Background

The endosymbiotic relationship described between Wolbachia and filarioid nematodes (families Onchocercinae, Dirofilariinae and Splendidofilariinae) has important implications in the biological processes of reproduction, development, molting and embryogenesis of filarioids [1,2,3,4]. Dirofilaria immitis, the causative agent of the canine heartworm disease (HWD), harbors Wolbachia endosymbiont in all its developmental stages, from adult to microfilariae (mfs) [5]. In addition, clinical studies indicate that Wolbachia is released into the bloodstream of hosts after death of D. immitis [6], also because of filaricidal treatment [7, 8]. Furthermore, Wolbachia associated to D. immitis infection may increase the severity of the clinical signs of HWD by triggering inflammatory response [7, 9,10,11]. As observed in other filarioids, such as Onchocerca volvulus [12], D. immitis may stimulate Th2 anti-inflammatory response, while Wolbachia triggers the Th1 pro-inflammatory response [10, 13, 14]. The latter is likely due to the effect that Wolbachia surface protein (WSP) and other molecules exert on antigen presenting cells and cytokine production [6, 7, 12, 15,16,17,18]. In addition, in vitro experiment using Asaia-WSP engineered bacterium was shown to elicit the Th1 cellular response against Leishmania infantum, with a protozoal killing effect [19].

Given the sympatric occurrence of L. infantum and D. immitis in many geographical areas around the world [20,21,22,23,24], the association of both vector-borne pathogens is of major interest from a diagnostic and clinical perspective [25]. For example, the prevalence of D. immitis infection increased in areas of southern Europe, where canine leishmaniosis (CanL) was historically endemic, as a consequence of pets traveling with their owners (i.e. from endemic to previously non-endemic regions) and changes in arthropod vector ecology and distribution [20, 21, 24, 26,27,28,29,30]. This overlapping geographical distribution of D. immitis and L. infantum led to potential immune interactions [20, 21, 24, 27, 28]. Indeed, the prevalence of dogs without clinical signs [31] depends on the balance between Th1 and Th2 immune response [32,33,34,35], which may be regulated by a plethora of factors (e.g. animal genetic, health and nutritional status, concurrent infections) [36]. In this context, the clinical manifestations of a patent leishmaniosis in dogs co-infected by D. immitis may be affected by the presence of Wolbachia endosymbiont [21, 27, 32]. Thus, the aim of this study was to investigate the interactions of Wolbachia in dogs naturally infected by D. immitis and/or L. infantum and to assess the relationship between pathogen infection and cytokine circulation.

Methods

Enrolled animals, parasitological and serological examination

A total of 187 dogs presenting no apparent clinical signs were selected to reduce other factors that could interfere with the analyses. All dogs, living in two municipal shelters in southern Italy (Lecce: 40.419326N, 18.165582E; Casarano 40.0126N, 18.1606E) were subjected to physical examination to establish their health status. Among them, a cohort of dogs (n = 84) was enrolled from a previous study aiming to control D. immitis and L. infantum infection [24].

Blood samples were collected from either the cephalic or jugular veins and placed in a K3 EDTA tube (2 ml) and in a clot activator tube (5 ml) to obtain serum after centrifugation (15 min at 1500 × g). Individual blood samples were screened by modified Knott test for the detection of mfs of D. immitis, as previously described [37]. The mfs identification was performed measuring the body length and width of specimens and by morphological analysis of the front end and the tail according to [38], using a digitally captured image software LAS V4.5 (Leica Microsystems). The count of the mfs (2 × 50 μl) was based on the average of the counting in the two slides. Dogs with a mfs load ≥ 600 mfs/20 μl were considered highly microfilaremic [24]. Serum samples were tested for the antigen detection of adult females of D. immitis by SNAP® 4Dx® Plus Test (IDEXX Laboratories, Inc.), according to the manufacturer's instructions.

To assess the exposure of dogs to L. infantum infection, sera were tested by immunofluorescence antibody test (IFAT) using as antigen the promastigotes of L. infantum zymodeme MON-1, as previously described [39]. Samples were considered positive when they produced a clear cytoplasmic and membrane fluorescence of promastigotes from a cut-off dilution of 1:80. Positive sera were titrated by serial dilutions until negative results were obtained.

Subsequently, all dogs were divided into four groups based on their positivity/negativity for mfs of D. immitis and to anti-Leishmania antibodies. Specifically, dogs positive for mfs and positive and/or negative to IFAT were grouped in G1 (n = 47) and G2 (n = 46), respectively, while dogs negative for mfs and for female antigens of D. immitis and positive or negative to IFAT were included in G3 (n = 47) and G4 (n = 47), respectively (Table 1).

Table 1 Dogs included in the study divided according to their positivity/negativity to Dirofilaria immitis and Leishmania infantum, microfilariae (mfs) load, IFAT titer and  positivity for Wolbachia and L. infantum by qPCR 
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In addition, a group of dogs (n = 65), selected based on their negativity and/or positivity to IFAT with a titer ≥ 1:160 and for mfs, were examined by analyzing the production of pro-inflammatory cytokines such as tumor necrosis factor α (TNFα) and interferon-gamma (IFNγ) and of anti-inflammatory cytokines such as interleukin-4 (IL-4), interleukin-6 (IL-6) and interleukin-10 (IL-10) using an ELISA kit, (Biolegend, USA, and Thermo Fisher, USA) (Table 2). The positivity of the sera was determined based on specific standard curves.

Table 2 Prevalence of cytokine production, calculated as the percentage of dogs from each group producing one cytokine, divided according to groups
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Genomic DNA blood extraction and molecular procedures

Genomic DNA (gDNA) was extracted from each blood sample using the GenUPgDNA commercial kit (Biotechrabbit GmbH, Hennigsdorf, Germany) according to the manufacturer’s instructions. All samples were tested for L. infantum kDNA minicircle by real time-PCR (qPCR) using the primers, probes and cycle protocol described elsewhere [40]. gDNA from L. infantum isolate cultured zymodeme MON-1 was used as positive control, whereas gDNA extracted from blood sample from a healthy dog was used as negative controls.

Samples were tested for Wolbachia of D. immitis by qPCR using primers (111 bp, WDiro.ftsZ.490-F/WDiro.ftsZ.600-R) and probe wDimm.ftsZ.523p (6FAM-CGTATTGCAGAGCTCGGATTA-TAMRA) targeting the ftsZ gene as previously described [41] with minor modifications.

Briefly, all qPCR reactions were carried out in a final volume of 20 μl, consisting of 10 μl IQ Supermix (Bio-Rad Laboratories, Hercules, CA, USA), 6 μl diethyl pyrocarbonate (DEPC)-treated pyrogen-free DNase/RNase-free water (Invitrogen, Carlsbad, CA, USA), 2.5 μl of template DNA (except no-template control), primers and probe at 50 μM and 20 μM concentration, respectively. The run protocol consisted of a hot-start at 95 °C for 3 min and 40 cycles of denaturation (95 °C for 5 s) and annealing-extension (60 °C for 30 s). The qPCR was performed in a CFX96 Real-Time System (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and the increase in the fluorescent signal was registered during the extension step of the reaction and analyzed using CFX Manager Software, version 3.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). All the samples were tested in duplicate, and DNA of adult of D. immitis and that from blood samples of pathogen-free dogs were used as positive and negative controls. The positivity for Wolbachia was established based on the threshold cycle (Ct) value up to 38.5.

Statistical analysis

Associations between infections and variables were assessed through univariate analysis. Exact binomial test established the confidence intervals (CI) with 95% confidence level. The Chi-square χ2 test was used to compare percentages of positivity among categories of the same independent variables. Collinearity among independent variables was assessed using Pearsonʼs correlation coefficient. A P-value < 0.05 was considered as statistically significant. Statistical analyses were performed using StatLib for Windows (version 13.0, SPSS, Inc., Chicago, IL, USA) Quantitative Parasitology 3.0 [42] and GraphPad Prism 8 software.

Results

Of 93 mfs-positive dogs, 79 (85%) tested positive for Wolbachia DNA (Table 1), with 37 from G1 (46.8%; 95% CI: 0.355–0.584) and 42 from G2 (53.2%; 95% CI: 0.416–0.645). The highest amount of Wolbachia DNA (Ct value 22.1) was detected in a dog from G1 (mfs load ≥ 600) and the lowest (Ct value 37.4) in dogs with low mfs load (i.e. 1 ≤ mfs ≤ 200) in both G1 and G2 (Table 1). No statistically significant difference in Wolbachia prevalence was observed between dogs from G1 and G2, though an overall higher value (37.6%) was recorded in dogs with a Wolbachia Ct values ranging from 25 to 30 (Table 3). In G1, a statistical difference was observed between dogs with Wolbachia Ct value of 25–30 vs. 30–34 (χ2 = 6, P < 0.0138) and vs. Ct > 35 (χ2 = 12.3, P = 0.0004) having a high mfs load and between low vs. high mfs score (χ2 = 10.64, P = 0.011) (Table 3). In G2, a statistically significant difference (χ2 = 9.8, P = 0.00178) was observed between dogs with Wolbachia Ct value of 25–30 and 30–34 vs. > 35, having high mfs load (Table 3).

Table 3 Prevalence of Wolbachia in dogs positive for Dirofilaria immitis divided according to the microfilaria load and the range of Ct value of qPCR
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Of 94 dogs positive for anti-Leishmania antibodies, 31 were from G1 (66%; 95% CI: 0.507–0.791) with low antibody titers (up to 1:160), followed by 16 dogs (34%; 95% CI: 0.209–0.49) with higher titers (from 1:320 to 1:2560). In G3, 23 dogs (48.9%; 95% CI: 0.341–0.639) had a high antibody titer (from 1:320 to 1:2560) (Table 1). The qPCR screened eight positive dogs of 187 blood samples examined for kDNA of L. infantum (4.3%) with a minimum and maximum Ct value (min = 24.7, max = 37.8) detected for dogs from G1 (Table 1).

Of 37 dogs positive for Wolbachia in G1, 26 (70.3%; 95% CI: 0.530–0.841) had low antibody titers (n = 20 1:80, n = 6 1:160) against L. infantum (Table 4). A statistical difference was observed between dogs showing the highest amount of Wolbachia DNA (Ct value range of 25–30) and those with antibody titers ranging from 1:320 to 1:2560 (χ2 = 6.8, P = 0.0089) (Table 4).

Table 4 Wolbachia-positive dogs divided according to Ct value of endosymbiont DNA and Leishmania infantum positivity to different tests
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IL-10 (55.4%) and IFNγ (44.6%) were the most prevalent cytokines produced throughout dogs examined (Table 2 and Fig. 1). In particular, the percentage of dogs producing TNFα was higher in G2 (42.8%) than G1 (28.6%) and G4 (14.3%). However, the highest mean of TNFα concentration was detected in G1 (246.5 pg/ml) and the lowest in G2 (65.1 pg/ml) and G4 (13.3 pg/ml) (Table 2, Fig. 1). A statistically significant difference (χ2 = 7.3, P = 0.007) was recorded analyzing the percentages of IFNγ-positive sera between dogs from G1 (64.3%) and G4 (14.2%), with the same percentage of positive dogs in G2 and G3 (50%) (Table 2). The highest mean of concentration of IFNγ production was observed in G2 (105.4 pg/ml) and G3 (97.23 pg/ml) (Table 2, Fig. 1). The highest percentage of dogs producing IL-10 (71.4%) was observed in G1 (mean concentration of 1809.5 pg/ml) followed by G4 (57.1%) and G3 (42.8%) (Table 2). The lowest percentage of dogs producing IL-4 was observed in G1 (7.1%), G2 and G3 (28.5%) (Table 2, Fig. 1). The highest mean concentration of IL-4 was observed in G2 (2794.5 pg/ml). IL-6 showed the same percentage of positivity in G1 and G4 (35.7%) and in G2 and G3 (50%), respectively, with the highest mean concentration registered in G1 (123.5 pg/ml) and a similar concentration observed in G2 (30.2 pg/ml) and G3 (29.7 pg/ml) (Table 2, Fig. 1).

Fig. 1
figure 1

Concentrations of A tumor necrosis factor-α (TNFα), B interleukin-10 (IL-10), C interleukin-6 (IL-6), D interleukin-4 (IL-4) and E interferon-γ (IFNγ) in sera from dogs positive for Leishmania infantum and Dirofilaria immitis (Group 1), positive only for Dirofilaria immitis (Group 2), positive only for Leishmania infantum (Group 3) and negative to both pathogens (Group 4)

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Except for IFNγ, no statistically significant differences were observed in cytokine production between groups, but within groups. In particular, in G1 a statistically significant difference was observed between dogs producing TNFα vs. IL-10 (χ2 = 5.1, P = 0.023), IFNγ vs. IL-4 (χ2 = 9.9, P = 0.001), IL-10 vs. IL-4 (χ2 = 12.1, P = 0.005) and IL-4 vs. IL-6 (χ2 = 17.4, P = 0.00003). Similarly, in G4 a statistically significant difference was observed between IFNγ and TNFα vs. IL-10 and between IL-10 vs. IL-4 (χ2 = 5.6, P = 0.018). No statistically significant difference was observed in cytokine production within groups G2 and G3 (Table 2). In addition, a statistically significant difference was observed in G1 between dogs positive for Wolbachia vs. TNFα (χ2 = 9.3, P = 0.022), IL-4 (χ2 = 17.4, P = 0.00003) and IL-6 (χ2 = 7.3, P = 0.0067) (Table 2).

Discussion

Data herein presented suggest that Wolbachia, associated with D. immitis, may affect the immune response against L. infantum of naturally co-infected dogs through a stimulatory or immune-suppressive mechanism [4]. Indeed, the high molecular prevalence of Wolbachia in co-infected dogs (78.7%) is coherent with the hypothesis that this endosymbiont might control the Leishmania infection. Overall, the prevalence of Wolbachia observed in co-infected dogs is in line with data described in a previous study [21], where the endosymbiont was detected in 68.8% dogs from Portugal. Nevertheless, the high amount of Wolbachia DNA in relationship to the mfs load (i.e. mfs > 600; Ct 25–30) exclusively observed in co-infected dogs (63.2%) differed from a previous study from Spain, where Wolbachia was more frequently detected in microfilaraemic dogs not infected with L. infantum [27]. In addition, in this previous study, an increased severity of clinical leishmaniotic signs was observed in microfilaraemic dogs with a lower prevalence of Wolbachia [27]. Furthermore, the role of the endosymbiont in stimulating a Th1 immune response was also suggested by the low number (3/11) of co-infected dogs with clinical signs, as previously described [21].

The role of Wolbachia in controlling the development of CanL may be highlighted by the high prevalence of co-infected dogs (70.3%) showing a low anti-Leishmania antibody titer (up to 1:160), likely due to the low parasitic load detected by qPCR in the blood [43], while a high value of IFAT titers was observed in most of dogs (48.9%) infected only with L. infantum.

Furthermore, the absence of clinical signs in dogs may be also determined by the stimulation of a Th1 immune response triggered by the endosymbiont bacterium by production of high levels of pro-inflammatory cytokines [12, 19, 21, 27, 44]. For example, the high Wolbachia amount recorded in co-infected dogs may trigger an elevated TNFα production (mean value 246.5 pg/ml), which was not recorded in dogs infected with only L. infantum. The strong effect of Wolbachia in producing this pro-inflammatory cytokine has also been demonstrated in an in vitro experiment [19], regardless of the presence of L. infantum. However, the absence of TNFα observed in dogs positive for L. infantum agrees with previous studies where the cytokine was detected only in a few dogs with active leishmaniosis [45, 46].

The Wolbachia amount detected in co-infected dogs, and consequently its effect on controlling the L. infantum infection, may also be supported by the high prevalence of dogs (64.3%) producing IFNγ, a similar mean cytokine concentration value being observed between these dogs (88.2 pg/ml) and those positive for L. infantum (97.23 pg/ml) or D. immitis (105.4 pg/ml). Accordingly, IFN-γ was associated with the absence or low antibody titer against L. infantum [47]. Besides TNFα and IFNγ, the detection of IL-4, IL-6 and IL-10 in infected dogs indicates a mixed Th1/Th2 immune response. Indeed, in previous studies using peripheral blood mononuclear cells (PBMCs) of dogs experimentally infected with L. infantum or stimulated with L. infantum antigen, an increase in IFNγ, IL-10 and IL-4 mRNA expression levels were recorded [35, 48, 49]. In addition, the expression of cytokines related to both Th1/Th2 responses was also detected in dogs naturally infected with D. immitis with circulating mfs associated to the presence of Wolbachia [10, 50]. All the above considerations may justify the high mean concentration (1809.5 pg/ml) of IL-10 recorded in co-infected dogs as well as its even production in dogs infected with D. immitis (507.8 pg/ml) or L. infantum (677.4 pg/ml). However, though IL-10 is considered a predictive parameter of the evolution of CanL and active visceral leishmaniosis in humans [51,52,53], other studies described the expression of this cytokine in dogs without clinical signs [48, 54,55,56]. Furthermore, the detection of IL-10 in uninfected dogs (863.8 pg/ml) agrees with the results described elsewhere [54], where the mRNA accumulation of this cytokine in dogs with severe disease was comparable with that of uninfected control dogs. Similarly, the detection of IL-4 in all groups of dogs examined is not surprising, considering that the role of this cytokine in the pathogenesis of CanL is still debated [54]. Indeed, in the current study a very low mean concentration (77.2 pg/ml) of IL-4 was detected in co-infected dogs compared with those recorded in L. infantum infected (254.9 pg/ml) or uninfected dogs (111.7 pg/ml). However, other studies also described these contrasting results, IL-4 being detected readily or not in L. infantum infected or asymptomatic dogs [48, 54, 57, 58]. The Wolbachia amount recorded in the co-infected dogs may have affected the IL-6 production (123.5 pg/ml) in these dogs. Indeed, the role of this endosymbiont in stimulation of IL-6 production was also supported by an in vitro experiment [19]. However, though IL-6 is generally regarded as a Th2 cytokine with disease progression, other studies described its protective role in some forms of leishmaniosis [59], thus indicating an imperfect fitting of this cytokine into the Th1/Th2 paradigm [45, 48, 60].

Overall, interpreting the cytokine expression profile in CanL is still problematic, and the differences observed regarding the role of cytokines may be due to the different methods used for the analyses (i.e. dosage by ELISA or evaluation of mRNA expression) [48, 54, 56]. In addition, immune response mediated by cytokines may be influenced by several variables (i.e. age, tissues examined) [35, 53]. Thus, the comprehensive knowledge of cytokine response and their interaction is a very crucial step to understand disease progression, mainly in co-infected dogs.

Conclusions

Though the present work has some limitations, such as the low number of dogs included and their heterogeneity, and the lack of a follow-up study, the results presented suggest the involvement of Wolbachia in clinical leishmaniosis, pointing at a possible role of this endosymbiont in the modulation of the Th1 immune response. However, future studies based on the simultaneous combination of different approaches of analysis (i.e. expression of the mRNA vs. quantification of cytokine production) is mandatory.