 – 866) γ, ζ 7.9 (7.0 – 8.4) ε, ι 2.1 (1.8 – 2.8) γ, η, κ 5.5 (4.6 – 6.5) ε, ι, λ 261 (197 – 377) 6.6 (6.2 – 7.1) 2.7 (2.5 – 3.0) 3.7 (3.5 – 4.4) 224 (166 – 443) 6.5 (5.6 – 7.0) 2.8 (2.4 – 3.2) 3.5 (3.2 – 4.3) 222 (167 – 338) 6.6 (6.2 – 7.3) 2.8 (2.5 – 3.1) 3.7 (3.2 – 4.6) 261 (187 – 375) 6.5 (5.9 – 7.2) 2.7 (2.3 – 3.1) 3.8 (3.3 – 4.6) Serum ferritin (μg/L) Total proteins (g/dL) Albumin (g/dL) Globulins (g/dL)
25.5 (5.1 – 60.9) η, κ 4.3 (2.9 – 4.8) 2.6 (2.2 – 3.2) η, ν 631 (312 – 874) δ, ι, μ 7.9 (6.9 – 8.9) ε, ι, κ 2.2 (1.8 – 2.7) ѱ, θ, μ 5.7 (4.2 – 6.8) ε, ι, λ 5.0 (5.0 – 20.2) 4.1 (2.2 – 4.8) 3.4 (2.9 – 3.9) α
1463 (543 – 3541) ε, ι, ν 129 (72.0 – 198) β, †
13.3 (5.0 – 57.7) 3.9 (2.0 – 4.8) 3.0 (2.4 – 3.7) 5.3 (5.0 – 14.4) 3.6 (1.5 – 4.8) 3.2 (2.7 – 3.8) 13.4 (5.0 – 64.2) 3.9 (2.1 – 4.6) 3.0 (2.4 – 3.5) CRPc(μg/mL) Haptoglobin (g/L) PON-1d(UI/mL)
6.6 (5.0 – 28.6) 3.1 (1.8 – 4.7) 3.0 (2.6 – 3.5)
245 (177 – 587) 7.3 (6.5 – 7.7) Ͼ, Ͽ 2.7 (2.2 – 3.0) 4.3 (3.6 – 5.1) Ͼ
< 12 < 3 3-4.3 (5.0 – 20.2) (1.5 – 4.8) (2.7 – 3.9)
4.4 (3.9 – 5.4) ε, ι 20.3 (5.0 -56.0) κ 4.4 (3.8 – 4.8) ζ 2.8 (2.4 – 3.5) 2.7 Ͻ 5.5 3.7 3.3 0.9 (0.7 – 1.3) 12.1 (8.2 – 25.0) * 4.8 (4.8 – 4.8) Anti-Leishmania antibodies (UFLa; S/P ratiob)
0.3 (0.3 – 0.4)
1 3
4 2 1
2
3
(2.1 – 3.1)
4
Reference valuese Quartiles (ELISA) Quartiles (TR-IFMA) Analyte (units)
Table 1 Values of the different analytes (median and 25th–75th percentiles within parenthesis) of the population study stratified by quartiles of anti-Leishmania antibody levels measured using a time-resolved immunofluorometric assay (TR-IFMA) and a commercially-available ELISA test.
A. Cantos-Barreda et al.
p < 0.001), total proteins (r = 0.481, p < 0.01), albumin (r = -0.548, p < 0.0001) and globulins (r = 0.649, p < 0.0001). The ELISA was correlated with CRP (r = 0.321, p < 0.05), haptoglobin (r = 0.526, p < 0.001), total proteins (r = 0.582, p < 0.001), albumin (r = -0.465, p < 0.01) and globulins (r = 0.679, p < 0.0001). 4. Discussion This study evaluated the possible relationships between two serological assays and some inflammatory biomarkers in 205 canine serum samples. The intention was to assess this relationship in three ways: (1) considering only if the dogs are Leishmania-seronegative or Leishmaniaseropositive, (2) taking into account the different amounts of antiLeishmania antibodies, and (3) considering if this relationship could be influenced if it is studied at the time of diagnosis or during treatment. These results will help to clarify the association between serological and APPs results in the case of CanL. When samples were assigned in positive and negative based on serological assays, no significant differences in the selected biochemical analytes were found between the groups. This presumably reflects the fact that in many cases a positive serology would not imply the existence of an active inflammation, and that the animal would be considered to be a clinically healthy seropositive dog (Solano-Gallego et al., 2016). In the other hand, the presence of a negative serology but changes in APPs may reflect the occurrence of undetected other diseases or infections that may alter the concentration of the biochemical analytes (Cerón et al., 2005). When the relationship between serology and biochemical analytes was studied by quartiles of anti-Leishmania antibodies, it was observed that dogs belonging to the quartile 4 with the highest anti-Leishmania antibody levels showed the highest concentration of total proteins and globulins, the highest median values of positive APPs, and lowest median values of negative APPs in relation to the lower quartiles. Increases in the concentration of total proteins and globulins would be related to augmentation in anti-Leishmania antibody levels since, as immunoglobulins, they are a fraction of the total globulins produced by the individual (Shenton et al., 2015). It is worthy of note that increases in specific antibodies are usually associated with an active disease (Solano-Gallego et al., 2011) and, as a result, abnormalities in the concentration of APPs may be observed as previously reported (Martínez-Subiela et al., 2016; Cantos-Barreda et al., 2018a). In addition, the differences in the biochemical analytes were more significant when TR-IFMA was used compared to the ELISA, in agreement with previous reports (Cantos-Barreda et al., 2017). A higher correlation between serology and biochemical analytes for treatment monitoring was observed using TR-IFMA. A debate exists about the use of serology for treatment monitoring. Some authors suggest that measuring Leishmania-specific antibodies for treatment monitoring of CanL is useful (Solano-Gallego et al., 2016; Segarra et al., 2017) whereas others are of the opposite opinion (Ferrer et al., 1995; Rougier et al., 2012). Our results would suggest that the use of serology for the treatment monitoring of CanL could depend on the assay. For instance, moderate correlations between TR-IFMA and serum ferritin and CRP were found, suggesting that TR-IFMA could be of use for monitoring the treatment of CanL (Cantos-Barreda et al., 2018b). In any case, the correlations between the TR-IFMA and APPs were moderate, and therefore they could not substitute for each other. One limitation of the research reported in this paper is that there was not enough clinical data to evaluate the scores of dogs’ clinical signs. This might prove to be a limitation with regard to the interpretation of the results, as the degree of severity of the disease in relation to biochemical analysis could not be evaluated. 5. Conclusion In conclusion, in a set of serum samples that were evaluated for the 66
Veterinary Parasitology 260 (2018) 63–68
A. Cantos-Barreda et al.
Table 2 Distributions of APPs and analytes related to protein concentration values by quartiles of anti-Leishmania antibody levels measured using the time-resolved immunofluorometric assay (TR-IFMA) and the commercially-available ELISA test for all dogs included in the study (n = 205). Quartile (TR-IFMA) Analyte
CRPa (n = 205) Below Above Haptoglobin (n = 205) Below Above PON-1b (n = 205) Below In range Above Serum ferritin (n = 205) Below In range Above Total proteins (n = 205) Below In range Above Albumin (n = 205) Below In range Above Globulins (n = 205) Below In range Above a b c
Reference valuesc (units)
Quartile (ELISA)
1 No (%)
2 No (%)
3 No (%)
4 No (%)
1 No (%)
2 No (%)
3 No (%)
4 No (%)
23 (45.1) 28 (54.9)
39 (75.0) 13 (25.0)
34 (66.7) 17 (33.3)
19 (37.3) 32 (62.7)
25 (49.0) 26 (51.0)
34 (65.4) 18 (34.6)
34 (66.7) 17 (33.3)
22 (43.1) 29 (56.9)
22 (43.1) 29 (56.9)
21 (40.4) 31 (59.6)
19 (37.3) 32 (62.7)
13 (25.5) 38 (74.5)
20 (39.2) 31 (60.8)
25 (40.1) 27 (51.9)
21 (41.2) 30 (58.8)
9 (17.7) 42 (82.3)
26 (51.0) 24 (47.0) 1 (2.0)
21 (40.4) 29 (55.8) 2 (3.8)
15 (29.4) 30 (58.8) 6 (11.8)
37 (72.5) 11 (21.6) 3 (5.9)
25 (49.0) 23 (45.1) 3 (5.9)
26 (50.0) 24 (46.2) 2 (3.8)
20 (39.2) 28 (54.9) 3 (5.9)
28 (54.9) 19 (37.3) 4 (7.8)
0 (0.0) 13 (25.5) 38 (74.5)
0 (0.0) 17 (32.7) 35 (67.3)
0 (0.0) 15 (29.4) 36 (70.6)
0 (0.0) 7 (13.7) 44 (86.3)
0 (0.0) 16 (31.4) 35 (68.6)
0 (0.0) 12 (23.1) 40 (76.9)
0 (0.0) 14 (27.5) 37 (72.5)
0 (0.0) 10 (19.6) 41 (80.4)
8 (15.7) 30 (58.8) 13 (25.5)
1 (1.9) 36 (69.2) 15 (28.9)
1 (2.0) 26 (51.0) 24 (47.0)
0 (0.0) 16 (31.4) 35 (68.6)
6 (11.8) 33 (64.7) 12 (23.5)
3 (5.8) 37 (71.1) 12 (23.1)
1 (1.9) 23 (45.1) 27 (53.0)
0 (0.0) 15 (29.4) 36 (70.6)
19 (37.3) 32 (62.7) 0 (0.0)
12 (23.1) 39 (75.0) 1 (1.9)
13 (25.5) 38 (74.5) 0 (0.0)
32 (62.8) 18 (35.3) 1 (1.9)
15 (29.4) 35 (68.7) 1 (1.9)
15 (28.8) 37 (71.2) 0 (0.0)
15 (29.4) 36 (70.6) 0 (0.0)
31 (60.8) 19 (37.3) 1 (1.9)
1 (1.9) 27 (53.0) 23 (45.1)
2 (3.8) 28 (53.9) 22 (42.3)
0 (0.0) 18 (35.3) 33 (64.7)
1 (1.9) 6 (11.8) 44 (86.3)
1 (1.9) 33 (64.8) 17 (33.3)
1 (1.9) 26 (50.0) 25 (49.1)
1 (1.9) 15 (29.4) 35 (68.2)
1 (1.9) 5 (9.8) 45 (88.2)
< 12 (μg/mL)
< 3 (g/L)
3-4.3 (UI/mL)
60-190 (μg/L)
5.4-7.2 (g/dL)
2.5-3.6 (g/dL)
2.6-3.8 (g/dL)
C-reactive protein. Paraoxonase 1. Interlab-UMU biochemical laboratory reference values.
initial diagnosis and treatment monitoring of CanL, the APPs and the analytes related to protein concentration did not show significant changes depending on whether the serological assays were positive or negative. However, they showed changes indicating an inflammatory status when the titer was high, and also showed moderate correlations with the TR-IFMA in treatment monitoring. These results may help to clarify the relationship between the measurement of anti-Leishmania antibody levels and the concentration of APPs and analytes related to proteins in CanL.
Acad. Sci. U. S. A. 90, 775–779. Cantos-Barreda, A., Escribano, D., Bernal, L.J., Pardo-Marín, L., Cerón, J.J., MartínezSubiela, S., 2017. New wide dynamic range assays for quantification of antiLeishmania IgG2 and IgA antibodies in canine serum. Vet. Immunol. Immunopathol. 189, 11–16. Cantos-Barreda, A., Escribano, D., Cerón, J.J., Tecles, F., Bernal, L.J., Martínez-subiela, S., 2018a. Changes in the concentration of anti-Leishmania antibodies in saliva of dogs with clinical leishmaniosis after short-term treatment. Vet. Parasitol. 254, 135–141. Cantos-Barreda, A., Escribano, D., Martínez-Subiela, S., Pardo-Marín, L., Segarra, S., Cerón, J.J., 2018b. Changes in serum anti-Leishmania antibody concentrations measured by time-resolved immunofluorometric assays in dogs with leishmaniosis after treatment. Vet. Immunol. Immunopathol. 198, 65–69. Cerón, J.J., Eckersall, P.D., Martínez-Subiela, S., 2005. Acute phase proteins in dogs and cats: current knowledge and future perspectives. Vet. Clin. Pathol. 34, 85–99. Eckersall, P.D., Bell, R., 2010. Acute phase proteins: biomarkers of infection and inflammation in veterinary medicine. Vet. J. 185, 23–27. Ferrer, L., Aisa, M.J., Roura, X., Portús, M., 1995. Serological diagnosis and treatment of canine leishmaniasis. Vet. Rec. 136, 514–516. Kato, H., Uezato, H., Katakura, K., Calvopiña, M., Marco, J.D., Barroso, P.A., Gomez, E.A., Mimori, T., Korenaga, M., Iwata, H., Nonaka, S., Hashiguchi, Y., 2005. Detection and identification of Leishmania species within naturally infected sand flies in the andean areas of ecuador by a polymerase chain reaction. Am. J. Trop. Med. Hyg. 72, 87–93. Maia, C., Campino, L., 2008. Methods for diagnosis of canine leishmaniasis and immune response to infection. Vet. Parasitol. 158, 274–287. Martínez-Subiela, S., Strauss-Ayali, D., Cerón, J.J., Baneth, G., 2011. Acute phase protein response in experimental canine leishmaniosis. Vet. Parasitol. 180, 197–202. Martínez-Subiela, S., Cerón, J.J., Strauss-Ayali, D., García-Martinez, J.D., Tecles, F., Tvarijonaviciute, A., Caldin, M., Baneth, G., 2014. Serum ferritin and paraoxonase-1 in canine leishmaniosis. Comp. Immunol. Microbiol. Infect. Dis. 37, 23–29. Martínez-Subiela, S., Pardo-Marín, L., Tecles, F., Baneth, G., Cerón, J.J., 2016. Serum Creactive protein and ferritin concentrations in dogs undergoing leishmaniosis treatment. Res. Vet. Sci. 109, 17–20. Mettler, M., Grimm, F., Capelli, G., Camp, H., 2005. Evaluation of enzyme-linked immunosorbent assays, an immunofluorescent-antibody test, and two rapid tests and gel tests for serological diagnosis of symptomatic and asymptomatic Leishmania infections in dogs. J. Clin. Microbil. 43, 5515–5519. Muñoz-Prieto, A., Martínez-Subiela, S., Tvarijonaviciute, A., Cerón, J.J., 2017. Use of heterologous immunoassays for quantification of serum proteins: the case of canine C-reactive protein. PLoS One 12, 1–14. Murata, H., Shimada, N., Yoshioka, M., 2004. Current research on acute phase proteins in veterinary diagnosis: an overview. Vet. J. 168, 28–40. Proverbio, D., Spada, E., Giorgi De, G.B., Perego, R., Valena, E., 2014. Relationship
Acknowledgements Ana Cantos Barreda was awarded a pre-doctoral fellowship “FPU” by the University of Murcia, Spain. Damián Escribano held a postdoctoral fellowship “Juan de la Cierva” supported by the “Ministerio de Economía y Competitividad”, Spain. This study was funded by the Program for Research Groups of Excellence of the Seneca Foundation (grant 19894/GERM/15). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.vetpar.2018.08.010. References Baneth, G., Aroch, I., 2008. Canine leishmaniasis: a diagnostic and clinical challenge. Vet. J. 175, 14–15. Barbosa, A., Martins-Filho, O.A., Mayrink, W., Tafuri, W.L., Corre, R., 2009. Systemic and compartmentalized immune response in canine visceral leishmaniasis. Vet. Immunol. Immunopathol. 128, 87–95. Burns, J.M., Shreffler, W.G., Benson, D.R., Ghalib, H.W., Badaro, R., Reed, S.G., 1993. Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis. Proc. Natl.
67
Veterinary Parasitology 260 (2018) 63–68
A. Cantos-Barreda et al.
Solano-Gallego, L., Morell, P., Arboix, M., Alberola, J., Ferrer, L., 2001. Prevalence of Leishmania infantum infection in dogs living in an area of canine leishmaniasis endemicity using PCR on several tissues and serology. J. Clin. Microbiol. 39, 560–563. Solano-Gallego, L., Miró, G., Koutinas, A., Cardoso, L., Pennisi, M.G., Ferrer, L., Bourdeau, P., Oliva, G., Baneth, G., The LeishVet Group, 2011. LeishVet guidelines for the practical management of canine leishmaniosis. Parasit. Vectors 4, 86. Solano-Gallego, L., Di Filippo, L., Ordeix, L., Planellas, M., Roura, X., Altet, L., MartínezOrellana, P., Montserrat, S., 2016. Early reduction of Leishmania infantum-specific antibodies and blood parasitemia during treatment in dogs with moderate or severe disease. Parasit. Vectors 9, 235. Tvarijonaviciute, A., Tecles, F., Caldin, M., Tasca, S., Cerón, J., 2012. Validation of spectrophotometric assays for serum paraoxonase type-1 measurement in dogs. Am. J. Vet. Res. 73, 34–41. Wolf, D., Failing, K., Taubert, A., Pantchev, N., 2014. Serological diagnosis of canine leishmaniosis: comparison of three commercially available tests. Parasitol. Res. 113, 1997–2002.
between Leishmania IFAT titer and clinicopathological manifestations (clinical score) in dogs. Biomed Res. Int. 2014, 412808. Ready, P.D., 2010. Leishmaniasis emergence in Europe. Euro Surveill. 15, 19505. Rougier, S., Hasseine, L., Delaunay, P., Michel, G., Marty, P., 2012. One-year clinical and parasitological follow-up of dogs treated with marbofloxacin for canine leishmaniosis. Vet. Parasitol. 186, 245–253. Segarra, S., Miró, G., Montoya, A., Pardo-Marín, L., Boqué, N., Ferrer, L., Cerón, J., 2017. Randomized, allopurinol-controlled trial of the effects of dietary nucleotides and active hexose correlated compound in the treatment of canine leishmaniosis. Vet. Parasitol. 239, 50–56. Shenton, J.M., Henson, K.L., Rebelatto, M.C., 2015. Consequences and indicators of test article-related immune suppression in nonhuman primates. In: Bluemel, J., Korte, S., Schenck, E., Weinbauer, G. (Eds.), The Nonhuman Primate in the Nonclinical Drug Development and Safety Assessment. Academic Press, Amsterdam, pp. 215–234. Silvestrini, P., Planellas, M., Roura, X., Brien, P.J.O., Pastor, J., 2012. Serum cardiac troponin I concentrations in dogs with leishmaniasis: correlation with age and clinicopathologic abnormalities. Vet. Clin. Pathol. 4, 568–574.
68