Martínez‑Orellana et al. Parasites Vectors https://doi.org/10.1186/s13071-019-3384-0 (2019) 12:119 Parasites & Vectors RESEARCH Open Access Total serum IgD from healthy and sick dogs with leishmaniosis Pamela Martínez‑Orellana1, Cristina Maristany1, Marta Baxarias1, Alejandra Álvarez‑Fernández1, Antonella Baldassarre2, Laura Ordeix1,3 and Laia Solano‑Gallego1* Abstract Background: Canine leishmaniosis (CanL) due to Leishmania infantum is characterized by the development of both cellular and humoral immune responses. The dysfunction of T cell-mediated immunity leads to a lack of proliferation of T cells in response to Leishmania antigens with the consequence of parasite dissemination that seems to be related to a T cell exhaustion mediated by regulatory B cells expressing immunoglobulin D (IgD). The aim of this study was to determine and compare the total serum IgD in dogs with clinical leishmaniosis and in clinically healthy dogs. Results: A total of 147 dog sera were studied. All dogs were tested for L. infantum-specific antibodies by quantita‑ tive ELISA. Interferon-gamma (IFN-γ) production was also determined by sandwich ELISA after blood stimulation with L. infantum soluble antigen (LSA) or concanavalin A (ConA). The quantification of total IgD was performed using a human IgD sandwich ELISA quantification set. Dogs were classified in three different groups. Group 1 included 40 clinically healthy non-infected dogs, all serologically negative to L. infantum-specific antibodies and non-producers of IFN-γ upon LSA stimulation. Group 2 included 63 clinically healthy infected dogs that were LSA IFN-γ producers (n = 61) and/or IFN-γ non-producers (n = 2) as well as negative to medium seropositive to L. infantum antigen. Finally, Group 3 included 44 dogs with clinical leishmaniosis (IFN-γ producers, n = 23; and IFN-γ non-producers, n = 21) that were negative to highly positive to L. infantum-specific antibodies. No significant differences were observed when the total IgD concentration was compared within groups. Additionally, total IgD of sick IFN-γ producers and IFN-γ non- producers was not significantly different. Finally, total IgD concentration was not statistically related to demographic parameters such as age, sex and breed. Conclusions: The results of this study demonstrated that there were no differences between groups in total serum IgD. Total serum IgD does not appear to be a marker of disease in CanL. Keywords: Leishmania infantum, Canine leishmaniosis, IFN-γ, IL-10, IgD, PD-1 Background of L. infantum promastigotes, both innate and adap- Canine leishmaniosis (CanL) is a vector-borne disease tive immune responses occur and their balance plays caused by the protozoan Leishmania infantum, which an important role in the outcome of this infection [4]. is mainly transmitted from dog to dog by female sand The adaptive response includes the activation of B and flies of the genus Phlebotomus in Europe [1] and Lutzo- T lymphocytes that are always present during Leishma- myia spp. in the New World [2]. The clinical manifesta- nia infection. B lymphocytes induce a humoral response tions of this infection are strongly associated with the with exacerbated antibodies production that seems to immune responses developed [3]. After the inoculation be related to a T cell exhaustion resulting in failure to control the infection, meanwhile T lymphocytes play an important role in the development of a cellular response. *Correspondence: laia.solano@uab.cat Dogs with severe disease have a very strong humoral 1 Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, response together with a reduction or absence of the Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain Full list of author information is available at the end of the article T cell-mediated immunity [1]. © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Martínez‑Orellana et al. Parasites Vectors (2019) 12:119 Page 2 of 9 Leishmania infantum infection does not always indi- sick dogs have high levels of I­gM+/IgD+ and I­gM-/IgD+ cate the development of a clinical disease [5]. This and low levels of ­IgM+/IgD- [17]. The population of ­IgD+ infection can manifest as a chronic subclinical infec- B cells increases with disease progression that conduces tion, self-limited disease or a severe fatal disease. Dogs to IL-10 release and an increase of the inhibitory receptor that develop clinical disease are divided into four clini- PD-1, leading to T cell function suppression and to cel- cal stages according to clinical signs, clinicopathological lular exhaustion. Furthermore, these cells induce other B abnormalities and the levels of specific antibodies [6, 7]. and T lymphocytes to produce IL-10 and supress IFN-γ Moreover, dogs with mild to moderate clinical stages pre- through PD-1 [17]. In addition, a higher concentration of sent higher interferon-gamma (IFN-γ) production, low total serum IgD in L. infantum infected sick dogs when parasite load and antibody response when compared to compared with healthy non-infected dogs was demon- the more severe stages [8, 9]. strated [17]. However, a very limited number of dogs Interestingly, some breeds appear to be more likely to were investigated [17]. We hypothesized that dogs with control infection while other breeds are more suscep- moderate to severe disease with a high antibody response tible. For example, the Ibizan Hound rarely develops and absence of production of L. infantum specific IFN-γ clinical disease [10] in contrast breeds such as bulldogs, in ex vivo blood [8] might have a higher concentration of dobermanns [11], German shepherds, rottweilers, boxers serum total IgD when compared with sick dogs with mild or cocker spaniels commonly develop clinical leishmani- to moderate disease but able to produce parasite specific osis [1, 12]. Other factors that might contribute in the IFN-γ and when compared with clinically healthy dogs as progression of clinical leishmaniosis are sex, nutritional previously described [17]. Therefore, the aim of this study state, virulence of Leishmania, parasitic load, co-infec- was to determine total serum IgD in dogs with clinical tions, other non-infectious debilitating diseases, stress leishmaniosis at the time of diagnosis (IFN-γ produc- and immunosuppressive treatments [6, 13–15]. ers and non-producers) and to compare with clinically As mentioned above, dogs with moderate to severe healthy non-infected and infected dogs and also to cor- clinical leishmaniosis present a reduced or absent par- relate with clinical and immunological parameters. asite-specific T cell response. The cellular basis and mechanisms for the development of antigen-specific T Methods cell unresponsiveness in CanL are not fully understood. Dogs and sampling However, a study demonstrated a concurrent C ­ D4+ A total of 147 canine archived serum samples were ret- + and ­CD8 T cell exhaustion in dogs with clinical leish- rospectively used. The study was conducted during 2015 maniosis by a surface overexpression of programmed and 2017. The dogs were classified into three differ- death 1 protein (PD-1) that inhibited their function and ent groups. Group 1 included 40 control non-infected co-induced a low production of IFN-γ and an increase of clinically healthy dogs that were negative to L. infan- interleukin-10 (IL-10) production. Blockade of IL-10 and tum-specific antibodies and parasite-specific IFN-γ non- PD-1 recovered both ­CD4+ and ­CD8+ T cell proliferation producers. Group 2 included samples collected from and ­CD4+ IFN-γ production but also reduced IL-10 pro- 63 apparently clinically healthy infected dogs that were duction [16]. negative, low- or medium-positive to L. infantum-spe- The activation of B lymphocytes and plasmatic cells cific antibodies and the majority were IFN-γ producers leads to a massive anti-Leishmania antibodies produc- (n = 61) with the exception of two dogs that were IFN-γ tion, mainly immunoglobulin G (IgG), but also other non-producers but low seropositive. Samples of both immunoglobulins like IgM, IgE and IgA [17–19]. The control groups were collected in different locations: Fun- levels of L. infantum-specific immunoglobulin isotypes dació Hospital Clínic Veterinari (Bellaterra, Barcelona) are higher in sick dogs than in clinically healthy infected (Group 1: 10 dogs; Group 2: 6 dogs), island of Mallorca dogs [18–20]. Immunoglobulin D (IgD) was first discov- (Balearic islands, Spain) (Group 1: 10 dogs; Group 2: 48 ered in 1965 as a human myeloma protein [21, 22] but dogs), Asturias (Spain) (Group 1: 17 dogs; Group 2: 4 then was also detected in mice, rats, rabbits and guinea dogs) and Facoltà de Veterinaria of Università degli Studi pigs [23]. In 1994, a study identified an IgD-like molecule di Bari “Aldo Moro” (Bari, Italy) (Group 1: 3 dogs; Group in dogs [24]. IgD is a surface marker for naïve B cells 2: 5 dogs). before isotype switching and for cells with B cell regula- Group 3 was composed of 44 dogs with clinical leish- tory dysfunction [15, 20]. IgD secretion may be a marker maniosis at the time of diagnosis that were negative to of a regulatory phenotype during any chronic infection, highly positive to L. infantum-specific antibodies and but is unlikely to be antigen- or disease-specific. As men- were further classified as IFN-γ producers or IFN-γ tioned before, normal canine B cells co-express IgM and non-producers. Additionally, sick dogs were also stud- IgD [17, 22] and produce IL-10 but Leishmania-infected ied depending on their clinical staging (I, IIa, IIb, III, Martínez‑Orellana et al. Parasites Vectors (2019) 12:119 Page 3 of 9 IV) according to LeishVet guidelines [6]. The samples Statistical analysis were taken at diagnosis in Fundació Hospital Clínic A descriptive study of signalment (sex, age and breed), Veterinari (Bellaterra, Barcelona) (31 dogs), Hospital total serum IgD concentration, clinical status, level of Ars Veterinària (Barcelona) (8 dogs), Consultori Mont- L. infantum antibodies and IFN-γ production was per- sant (Falset, Tarragona) (4 dogs) and Facoltà de Vet- formed. Chi-square and Fisher’s exact test were used erinaria of Università degli Studi di Bari “Aldo Moro” to compare proportions of categorical data (sex, age, (Bari, Italy) (1 dog). breed, seropositivity to L. infantum and IFN-γ produc- All serum samples of all dogs studied were treated ers or non-producers) by dog groups and clinical stages. equally and stored at -80 °C. Leishmania infantum-spe- Dogs were classified as young if they were equal to or cific antibodies were measured by endpoint ELISA in all less than 18 months of age or as adults if they were more dogs studied as previously described [25]. Determination than 18 months of age, in order to change the numeric of IFN-γ production was measured by sandwich ELISA data to categorical data. Comparisons of IgD concentra- after heparin whole blood stimulation with L. infantum tion between signalment binomial data (sex, age, breed, soluble antigen (LSA) and concanavalin A (ConA). Dogs seropositivity to L. infantum and IFN-γ producer or non- were considered producers when IFN-γ was detectable producer) and between dog groups and clinical stages for after subtracting medium alone [8]. quantitative variables (parasite specific antibody levels, IgD concentration and LSA and ConA IFN-γ produc- tion) were performed using the non-parametric Mann- Detection of total serum IgD by sandwich ELISA Whitney U-test. Association between parasite-specific Quantification of canine IgD was performed according antibody response, IgD concentration and age, LSA and ConA IFN- γ production was assessed with Spearman’s Laboratories® (Montgomery, USA) with slightly modifi- to the human IgD ELISA Quantitation Set from Bethyl correlation. cations from Schaut et al. [17]. A P-value of < 0.05 was considered statistically sig- Briefly, 100 µl of coating antibody at a dilution of 1:5000 nificant. The statistical analysis was performed using the in carbonate-bicarbonate buffer was added to each well moments and mass packages [26, 27] for the software R and incubated at room temperature for 1 h. Then, five i386 v.3.4.2 [28] and Deducer v.1.8-4 [29] for Windows. washes were performed with 200 µl of phosphate-buff- Graphics were built using GraphPad Prism 8 (GraphPad ered saline (PBS) with 0.01% of Tween-20. Then, 200 µl Software, San Diego, Canada). of blocking solution composed of PBS and 1% of bovine serum albumin (BSA) was added to each well and incu- bated for 30 min at room temperature. After washing Results five times in PBS 0.01% Tween-20, 100 µl of standards Clinical findings, L. infantum antibodies and IFN‑γ and undiluted serum samples was added per duplicate production and incubated for 1 h at room temperature. Then, five In the control non-infected Group 1, there were a total PBS 0.01% Tween-20 washes were repeated and 100 µl of of 40 dogs. The average age at recruitment was 4.2 years diluted horseradish peroxidase (HRP) detection antibody with a range from 4 months to 13 years. Twenty were diluted at 1:20,000 in PBS was added and incubated for 1 female, 15 were male and five were not defined due to h at room temperature. After washing five times in PBS lack of information. Mixed breeds and 15 pure breeds 0.01% Tween-20, 100 µl of 3,3′,5,5′-tetramethylbenzidine were represented. All 40 dogs were seronegative to L. (TMB) substrate solution was added to each well and infantum-specific antibodies. incubated for 15 min. Then, 100 µl of stop solution (HCL Control healthy non-infected dogs were all IFN-γ 5M) was added. Finally, the absorbance was measured non-producers with lower production after LSA (Mann- using an automatic micro ELISA reader Anthos 2020 Whitney U-test: Z = -8.31, P < 0.0001) and ConA (Mann- (Biochrom, Cambridge, UK) at 450 nm [17]. Whitney U-test: Z = -2.67, P = 0.008) stimulation when Standards were prepared by mixing human reference compared with control healthy infected IFN-γ producers serum included in the IgD ELISA kit with buffer diluent (Fig. 1a, b). (blocking buffer plus 0.01% of Tween-20). Each plate had A total of 63 apparently clinically healthy infected dogs a total of eight standards per duplicate with concentra- were included in Group 2. The average age was 4.4 years tions between 3.9 and 500 ng/ml. Quantification was with a range from 4 months to 10 years. Thirty-six were female, 16 were male and 11 were not defined due to a Assays® (https​://www.myass​ays.com/). Furthermore, performed using a four-parameter logistic curve in My lack of information. Mixed breeds and 11 pure breeds plates were repeated when the R2-value of the standard were represented. The results of the antibody levels curve was below 0.98. of Group 2 are shown in Fig. 2a. Fifteen out of 63 dogs Martínez‑Orellana et al. Parasites Vectors (2019) 12:119 Page 4 of 9 Fig. 1 a Specific L. infantum IFN-γ concentration based on IFN-γ producers and IFN-γ non-producers in healthy non-infected, healthy infected and sick dogs. b IFN-γ concentration after ConA stimulation based on IFN-γ producers and IFN-γ non-producers in healthy non-infected, healthy infected and sick dogs. White circles represent individual data of each dog, boxes represent minimum and maximum values of quartiles, the bands inside represent the second quartile (median). Statistical results of LSA IFN-γ: Group 1 (healthy non-infected) < Group 2 (healthy infected) (Mann-Whitney U-test: Z = -8.31, P < 0.0001), Group 1 (healthy non-infected) < Group 3 (sick) (Mann-Whitney U-test: Z = -3.81, P = 0.0001), Group 2 (healthy infected) < Group 3 (sick) (Mann-Whitney U-test: Z = -3.28, P = 0.001). ConA IFN-γ: Group 1(healthy non-infected) < Group 2 (healthy infected) (Mann-Whitney U-test: Z = -2.67, P = 0.008). Abbreviations: LSA, Leishmania soluble antigen; ConA, concanavalin A; IFN-γ, interferon gamma Fig. 2 a Specific L. infantum antibodies levels in healthy non-infected, healthy infected and sick dogs classified based on LeishVet clinical staging. b Total serum IgD concentrations in healthy non-infected, healthy infected and sick dogs classified based on LeishVet clinical staging. White circles represent individual data of each animal, boxes represent minimum and maximum values of quartiles, the bands inside represent the second quartile (median). Statistical results of L. infantum specific antibodies by ELISA: Group 1 (healthy non-infected) < Group 2 (healthy infected) (Mann-Whitney U-test: Z = -4.46, P < 0.0001), Group 1 (healthy non-infected) < Group 3 (sick) (Mann-Whitney U-test: Z = −9.59, P < 0.0001), Group 2 (healthy infected) < Group 3 (sick) (Mann-Whitney U-test: Z = -7.84, P < 0.0001), mild stages (I and IIa) < severe stages (IIb, III and IV) (Mann-Whitney U-test: Z = -3.07, P = 0.002), stage I < stage IIa (Mann-Whitney U-test: Z = -4.45, P < 0.0001), stage I < stage IIb (Mann-Whitney U-test: Z = -3.07, P = 0.002), stage I < Stage III (Mann-Whitney U-test: Z = -3.25, P = 0.001), stage I < stage IV (Mann-Whitney U-test: Z = -2.85, P = 0.004), stage IIa < stage IV (Mann-Whitney U-test: Z = -2.59, P = 0.01) Martínez‑Orellana et al. Parasites Vectors (2019) 12:119 Page 5 of 9 presented low to medium levels of L. infantum antibod- ies and the remaining dogs were seronegative. The pro- portion of IFN-γ producers and IFN-γ non-producers on this group of healthy infected dogs was 96.8% (n = 61) and 3.1% (n = 2), respectively (Fig. 1a). The two IFN-γ non-producers were low seropositive. In the group of sick animals (Group 3) there were a total of 44 dogs with an average age of 4.7 years with a range from 5 months to 17 years. Nineteen were female, 24 were male and sex was not identified in one dog. Mixed breeds and 19 pure breeds were represented. Dogs from Group 3 were classified in four clinical stages. Leishmania-infantum antibody levels and statisti- cally significant differences are described in Fig. 2a. Sick dogs (n = 44) presented statistically significant higher levels of antibodies against L. infantum (Mann-Whitney U-test: Z = -8.9, P < 0.0001) when compared with control dogs (Groups 1 and 2, n = 103). A tendency of higher L. Fig. 3 Total serum IgD concentrations in IFN-γ producers and IFN-γ infantum antibody levels was found in IFN-γ non-pro- non-producers sick dogs classified based on LeishVet clinical staging. ducers when compared with IFN-γ producers sick dogs Circles represent individual data of each dog, the horizontal line represents the mean and the vertical lines represent the standard (Mann-Whitney U-test: Z = -1.91, P = 0.056). Higher deviation. No statistical differences were observed within LeishVet concentration of IFN-γ after LSA (Mann-Whitney clinical stages. Abbreviations: LSA, Leishmania soluble antigen; ConA, U-test: Z = -5.96, P < 0.0001) and ConA (Mann-Whitney concanavalin A; IFN-γ, interferon gamma U-test: Z = -2.53, P = 0.01) was found in sick dogs IFN-γ producers when compared with IFN-γ non-producers no statistical differences were found when IgD concen- (Fig. 1a, b). trations were compared between the different LeishVet IFN-γ producer sick dogs presented significantly higher clinical stages (Fig. 3). Furthermore, statistically signifi- L. infantum antibody levels (Mann-Whitney U-test: cant differences of total IgD concentrations were also not Z =-7.94, P < 0.0001), and IFN-γ production after LSA found when sick dogs were grouped based on LeishVet (Mann-Whitney U-test: Z = -6.95, P < 0.0001) and ConA clinical stages (stages I-IIa versus stages IIb-III-IV). (Mann-Whitney U-test: Z = -2.57, P = 0.01) when com- IgD concentration based on dichotomous parameters pared with IFN-γ non-producer healthy dogs (Fig. 1a, b). such as signalment (sex, age and breed), L. infantum serol- Differences between sex, age and breed were not found ogy and IFN-γ production are summarized in Table 1. between all the three groups studied. Furthermore, when all dogs studied were divided into seropositive or seroneg- ative, no differences were found regarding signalment (sex Correlations and breed). However, a statistically significant difference A positive correlation between L. infantum antibod- was found between age and serology with young dogs ies, age and IFN-γ production was found when all dogs more likely to be seronegative (Chi-square test: χ2 = 9.37, from the three groups studied (n = 147) were analysed df = 1, P = 0.002). Nevertheless, the Mann-Whitney U-test (Table 2). However, no significant correlation of the anti- revealed no significant differences for age between seron- body response to L. infantum antigen or IFN-γ produc- egative and seropositive dogs (Z = -1.23, P = 0.22). tion and IgD concentration was encountered. A similar positive correlation was found between L. infantum antibodies and IFN-γ production when the IgD concentration control healthy dogs included in Groups 1 and 2 (n = 103) The results of IgD concentration for each group are and also when only Group 2 (healthy infected) were ana- shown in Figs. 2b, 3. IgD concentration between groups lysed (Table 2). No correlation was observed with any of did not show any significant differences when sick dogs the parameters studied (L. infantum antibodies or IFN-γ were compared with control healthy non-infected and production or age) and IgD concentration in Group 1 healthy infected dogs as well when both groups of con- (healthy non-infected). trol animals were compared (Fig. 2b). Moreover, when Finally, sick dogs (Group 3; n = 44) were studied sepa- IgD concentrations were compared between sick dogs rately and a negative correlation was found between IFN-γ producers and sick dogs IFN-γ non-producers no L. infantum antibodies and LSA IFN-γ production significant differences were detected (Fig. 3). In addition, Martínez‑Orellana et al. Parasites Vectors (2019) 12:119 Page 6 of 9 Table 1 IgD concentration based on dichotomous parameters such as signalment (sex, age and breed), L. infantum serology and IFN-γ production Variable Group IgD concentration (ng/ml) Mann–Whitney test Mean ± SD U-value Z-value P-value Sex Male (n = 54) 391.33 ± 1873.22 1922 -0.37 0.7136 Female (n = 74) 41.4 ± 124.27 Age ≤ 18 months (n = 35) 14.85 ± 31.46 1960 -1.89 0.0583 > 18 months (n = 92) 259 ± 1442.28 Breed Purebred (n = 108) 177.81 ± 1302.62 1588 -0.44 0.6632 Mixed breed (n = 31) 168.69 ± 568.43 L. infantum serology Positive (n = 54) 351.08 ± 1855.6 2383 -0.51 0.6068 Negative (n = 93) 115.76 ± 534.48 IFN-γ production Producer (n = 63) 62.32 ± 194.48 2599 -0.18 0.8555 Non-producer (n = 84) 388.71 ± 1812.55 Abbreviations: IgD, immunoglobulin D; IFN-γ, interferon gamma; n, number of dogs; SD, standard deviation Table 2 Spearman’s correlation between age, IgD concentration, LSA IFN-γ concentration and serology by groups studied Spearman’s rs (P-value) IgD concentration (ng/ml) L. infantum antibodies (EU) All dogs (n = 147) Age (months) 0.0220 (0.8096) 0.2024 (0.0254)* IgD concentration (ng/ml) 1 0.0307 (0.7120) L. infantum antibodies (EU) 0.0307 (0.7120) 1 LSA IFN-γ concentration (pg/ml) 0.0438 (0.5983) 0.1742 (0.0348)* All healthy dogs (n = 103) Age (months) 0.1664 (0.1401) 0.0713 (0.5300) IgD concentration (ng/ml) 1 0.0067 (0.9468) L. infantum antibodies (EU) 0.0067 (0.9468) 1 LSA IFN-γ concentration (pg/ml) 0.0481 (0.6297) 0.4872 (< 0.0001)* Sick dogs (n = 44) Age (months) -0.2665 (0.0881) 0.1104 (0.4862) IgD concentration (ng/ml) 1 0.0297 (0.8483) L. infantum antibodies (EU) 0.0297 (0.8483) 1 LSA IFN-γ concentration (pg/ml) 0.0549 (0.7235) -0.3233 (0.0323)* Control healthy dogs (non-infected) (n = 40) Age (months) 0.1206 (0.5038) 0.0609 (0.7366) IgD concentration (ng/ml) 1 -0.1898 (0.2408) L. infantum antibodies (EU) -0.1898 (0.2408) 1 LSA IFN-γ concentration (pg/ml) -0.0104 (0.9493) -0.2315 (0.1506) Control healthy dogs (infected) (n = 63) Age (months) 0.2145 (0.1476) 0.0735 (0.6235) IgD concentration (ng/ml) 1 0.0692 (0.5899) L. infantum antibodies (EU) 0.0692 (0.5899) 1 LSA IFN-γ concentration (pg/ml) 0.0124 (0.9232) 0.4212 (0.0006)* *Statistically significant P-values Abbreviations: LSA, Leishmania soluble antigen; ConA, concanavalin A; IFN-γ, interferon gamma; IgD, immunoglobulin D; EU, ELISA units Martínez‑Orellana et al. Parasites Vectors (2019) 12:119 Page 7 of 9 (Table 2). Once again, no correlation was found with is called Moraxella IgD binding protein/hemagglutinin. any of the parameters studied (L. infantum antibodies or In human patients, IgD is increased in many pathologic IFN-γ production or age) and IgD concentration. problems, including infections, autoimmune diseases, immunodeficiencies and allergies [23]. It is important to Discussion highlight that, so far, there is limited information regard- IgD is expressed on the surface of B cells and is usually ing IgD in dogs and its release under clinicopathologic accompanied by IgM [22]. Knowledge about the role of conditions. To the best of our knowledge, IgD has only IgD in dogs has progressed little since the discovery of been investigated in one study in CanL [17] other than the molecule IgD-like in 1994 [24]. the present study. It is likely that the total serum IgD The progression of clinical CanL has been related to might increase in other clinical conditions such as other T cell exhaustion by an overexpression of PD-1, which infections in dogs as previously reported in humans [23]. increases the levels of IL-10 and decreases the produc- Therefore, the present results might be influenced by tion of IFN-γ [16]. A previous study demonstrated that the presence of concomitant diseases (such as atopy or all dogs have regulatory B cells expressing IgD and pro- other allergies) or infections, which would increase the ducing IL-10, but dogs with clinical leishmaniosis tend total serum IgD concentration without being related to L. to have higher levels of B cells expressing IgD, leading to infantum infection. Unfortunately, in the present study it an increase of PD-1 and IL-10 and a progression of the was not possible to investigate the possibility of concomi- disease [17]. In addition, the same group showed a sig- tant infections or levels of IgE against different allergens nificant higher concentration of total serum IgD in sick present in all the dogs studied. In addition, it remains dogs (n = 16) when compared with healthy non-infected unknown if the concentration of total serum IgD might dogs (n = 10) [17]. In the present study, we aimed to be different depending on provenience, age, sex, breed or investigate and compare the total serum IgD in clinically clinical background. However, in the present study, the healthy and sick dogs. Unfortunately, results presented total serum IgD concentration appears not to be different here did not show a significant higher concentration of based on sex and breed while age might have influenced serum total IgD in all sick dogs when compared with all this immunoglobulin. In this study, young dogs presented healthy dogs (non-infected and infected) or when com- a trend of lower concentration of serum total IgD when pared healthy non-infected dogs or healthy infected dogs compared with adult dogs. However, Spearman’s correla- alone. Nevertheless, an increase of total IgD concentra- tion did not support this finding. Studies at least in other tion in all sick dogs was found when compared with both canine clinical conditions would be of interest. groups of healthy infected and non-infected dogs (see In the present study and based on the findings previ- Fig. 2b). However, those differences were not statisti- ously described [17], we hypothesized that dogs with cally significant. For this reason, results presented in this a pattern of moderate to severe disease, high antibody study do not fully confirm previous work [17] but suggest response and absence of specific L. infantum IFN-γ a similar tendency of high concentration of total IgD in response might have higher IgD serum concentration dogs with clinical leishmaniosis. when compared with sick dogs with mild to moderate Furthermore, when control groups were compared, disease, low parasite load and parasite-specific IFN-γ a higher but not significant level of total IgD was found response [8]. Unfortunately, we did not find significant in clinically healthy non-infected dogs when compared differences on IgD concentrations between sick dogs L. with clinically healthy infected dogs (Fig. 2b). Conversely, infantum IFN-γ producers and IFN-γ non-producers. Schaut et al. [17] observed an opposite trend, although In addition, significant differences of total IgD concen- this difference was also not significant. Our findings trations were also not encountered when sick dogs were might suggest that total IgD does not appear to increase grouped based on LeishVet clinical stages (stages I-IIa in healthy dogs infected with L. infantum. versus stages IIb-III-IV). Therefore, serum total IgD Since human IgD was discovered [23], it has been iden- does not seem to be a useful marker of disease severity tified in many other diseases and infections such as the in clinical leishmaniosis in dogs. In contrast, our find- immunodeficiency virus [30], leprosy [31], tuberculosis ings showed L. infantum specific IFN-γ in ex vivo blood [32], malaria [33, 34], hepatitis [35] and patients suffer- and L. infantum specific antibody levels are useful mark- ing from hyper-IgD and periodic fever syndrome [36]. ers to assess disease severity in agreement with previous The production of pathogen-specific IgD in response to reports [8, 9]. IFN-γ producer dogs are strongly associ- infection [22, 23] as it binds to pathogenic microorgan- ated with mild to moderate clinical staging (stages I and isms and their virulence factors has been associated to IIa) while IFN-γ non-producer dogs have been associated Streptococcus streptolysin O [37], Moraxella catarrhalis with more severe clinical stages (stages IIb, III and IV). [38] and Haemophilus influenzae adhesion [39] which On the contrary to the clinical analysis performed here, Martínez‑Orellana et al. Parasites Vectors (2019) 12:119 Page 8 of 9 the previous study compared the total IgD measured in Funding This study was supported by Spanish ministry grants, Ministerio de Economía sick dogs with leishmaniosis, based on physical exami- y competitividad and Fondo Europeo de Desarrollo Regional (FEDER, EU) nation and classification of clinical staging was not per- (AGL2012-32498 and AGL2015-68477). formed, which means that sick dogs were classified as Availability of data and materials PCR positive with high serological levels as symptomatic Data supporting the conclusions of this article are included within the article. [17]. In addition, the IFN-γ concentration after stimula- The datasets used and/or analysed during the present study available from tion with Leishmania antigen and its relation with IgD the corresponding author upon reasonable request. levels was not described [17]. Authors’ contributions Limitations of this study include its retrospective LSG designed the research study. LSG and PMO supervised technical work. nature that hindered having all clinical data of the dogs; LSG, PMO and MB contributed with data analysis and interpretation. MB developed the statistical analysis. LSG, PMO and CM wrote the manuscript. for example, the existence of concomitant infections or LSG, AAF, AB and LO coordinated the enrolled veterinary clinics. LSG, LO, AAF the effect of chronic clinical conditions such as atopy or and AB examined and collected samples. PMO and AB performed whole other allergies. The ELISA employed was for the detec- blood assay and collection of supernatants. PMO and CM performed all IgD detection, serological and cytokine testing. All authors read and approved the tion of human IgD and it seemed to work well as dem- final manuscript. onstrated here and previously [17]. However, the use of proper canine antibodies might increase the detec- Ethics approval and consent to participate A signed informed consent was obtained from all owners. Residual samples of tion and performance of canine IgD. Furthermore, it blood were used in this study; therefore, ethical approval was not required. is important to remark that it is also likely that the pre- sent study lacks statistical power to detect subtle differ- Consent for publication Not applicable. ences between groups. Ultimately, the lack of tools to understand how Leishmania-specific IgD behaves during Competing interests infection and disease hampers a greater interest in the The authors declare that they have no competing interests. presented data as many confounding factors associated with IgD evaluation exist. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. Conclusions This study demonstrates that there is no clear increase of Author details 1 Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, total serum IgD in dogs with clinical leishmaniosis when Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain. 2 Università compared with healthy dogs, although a trend was found. degli Studi di Bari “Aldo Moro”, Bari, Italy. 3 Hospital Clínic Veterinari, Universitat Interestingly, higher but not statistically significant levels Autònoma de Barcelona, Bellaterra, Spain. of total serum IgD were found in clinically healthy non- Received: 24 November 2018 Accepted: 7 March 2019 infected dogs when compared with infected dogs. Addi- tionally, higher but not significant total serum IgD was observed in sick dogs classified as IFN-γ non-producers comparing to IFN-γ poducers. Therefore, total serum IgD References does not seem to be a useful marker for establishing dis- 1. 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