Latrofa et al. Parasites & Vectors (2023) 16:77 Parasites & Vectors https://doi.org/10.1186/s13071-023-05662-y RESEARCH Open Access Interaction between Wolbachia pipientis and Leishmania infantum in heartworm infected dogs Maria Stefania Latrofa1, Ilaria Varotto‑Boccazzi2, Viviane Noll Louzada‑Flores1, Roberta Iatta3, Jairo Alfonso Mendoza‑Roldan1, Xavier Roura4, Andrea Zatelli1, Sara Epis2,5, Claudio Bandi2,5 and Domenico Otranto1,6* Abstract Background Wolbachia is a Gram-negative endosymbiont associated with several species of arthropods and filarioid nematodes, including Dirofilaria immitis. This endosymbiont may elicit a Th1 response, which is a component of the immunity against Leishmania infantum. Methods To investigate the interactions between Wolbachia of D. immitis and L. infantum in naturally infected dogs and cytokine circulation, dogs without clinical signs (n = 187) were selected. Dogs were tested for microfilariae (mfs) by Knott, for female antigens of D. immitis by SNAP, and for anti-L. infantum antibodies by IFAT and assigned to four groups. Dogs of group 1 (G1) and 2 (G2) were positive for D. immitis and positive or negative to L. infantum, respec‑ tively. Dogs of group 3 (G3) and 4 (G4) were negative to D. immitis and positive or negative to L. infantum, respectively. Wolbachia and L. infantum DNA was quantified by real-time PCR (qPCR) in dog blood samples. A subset of dogs (n = 65) was examined to assess pro- and anti-inflammatory cytokine production using an ELISA test. Results Of 93 dogs positive to D. immitis with circulating mfs, 85% were positive to Wolbachia, with the highest amount of DNA detected in G1 and the lowest in dogs with low mfs load in G1 and G2. Among dogs positive to L. infantum, 66% from G1 showed low antibody titer, while 48.9% from G3 had the highest antibody titer. Of 37 dogs positive to Wolbachia from G1, 26 (70.3%) had low antibody titers to L. infantum (1:160). Among cytokines, TNFα showed the highest mean concentration in G1 (246.5 pg/ml), IFNγ being the one most represented (64.3%). IL-10 (1809.5 pg/ml) and IL-6 (123.5 pg/ml) showed the highest mean concentration in dogs from G1. A lower percentage of dogs producing IL-4 was observed in all groups examined, with the highest mean concentration (2794 pg/ml) recorded in G2. Conclusion Results show the association of D. immitis and Wolbachia with the lower antibody titers of L. infantum in co-infected dogs, suggesting the hypothesis that the endosymbiont may affect the development of the patent leishmaniosis. However, due to the limitations associated with the heterogeneity of naturally infected dogs in field conditions, results should be validated by investigation on experimental models. Keywords Canine, Dirofilariosis, TNFα, IFNγ, Immune response, IL-4, IL-6, IL-10, Leishmaniosis, Wolbachia *Correspondence: Domenico Otranto domenico.otranto@uniba.it Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. 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 in a credit line to the data. Latrofa et al. Parasites & Vectors (2023) 16:77 Page 2 of 11 Background Methods The endosymbiotic relationship described between Enrolled animals, parasitological and serological Wolbachia and filarioid nematodes (families Oncho- examination cercinae, Dirofilariinae and Splendidofilariinae) has A total of 187 dogs presenting no apparent clinical signs important implications in the biological processes of were selected to reduce other factors that could interfere reproduction, development, molting and embryogen- with the analyses. All dogs, living in two municipal shel- esis of filarioids [1–4]. Dirofilaria immitis, the causa- ters in southern Italy (Lecce: 40.419326N, 18.165582E; tive agent of the canine heartworm disease (HWD), Casarano 40.0126N, 18.1606E) were subjected to physi- harbors Wolbachia endosymbiont in all its develop- cal examination to establish their health status. Among mental stages, from adult to microfilariae (mfs) [5]. them, a cohort of dogs (n = 84) was enrolled from a pre- In addition, clinical studies indicate that Wolbachia vious study aiming to control D. immitis and L. infantum is released into the bloodstream of hosts after death infection [24]. of D. immitis [6], also because of filaricidal treatment Blood samples were collected from either the cephalic [7, 8]. Furthermore, Wolbachia associated to D. immi- or jugular veins and placed in a K3 EDTA tube (2 ml) and tis infection may increase the severity of the clinical in a clot activator tube (5 ml) to obtain serum after cen- signs of HWD by triggering inflammatory response [7, trifugation (15 min at 1500 × g). Individual blood samples 9–11]. As observed in other filarioids, such as Oncho- were screened by modified Knott test for the detection of cerca volvulus [12], D. immitis may stimulate Th2 anti- mfs of D. immitis, as previously described [37]. The mfs inflammatory response, while Wolbachia triggers the identification was performed measuring the body length Th1 pro-inflammatory response [10, 13, 14]. The latter and width of specimens and by morphological analysis of is likely due to the effect that Wolbachia surface protein the front end and the tail according to [38], using a digi- (WSP) and other molecules exert on antigen presenting tally captured image software LAS V4.5 (Leica Microsys- cells and cytokine production [6, 7, 12, 15–18]. In addi- tems). The count of the mfs (2 × 50 μl) was based on the tion, in vitro experiment using Asaia-WSP engineered average of the counting in the two slides. Dogs with a bacterium was shown to elicit the Th1 cellular response mfs load ≥ 600 mfs/20 μl were considered highly micro- detection of adult females of D. immitis by SNAP® 4Dx® against Leishmania infantum, with a protozoal killing filaremic [24]. Serum samples were tested for the antigen effect [19]. Given the sympatric occurrence of L. infantum and Plus Test (IDEXX Laboratories, Inc.), according to the D. immitis in many geographical areas around the manufacturer’s instructions. world [20–24], the association of both vector-borne To assess the exposure of dogs to L. infantum infection, pathogens is of major interest from a diagnostic and sera were tested by immunofluorescence antibody test clinical perspective [25]. For example, the prevalence (IFAT) using as antigen the promastigotes of L. infantum of D. immitis infection increased in areas of southern zymodeme MON-1, as previously described [39]. Sam- Europe, where canine leishmaniosis (CanL) was his- ples were considered positive when they produced a clear torically endemic, as a consequence of pets traveling cytoplasmic and membrane fluorescence of promastig- with their owners (i.e. from endemic to previously otes from a cut-off dilution of 1:80. Positive sera were non-endemic regions) and changes in arthropod vec- titrated by serial dilutions until negative results were tor ecology and distribution [20, 21, 24, 26–30]. This obtained. overlapping geographical distribution of D. immitis Subsequently, all dogs were divided into four groups and L. infantum led to potential immune interactions based on their positivity/negativity for mfs of D. immitis [20, 21, 24, 27, 28]. Indeed, the prevalence of dogs with- and to anti-Leishmania antibodies. Specifically, dogs pos- out clinical signs [31] depends on the balance between itive for mfs and positive and/or negative to IFAT were Th1 and Th2 immune response [32–35], which may be grouped in G1 (n = 47) and G2 (n = 46), respectively, regulated by a plethora of factors (e.g. animal genetic, while dogs negative for mfs and for female antigens of D. health and nutritional status, concurrent infections) immitis and positive or negative to IFAT were included in [36]. In this context, the clinical manifestations of a G3 (n = 47) and G4 (n = 47), respectively (Table 1). patent leishmaniosis in dogs co-infected by D. immitis In addition, a group of dogs (n = 65), selected based may be affected by the presence of Wolbachia endos- on their negativity and/or positivity to IFAT with a ymbiont [21, 27, 32]. Thus, the aim of this study was to titer ≥ 1:160 and for mfs, were examined by analyzing the investigate the interactions of Wolbachia in dogs natu- production of pro-inflammatory cytokines such as tumor rally infected by D. immitis and/or L. infantum and to necrosis factor α (TNFα) and interferon-gamma (IFNγ) assess the relationship between pathogen infection and and of anti-inflammatory cytokines such as interleukin-4 cytokine circulation. (IL-4), interleukin-6 (IL-6) and interleukin-10 (IL-10) Latrofa et al. Parasites & Vectors (2023) 16:77 Page 3 of 11 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 Groups Dogs Knott test Snap test qPCR Wolbachia IFAT qPCR L. infantum mfs load L. infantum n dogs Pos (%; Ct n dogs; titer n dogs Pos (%; Ct 95% CI) 95% CI) min–mean–max min–mean–max G1 Dim + /Leis + 18 5 < low ≤ 200 Na 11 (61.1; 29.2–33.1–37.1 n = 7; 1:80 1 (5.5; 0.001– 37.8 0.357–0.827) n = 2; 1:160 0.273) n = 4; 1:320 n = 3; 1:640 n = 2; 1:2560 10 200 < medium ≤ 300 7 (70; 0.348– 26.6–31.7–35.3 n = 7; 1:80 1 (10; 0.003– 33.9 0.933) n = 2; 1:320 0.445) n = 1; 1:640 19 High ≥ 600 19 (100; 22.1–29.5–35.4 n = 11; 1:80 4 (21; 0.061– 24.7–30.7–36.5 0.824–1.000) n = 4; 1:160 0.456) n = 2; 1:320 n = 2; 1:1280 Total 47 37 (78.7; 6 (12.8; – 0.643–0.893) 0.048–0.257) G2 Dim + /Leis− 20 1 < low ≤ 200 Na 16 (75; 28.8–32.9–37.4 Neg Neg Na 0.563–0.943) 11 200 < medium ≤ 300 11 (100; 28.3–30.6–32.8 0.715–1.000) 15 high ≥ 400 15 (100; 28.4–31.1–35.6 0.782–1.000) Total 46 – 42 (91.3; – – – 0.792–0.976) G3 Dim −/Leis + Neg Neg Na Na n = 15; 1:80 2 (4.2; 0.005– 32.59–32.8–33.1 n = 9; 1:160 0.145) n = 6; 1:320 n = 8; 1:640 n = 6; 1:1280 n = 3; 1:2560 Total 47 – – – – – 2 (4.2; 0.005– – 0.145) G4 Dim −/Leis − 47 Neg Neg Na Na Neg Neg Na Total 187 – 79 (85; 8 (4.3; 0.019– 0.760–0.915) 0.083) Minimum, mean and maximum of threshold value are indicated. Ct = threshold value, 95% CI = 95% confidence level using an ELISA kit, (Biolegend, USA, and Thermo Fisher, cultured zymodeme MON-1 was used as positive con- USA) (Table 2). The positivity of the sera was determined trol, whereas gDNA extracted from blood sample from a based on specific standard curves. healthy dog was used as negative controls. Samples were tested for Wolbachia of D. immitis by Genomic DNA blood extraction and molecular procedures qPCR using primers (111 bp, WDiro.ftsZ.490-F/WDiro. Genomic DNA (gDNA) was extracted from each blood ftsZ.600-R) and probe wDimm.ftsZ.523p (6FAM-CGT​ sample using the GenUPgDNA commercial kit (Bio- ATT​GCA​GAG​CTC​GGA​TTA-TAMRA) targeting techrabbit GmbH, Hennigsdorf, Germany) according to the ftsZ gene as previously described [41] with minor the manufacturer’s instructions. All samples were tested modifications. for L. infantum kDNA minicircle by real time-PCR Briefly, all qPCR reactions were carried out in a final (qPCR) using the primers, probes and cycle protocol volume of 20 μl, consisting of 10 μl IQ Supermix (Bio- described elsewhere [40]. gDNA from L. infantum isolate Rad Laboratories, Hercules, CA, USA), 6 μl diethyl Latrofa et al. Parasites & Vectors Table 2 Prevalence of cytokine production, calculated as the percentage of dogs from each group producing one cytokine, divided according to groups Groups qPCR Cytochine production (2023) 16:77 Wolbachia n dogs Pos (%) TNFα IFNγ IL-10 IL-4 IL-6 Concentration Prevalence Concentration Prevalence Concentration Prevalence Concentration Prevalence Concentration Prevalence (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) min–mean– n dogs Pos (%; min–mean– n dogs Pos (%; n dogs Pos min–mean– n dogs Pos (%; min–mean– n dogs Pos max 95%CI) max 95%CI) (%; 95%CI) max 95%CI) max (%;95%CI) G1 (n = 14) 12 (85.7)6,7,8 44.8–246.5– 4 (28.6; 3.6–88.2–475 9 (64.3; 5.97–1809.5– 10 (71.4; 77.2 1 (7.1; 0.002– 3.01–123.5 5 (35.7; 649.4 0.084–0.581)1,8 0.351–0.872)a,2 4120 0.419–0.916)1,3 0.339)2,3,6 -392.6 0.128–0.649)7 G2 (n = 14) 14 (100) 2.27 – 65.1 6 (42.8; 13.3–105.4– 7 (50; 0.230– 19.1–507.8– 7 (50; 501.1–2794.5– 4 (28.5; 5.05–30.2–105 7 (50; 0.230– -172.9 0.177–0.711) 265.1 0.770)b 1440 0.230–0.770) 5739.5 0.084–0.581) 0.770) G3 (n = 14) N/A – – 0.65–97.23– 7 (50; 0.230– 50.4–677.4– 6 (42.8; 62.5–254.9– 4 (28.5; 6.1–29.7–67.2 7 (5 0:0.230– 617.6 0.770)b 2644.8 0.177–0.711) 480.4 0.084–0.581) 0.770) G4 (n = 14) N/A 8.1- 13.3–18.6 2 (14.3; 3.6–3.7–23.7 2 (14.2; 0.018– 81.9–863.8– 8 (57.1; 62.5–111.7– 2 (14.3; 5.05–13.9–22.3 5 (35.7; 0.018–0.428)4 0.428)a,b,4 3320 0.289–0.823)4,5 160.9 0.018–0.428)5 0.128–0.649) Total (n = 56) 26 (46.4) – 12 (21.4; – 25 (44.6; 0.313– – 31 (55.4; – 11 (19.6; – 24 (42.9; 0.116–0.344) 0.585) 0.415–0.687) 0.102–0.324) 0.297–0.568) Minimum, mean and maximum of concentration (pg/ml) are indicated. Statistically significance differences in prevalence are marked with equal superscript letters. P-value < 0.05. 95% CI = 95% confidence level. Page 4 of 11 Latrofa et al. Parasites & Vectors (2023) 16:77 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 Groups Knott test n dogs qPCR Wolbachia mfs load Ct = 0 Prevalence (%); 95% CI 25 ≤ Ct ≤ 30 Prevalence (%); 95% CI 30 < Ct ≤ 34 Prevalence (%); 95% CI Ct > 35 Prevalence (%); 95% CI d G1 Dim + /Leis + 5 ≤ low ≤ 200 18 7 38.9; 0.173–0.643 2 11.1; 0.014–0.347 4 22.2; 0.064–0.476 5 27; 0.097–0.535 200 < medium ≤ 300 10 3 30; 0.067–0.652 3 30; 0.067–0.652 1 10; 0.003–0.445 3 30; 0.067–0.652 High ≥ 600 19 0 – 12c,d 63.2; 0.384–0.837 5c 26.3; 0.091–0.51 2c 30; 0.067–0.652 Tot 47 10 21.3; 0.107–0.357 17 36.2; 0.227–0.515 10 21.3; 0.107–0.357 10 21.3; 0.107–0.357 G2 Dim + /Leis− 1 ≤ low ≤ 200 20 5 25%; 0.087–0.491 5 25; 0.087–0.491 6 30; 0.119–0.543 4 20; 0.057–0.437 200 < medium ≤ 300 11 0 – 5 54.5; 0.234–0.833 6 54.5; 0.234–0.833 0 0 High ≥ 400 15 0 – 8e 53.3; 0.266–0.787 6e 40; 0.163–0.677 1e 6.7; 0.002–0.319 Tot 46 5 10.9; 0.036–0.236 18 39.1; 0.251–0.546 18 39.1; 0.251–0.546 5 10.9; 0.036–0.236 Total 93 15 16.1; 0.093–0.252 35a 37.6; 0.278–0.483 28b 30.1; 0.210–0.405 15a,b 16.1; 0.093–0.252 Ct = threshold value, 95% CI = 95% confidence level. Statistically significant differences in prevalence are marked with equal superscript letters. P-value < 0.05 Page 5 of 11 Latrofa et al. Parasites & Vectors (2023) 16:77 Page 6 of 11 pyrocarbonate (DEPC)-treated pyrogen-free DNase/ Of 94 dogs positive for anti-Leishmania antibodies, 31 RNase-free water (Invitrogen, Carlsbad, CA, USA), 2.5 μl were from G1 (66%; 95% CI: 0.507–0.791) with low anti- of template DNA (except no-template control), primers body titers (up to 1:160), followed by 16 dogs (34%; 95% and probe at 50 μM and 20 μM concentration, respec- CI: 0.209–0.49) with higher titers (from 1:320 to 1:2560). tively. The run protocol consisted of a hot-start at 95 °C In G3, 23 dogs (48.9%; 95% CI: 0.341–0.639) had a high for 3 min and 40 cycles of denaturation (95 °C for 5 s) and antibody titer (from 1:320 to 1:2560) (Table 1). The qPCR annealing-extension (60 °C for 30 s). The qPCR was per- screened eight positive dogs of 187 blood samples exam- formed in a CFX96 Real-Time System (Bio-Rad Labora- ined for kDNA of L. infantum (4.3%) with a minimum tories, Inc., Hercules, CA, USA), and the increase in the and maximum Ct value (min = 24.7, max = 37.8) detected fluorescent signal was registered during the extension for dogs from G1 (Table 1). step of the reaction and analyzed using CFX Manager Of 37 dogs positive for Wolbachia in G1, 26 (70.3%; Software, version 3.1 (Bio-Rad Laboratories, Inc., Hercu- 95% CI: 0.530–0.841) had low antibody titers (n = 20 les, CA, USA). All the samples were tested in duplicate, 1:80, n = 6 1:160) against L. infantum (Table 4). A statis- and DNA of adult of D. immitis and that from blood tical difference was observed between dogs showing the samples of pathogen-free dogs were used as positive highest amount of Wolbachia DNA (Ct value range of and negative controls. The positivity for Wolbachia was 25–30) and those with antibody titers ranging from 1:320 established based on the threshold cycle (Ct) value up to to 1:2560 (χ2 = 6.8, P = 0.0089) (Table 4). 38.5. IL-10 (55.4%) and IFNγ (44.6%) were the most prevalent cytokines produced throughout dogs examined (Table 2 Statistical analysis and Fig. 1). In particular, the percentage of dogs produc- Associations between infections and variables were ing TNFα was higher in G2 (42.8%) than G1 (28.6%) and assessed through univariate analysis. Exact binomial test G4 (14.3%). However, the highest mean of TNFα concen- established the confidence intervals (CI) with 95% confi- tration was detected in G1 (246.5 pg/ml) and the lowest dence level. The Chi-square χ2 test was used to compare in G2 (65.1 pg/ml) and G4 (13.3 pg/ml) (Table 2, Fig. 1). percentages of positivity among categories of the same A statistically significant difference (χ2 = 7.3, P = 0.007) independent variables. Collinearity among independent was recorded analyzing the percentages of IFNγ-positive variables was assessed using Pearsonʼs correlation coef- sera between dogs from G1 (64.3%) and G4 (14.2%), with ficient. A P-value < 0.05 was considered as statistically the same percentage of positive dogs in G2 and G3 (50%) significant. Statistical analyses were performed using (Table 2). The highest mean of concentration of IFNγ StatLib for Windows (version 13.0, SPSS, Inc., Chicago, production was observed in G2 (105.4 pg/ml) and G3 IL, USA) Quantitative Parasitology 3.0 [42] and Graph- (97.23 pg/ml) (Table 2, Fig. 1). The highest percentage of Pad Prism 8 software. dogs producing IL-10 (71.4%) was observed in G1 (mean concentration of 1809.5 pg/ml) followed by G4 (57.1%) Results and G3 (42.8%) (Table 2). The lowest percentage of dogs Of 93 mfs-positive dogs, 79 (85%) tested positive for producing IL-4 was observed in G1 (7.1%), G2 and G3 Wolbachia DNA (Table 1), with 37 from G1 (46.8%; 95% (28.5%) (Table 2, Fig. 1). The highest mean concentration CI: 0.355–0.584) and 42 from G2 (53.2%; 95% CI: 0.416– of IL-4 was observed in G2 (2794.5 pg/ml). IL-6 showed 0.645). The highest amount of Wolbachia DNA (Ct value the same percentage of positivity in G1 and G4 (35.7%) 22.1) was detected in a dog from G1 (mfs load ≥ 600) and and in G2 and G3 (50%), respectively, with the highest the lowest (Ct value 37.4) in dogs with low mfs load (i.e. mean concentration registered in G1 (123.5 pg/ml) and 1 ≤ mfs ≤ 200) in both G1 and G2 (Table 1). No statisti- a similar concentration observed in G2 (30.2 pg/ml) and cally significant difference in Wolbachia prevalence was G3 (29.7 pg/ml) (Table 2, Fig. 1). observed between dogs from G1 and G2, though an over- Except for IFNγ, no statistically significant differences all higher value (37.6%) was recorded in dogs with a Wol- were observed in cytokine production between groups, bachia Ct values ranging from 25 to 30 (Table 3). In G1, but within groups. In particular, in G1 a statistically sig- a statistical difference was observed between dogs with nificant difference was observed between dogs produc- Wolbachia Ct value of 25–30 vs. 30–34 (χ2 = 6, P < 0.0138) ing TNFα vs. IL-10 (χ2 = 5.1, P = 0.023), IFNγ vs. IL-4 and vs. Ct > 35 (χ2 = 12.3, P = 0.0004) having a high mfs (χ2 = 9.9, P = 0.001), IL-10 vs. IL-4 (χ2 = 12.1, P = 0.005) load and between low vs. high mfs score (χ2 = 10.64, and IL-4 vs. IL-6 (χ2 = 17.4, P = 0.00003). Similarly, in G4 P = 0.011) (Table 3). In G2, a statistically significant dif- a statistically significant difference was observed between ference (χ2 = 9.8, P = 0.00178) was observed between IFNγ and TNFα vs. IL-10 and between IL-10 vs. IL-4 dogs with Wolbachia Ct value of 25–30 and 30–34 (χ2 = 5.6, P = 0.018). No statistically significant difference vs. > 35, having high mfs load (Table 3). was observed in cytokine production within groups G2 Latrofa et al. Parasites & Vectors (2023) 16:77 Page 7 of 11 Table 4 Wolbachia-positive dogs divided according to Ct value of endosymbiont DNA and Leishmania infantum positivity to different tests Group n dogs Pos (%) qPCR IFAT qPCR Wolbachia L. infantum L. infantum n dogs Pos (%) 1:80 1:160 1:320 1:640 1:1280 1:2560 n dogs Pos (%) G1 15 (40.5)a,c 25 ≤ Ct < 30 10 (66.7) (1 (6.7) 2 (13.3) 0 2 (13.3) 0)a 3 (20)c b,d b 13 (35.1) 30 ≤ Ct < 34 5 (38.5) 2 (15.4) 3 (23.1) 2 (15.4) 0 1 (7.7) 3 (23.1)d 9 (24.3) 34 ≤ Ct < 35 5 (55.5) 3 (33.4) 0 1 (11.1) 0 0 0 Total 37 20 (54.1) 6 (16.2) 5 (13.5) 3 (8.1) 2 (5.4) 1 (2.7) 6 (16.2) and G3 (Table 2). In addition, a statistically significant production of high levels of pro-inflammatory cytokines difference was observed in G1 between dogs positive for [12, 19, 21, 27, 44]. For example, the high Wolbachia Wolbachia vs. TNFα (χ2 = 9.3, P = 0.022), IL-4 (χ2 = 17.4, amount recorded in co-infected dogs may trigger an ele- P = 0.00003) and IL-6 (χ2 = 7.3, P = 0.0067) (Table 2). vated TNFα production (mean value 246.5 pg/ml), which was not recorded in dogs infected with only L. infantum. Discussion The strong effect of Wolbachia in producing this pro- Data herein presented suggest that Wolbachia, associ- inflammatory cytokine has also been demonstrated in ated with D. immitis, may affect the immune response an in vitro experiment [19], regardless of the presence of against L. infantum of naturally co-infected dogs through L. infantum. However, the absence of TNFα observed in a stimulatory or immune-suppressive mechanism [4]. dogs positive for L. infantum agrees with previous studies Indeed, the high molecular prevalence of Wolbachia in where the cytokine was detected only in a few dogs with co-infected dogs (78.7%) is coherent with the hypothesis active leishmaniosis [45, 46]. that this endosymbiont might control the Leishmania The Wolbachia amount detected in co-infected infection. Overall, the prevalence of Wolbachia observed dogs, and consequently its effect on controlling the L. in co-infected dogs is in line with data described in a pre- infantum infection, may also be supported by the high vious study [21], where the endosymbiont was detected prevalence of dogs (64.3%) producing IFNγ, a simi- in 68.8% dogs from Portugal. Nevertheless, the high lar mean cytokine concentration value being observed amount of Wolbachia DNA in relationship to the mfs between these dogs (88.2 pg/ml) and those positive for load (i.e. mfs > 600; Ct 25–30) exclusively observed in co- L. infantum (97.23 pg/ml) or D. immitis (105.4 pg/ml). infected dogs (63.2%) differed from a previous study from Accordingly, IFN-γ was associated with the absence Spain, where Wolbachia was more frequently detected in or low antibody titer against L. infantum [47]. Besides microfilaraemic dogs not infected with L. infantum [27]. TNFα and IFNγ, the detection of IL-4, IL-6 and IL-10 In addition, in this previous study, an increased severity in infected dogs indicates a mixed Th1/Th2 immune of clinical leishmaniotic signs was observed in microfila- response. Indeed, in previous studies using periph- raemic dogs with a lower prevalence of Wolbachia [27]. eral blood mononuclear cells (PBMCs) of dogs experi- Furthermore, the role of the endosymbiont in stimulating mentally infected with L. infantum or stimulated with a Th1 immune response was also suggested by the low L. infantum antigen, an increase in IFNγ, IL-10 and number (3/11) of co-infected dogs with clinical signs, as IL-4 mRNA expression levels were recorded [35, 48, previously described [21]. 49]. In addition, the expression of cytokines related The role of Wolbachia in controlling the development to both Th1/Th2 responses was also detected in dogs of CanL may be highlighted by the high prevalence of naturally infected with D. immitis with circulating co-infected dogs (70.3%) showing a low anti-Leishmania mfs associated to the presence of Wolbachia [10, 50]. antibody titer (up to 1:160), likely due to the low parasitic All the above considerations may justify the high mean load detected by qPCR in the blood [43], while a high concentration (1809.5 pg/ml) of IL-10 recorded in co- value of IFAT titers was observed in most of dogs (48.9%) infected dogs as well as its even production in dogs infected only with L. infantum. infected with D. immitis (507.8 pg/ml) or L. infantum Furthermore, the absence of clinical signs in dogs may (677.4 pg/ml). However, though IL-10 is considered be also determined by the stimulation of a Th1 immune a predictive parameter of the evolution of CanL and response triggered by the endosymbiont bacterium by active visceral leishmaniosis in humans [51–53], other Latrofa et al. Parasites & Vectors (2023) 16:77 Page 8 of 11 Fig. 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) studies described the expression of this cytokine in control dogs. Similarly, the detection of IL-4 in all dogs without clinical signs [48, 54–56]. Furthermore, groups of dogs examined is not surprising, consider- the detection of IL-10 in uninfected dogs (863.8 pg/ml) ing that the role of this cytokine in the pathogenesis of agrees with the results described elsewhere [54], where CanL is still debated [54]. Indeed, in the current study the mRNA accumulation of this cytokine in dogs with a very low mean concentration (77.2 pg/ml) of IL-4 severe disease was comparable with that of uninfected was detected in co-infected dogs compared with those Latrofa et al. Parasites & Vectors (2023) 16:77 Page 9 of 11 recorded in L. infantum infected (254.9 pg/ml) or unin- (Project no. PE00000007, INF-ACT). S.E. and C.B. thank the UNIMI GSA-IDEA project. fected dogs (111.7 pg/ml). However, other studies also described these contrasting results, IL-4 being detected Author contributions readily or not in L. infantum infected or asymptomatic Conceptualization, MSL, DO; Methodology, MSL, IVB, VNLF, RI, SE; Formal Analy‑ sis, MSL, IVB, VNLF, RI; Writing Original Draft Preparation, MSL, DO; Writing- dogs [48, 54, 57, 58]. The Wolbachia amount recorded Review & Editing, MSL, IVB, VNLF, RI, JAMR, AZ, XR, SE, CB, DO; Supervision, MSL, in the co-infected dogs may have affected the IL-6 SE, CB, DO. All authors read and approved the final manuscript. production (123.5 pg/ml) in these dogs. Indeed, the Funding role of this endosymbiont in stimulation of IL-6 pro- Not applicable. duction was also supported by an in vitro experiment [19]. However, though IL-6 is generally regarded as a Availability of data and materials All data generated or analyzed during this study are included in this published Th2 cytokine with disease progression, other studies article. described its protective role in some forms of leish- maniosis [59], thus indicating an imperfect fitting of Declarations this cytokine into the Th1/Th2 paradigm [45, 48, 60]. Overall, interpreting the cytokine expression pro- Ethics approval and consent to participate Protocols for collection of dog samples were approved by the ethical com‑ file in CanL is still problematic, and the differences mittee of the Department of Veterinary Medicine of the University of Bari, Italy observed regarding the role of cytokines may be due (Prot. Uniba 12/20). to the different methods used for the analyses (i.e. dos- Consent for publication age by ELISA or evaluation of mRNA expression) [48, Not applicable. 54, 56]. In addition, immune response mediated by cytokines may be influenced by several variables (i.e. Competing interests The authors declare that they have no competing interests. age, tissues examined) [35, 53]. Thus, the comprehen- sive knowledge of cytokine response and their inter- Author details action is a very crucial step to understand disease 1 Department of Veterinary Medicine, University of Bari, Bari, Italy. 2 Department of Biosciences, University of Milan, Milan, Italy. 3 Interdisciplinary Department progression, mainly in co-infected dogs. of Medicine, University of Bari, Bari, Italy. 4 Hospital Clínic Veterinari, Universitat Autònoma de Barcelona, Barcelona, Spain. 5 Pediatric CRC ‘Fondazione Romeo ed Enrica Invernizzi’, University of Milan, Milan, Italy. 6 Faculty of Veterinary Sci‑ ences, Bu-Ali Sina University, Hamedan, Iran. Conclusions Though the present work has some limitations, such Received: 28 November 2022 Accepted: 10 January 2023 as the low number of dogs included and their hetero- geneity, 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 References 1. Taylor MJ, Bandi C, Hoerauf A. Wolbachia bacterial endosymbionts of endosymbiont in the modulation of the Th1 immune filarial nematodes. Adv Parasitol. 2005;60:245–84. response. However, future studies based on the simul- 2. Bandi C, Trees AJ, Brattig NW. Wolbachia in filarial nematodes: evolution‑ taneous combination of different approaches of analy- ary aspects and implications for the pathogenesis and treatment of filarial diseases. Vet Parasitol. 2001;98:215–38. sis (i.e. expression of the mRNA vs. quantification of 3. Martin C, Gavotte L. 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