Attipa et al. BMC Veterinary Research (2019) 15:331 https://doi.org/10.1186/s12917-019-2083-6 RESEARCH ARTICLE Open Access Associations between clinical canine leishmaniosis and multiple vector-borne co- infections: a case-control serological study Charalampos Attipa1,2,3,4* , Laia Solano-Gallego5, Christian M. Leutenegger6, Kostas Papasouliotis1,7, Francesca Soutter2, Jörg Balzer8, Scott Carver9, Jesse S. Buch10 and Séverine Tasker1,11,12 Abstract Background: Dogs that have clinical leishmaniosis (ClinL), caused by the parasite Leishmania infantum, are commonly co-infected with other pathogens, especially vector-borne pathogens (VBP). A recent PCR-based study found that ClinL dogs are more likely to be additionally infected with the rickettsial bacteria Ehrlichia canis. Further information on co- infections in ClinL cases with VBP, as assessed by serology, is required. The research described in this report determined if dogs with ClinL are at higher risk of exposure to VBP than healthy control dogs using a case-control serology study. Results: Of the 47 dogs with ClinL, anti-E. canis/ Ehrlichia ewingii antibodies were detected in 17 (36.2%), anti- Anaplasma phagocytophilum/Anaplasma platys antibodies in 5 (10.6%) and antigen for Dirofilaria immitis in 2 (4.3%). Of the 87 control dogs, anti-E. canis/E. ewingii antibodies were detected in 14 (16.1%) and anti-A. phagocytophilum/ A. platys antibodies in 2 (2.3%). No anti-Borrelia burgdorferi antibody tests were positive. No statistical differences between the ClinL dogs and control dogs regarding lifestyle or use of ectoparasitic prevention, were identified. The ClinL was significantly associated with anti-E. canis/E. ewingii antibodies (odds ratio = 2.9, 95% confidence interval: 1.3– 6.7, P = 0.010) compared to controls by both multivariable logistic regression and structural equation modelling. Conclusions: It was demonstrated that an increased risk for E. canis/E. ewingii seropositivity is present in dogs with ClinL compared to clinically healthy control dogs, despite similar ectoparasitic prevention use and lifestyle. Based on these findings it is suggested that dogs with ClinL should not only be tested for E. canis co-infection using PCR but also serologically for E. canis/E. ewingii. Keywords: Dog, Leishmania infantum, Ehrlichia canis, Borrelia burgdorferi, Acanthocheilonema reconditum, Vector- borne pathogen, Co-infection, Cyprus Background (ClinL) despite being transmitted by vectors different Canine leishmaniosis (CanL) is a significant zoonotic than these for L. infantum [2–4]. Such co-infections can disease in Mediterranean region and is caused by the result in an unexpected incubation time, atypical clinical kinetoplastid parasite Leishmania infantum that is trans- sings, more severe clinicopathological abnormalities and mitted by sand flies vectors belonging to the Phleboto- worse prognosis for the dogs with CanL, compared with mus genus [1]. Often vector-borne pathogens (VBP) dogs that have CanL alone [2, 3, 5]. Furthermore, a such as Anaplasma platys, Ehrlichia canis, Dirofilaria recent PCR-based case-control study found that dogs immitis, Hepatozoon canis and Babesia vogeli concur- with ClinL are in higher risk to be co-infected with E. rently infect dogs which have clinical leishmaniosis canis compared to healthy matched controls [6]. Additional information on co-infections in ClinL cases * Correspondence: attipacy@gmail.com with VBP, as assessed by serology in case-control stud- 1 Molecular Diagnostic Unit, Diagnostic Laboratories, Bristol Veterinary School ies, is required. and Langford Vets, University of Bristol, Langford, UK 2 Department of Pathobiology and Population Sciences, The Royal Veterinary The aim of this study was to examine if dogs with College, University of London, Hatfield, Hertfordshire, UK ClinL are more likely to be exposed to A. phagocytophilum/ Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/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://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Attipa et al. BMC Veterinary Research (2019) 15:331 Page 2 of 6 A. platys, B. burgdorferi and E. canis/Ehrlichia ewingii, or Table 1 Demographic characteristics of the study dog groups infected for D. immitis than clinically healthy controls. and serology results for the VBPs tested. All dogs tested negative for Borrelia burgdorferi antibodies Results Characteristic No. of cases ClinL (%) (n = 47) No. of Control (%) (n = 87) Serum was available in 47 dogs with ClinL and 87 Age in years dog controls that were included in this study. The Median 3.0 4.1 age of these 134 dogs ranged from 1 up to 12 years Interquartile range 3.3 3.0 (median 4 years, interquartile range 3 years) and 98 Sex (73%) were pedigree including Cocker spaniel, Segugio Male 23 (49) 47 (54) Italiano, Beagle, German Shepherd and other breeds Female 24 (51) 40 (46) (Additional file 1). Lifestyle In the ClinL group, anti-A. phagocytophilum/A. platys Outdoors 34 (72) 65 (75) antibodies were detected in 5 (10.6%), anti-E. canis/E. Mainly indoors 13 (28) 22 (25) ewingii antibodies in 17 (36.2%) and antigen for D. immitis in 2 (4.3%) dogs. Of the 87 control dogs, anti-A. Ectoparasitic prevention phagocytophilum/A. platys antibodies were detected in 2 Used 16 (34) 36 (41) (2.3%) and anti-E. canis/E. ewingii antibodies in 14 Not used 31 (66) 51 (59) (16.1%). No anti-B. burgdorferi antibody tests were Breed positive (Fig. 1). Table 1 summarizes the demographic Pedigree 33 (70) 65 (75) characteristics and the serology findings. The two dogs Crossbreed 14 (30) 22 (25) with D. immitis antigens underwent microfilaria PCR E. canis/E. ewingii specification which was positive for A. reconditum and Positive 17 (36) 14 (16) negative for D. immitis for both cases. Negative 30 (64) 73 (84) ClinL was significantly associated with anti-E. canis/E. A. phagocytophilum/A. platys ewingii antibodies [odds ratio (OR) = 2.9, 95% confidence Positive 5 (10) 2 (2) interval (CI): 1.3–6.7, P = 0.010], compared to healthy Negative 42 (90) 85 (98) controls using multivariable logistic regression. The D. immitis presence of anti-A. phagocytophilum/A. platys anti- Positive 2 (4) 0 (0) bodies was initially associated significantly with ClinL Negative 45 (96) 87 (100) compared to controls using univariable analysis (OR = Abbreviations: VBP Vector-borne pathogen, ClinL Clinical leishmaniosis, E. canis 5.1, 95% CI: 0.9–27.2, P = 0.038) but this association was Ehrlichia canis, E. ewingii Ehrlichia ewingii, D. immitis Dirofilaria immitis, A. phagocytophilum Anaplasma phagocytophilum, A. platys. Anaplasma platys not maintained during multivariable logistic regression Fig. 1 Comparison of VBP percentages detected by serology between dogs with ClinL (n = 47) and healthy control dogs (n = 87). Abbreviations: VBP, vector-borne pathogen; ClinL, clinical leishmaniosis; E. canis, Ehrlichia canis; E. ewingii, Ehrlichia ewingii; D. immitis, Dirofilaria immitis; A. phagocytophilum, Anaplasma phagocytophilum; A. platys. Anaplasma platys Attipa et al. BMC Veterinary Research (2019) 15:331 Page 3 of 6 analysis. The numbers of D. immitis were very low compared with healthy controls, but they did not found an hindering any further statistical analysis. Age, breed, sex, association between ClinL and E. canis seroreactivity. This lifestyle, and use of ectoparasitic prevention were not discrepancy probably reflects the different prevalence of statistically different between the ClinL and the control these pathogens in Cyprus and other Mediterranean areas dogs. in comparison to Catalonia. Two associations were identified based on SEM (Fig. 2, The seroprevalence of the various VBP in this specific Table 2). It was more likely for dogs with ClinL be E. canine population of 134 dogs from the area of Paphos, canis/E. ewingii seropositive and dogs seropositive for E. Cyprus, revealed a strikingly high seroreactivity to E. canis/E. ewingii are more likely to have be infected with canis/E. ewingii (23%) and anti-A. phagocytophilum/A. E. canis based on PCR. A trend was identified between platys (13%) antibodies compared to other studies from dogs with ClinL and A. phagocytophilum/A. platys Mediterranean countries using a similar in-house ELISA seropositive. kit as the one utilised in this study [8–10]. If quantitative ELISA or IFAT with higher sensitivity, compared to the Discussion in-house kit, were used in this study, then seropreva- The findings from this serology study are in agreement lences of the VBP could have been even higher than with previous studies [3, 7] and further support the these reported [11]. The area of Paphos, Cyprus, may be findings from the initial PCR based study, using a fairly Lyme disease free as no anti-B. burgdorferi antibodies similar cohort of samples, in which it was demonstrated were detected in any of the dogs tested in this study, that it is 12 times more likely for dogs with ClinL be and the tick vectors that transmit this pathogen, includ- co-infected with E. canis compared with healthy canine ing Ixodes Ricinus, have not yet been identified in controls (CI: 1.5–106.0, P = 0.022) [6]. A previous 3-year Cyprus [12]. In two dogs (1%) antigens for D. immitis longitudinal study, evaluating E. canis and L. infantum were detected but PCR failed to confirm this infection co-infection in naturally exposed dogs, found that E. and instead an infection with A. reconditum was identi- canis infection preceded L. infantum infection in dogs fied for both cases. These results may indicate that the with dual infections, thus suggesting that E. canis could dogs had dual infection with both D. immitis and A. contribute in the establishment of ClinL [7]. Interest- reconditum, and the negative PCR for D. immitis was ingly, a recent study by Baxarias et al. [5] from Catalonia as a result of low level microfilaraemia. However, (Spain) found that dogs with ClinL were four times more false positive D. immitis results cannot be ruled out likely to be seropositive for Rickettsia conorii and 14 entirely especially in light of a recent study from times most likely to be seropositive for A. phagocytophilum Cyprus in which, using a modified Knott’s testing for Fig. 2 Structural equation model showing predictors of vector-borne serological exposure status (except ClinL), and pathogen covariance (including ClinL), in domestic dogs. Values represent standardised coefficients among variables. Single headed arrows represent directional/causal relationships and double headed arrows covariance relationships among pathogens. For image clarity the serological status is in yellow boxes and the coefficients of host characteristics predicting pathogens are listed next to each host characteristic. The covariances E. canis, A. platys, Hepatozoon spp. and M. haemocanis were PCR-based diagnosed. In all cases, except age, variables are binomial (0 or 1) with 1 equal to male, outside, ectoparasitic prevention use, pedigree and positive pathogen status. Standardised coefficients with significant relationships of P ≤ 0.05 (also see Table 2) are denoted in bold. Abbreviations: ClinL, clinical leishmaniosis; E. canis, Ehrlichia canis; E. ewingii, Ehrlichia ewingii; A. phagocytophilum, Anaplasma phagocytophilum; A. platys. Anaplasma platys; M. haemocanis, Mycoplasma haemocanis Attipa et al. BMC Veterinary Research (2019) 15:331 Page 4 of 6 Table 2 Structural equation model statistical output showing host characteristics predicting serological exposure status for co- infecting pathogens (except ClinL), and the covariance among pathogens (including ClinL), in domestic dogs. The covariances E. canis, A. platys, Hepatozoon spp. and M. haemocanis were PCR based diagnosed. In all cases, except age, variables are binomial (0 or 1) with 1 equal to male, outside, ectoparasites controlled, pedigree and positive pathogen status Standardised coefficient/covariance z-value P-value E. canis/E. ewingii serology Age −0.081 −0.908 0.364 Sex −0.015 − 0.143 0.886 Lifestyle 0.059 0.515 0.606 Ectoparasite prevention −0.041 − 0.336 0.737 Pedigree 0.015 0.142 0.887 A. phagocytophilum/A. platys serology Age −0.035 −0.415 0.678 Sex 0.014 0.103 0.918 Lifestyle −0.037 − 0.363 0.717 Ectoparasite prevention −0.162 −1.532 0.126 Pedigree −0.111 −0.861 0.389 Covariances E. canis/E. ewingii ~~ ClinL 0.229 2.453 0.014 A. phagocytophilum/A. platys ~~ ClinL 0.183 1.654 0.098* E. canis/E. ewingii ~~ A. phagocytophilum/A. platys 0.180 1.443 0.149 E. canis/E. ewingii ~~ E. canis 0.317 2.164 0.030 E. canis/E. ewingii ~~ A. platys 0.009 0.092 0.926 E. canis/E. ewingii ~~ Hepatozoon spp. 0.068 0.747 0.455 E. canis/E. ewingii ~~ M. haemocanis 0.099 1.015 0.310 A. phagocytophilum/A. platys ~~ E. canis −0.015 −0.358 0.721 A. phagocytophilum/A. platys ~~ A. platys 0.349 1.290 0.197 A. phagocytophilum/A. platys ~~ Hepatozoon spp. −0.106 −1.167 0.243 A. phagocytophilum/A. platys ~~ M. haemocanis 0.000 −0.005 0.996 Significant relationships (P < 0.05) denoted by bold font and trending relationships (P < 0.1) denoted by * Abbreviations: VBP Vector-borne pathogen, ClinL Clinical leishmaniosis, E. canis Ehrlichia canis, E. ewingii Ehrlichia ewingii, A. phagocytophilum Anaplasma phagocytophilum, A. platys. Anaplasma platys, M. haemocanis Mycoplasma haemocanis morphological identification of microfilariae in a total of presented to a small animal veterinary hospital in Pa- 200 healthy dogs which did not receive any kind of heart- phos, Cyprus from April 2013 until March 2014. That worm prevention, only A. reconditum was identified in 9 area was selected since there are high numbers of dogs (4.5%) and no D. immitis was found [13]. CanL [14] and various canine VBP have been re- ported [15, 16]. Conclusions The exact recruiting criteria and the demographic It was demonstrated that dogs with ClinL are three characteristics recorded can be found in the previously times more likely to be exposed to E. canis/E. ewingii published study [6]. Briefly, the dogs that had ClinL were than clinically healthy control dogs in Paphos, Cyprus. naturally infected and matched with clinically healthy Furthermore, dogs from this area have a high seroreac- control dogs in-terms of breed, sex, age, living in the tivity to E. canis/E. ewingii and A. phagocytophilum/A. same geographical area as well as ideally lifestyle and platys while they are B. burgdorferi free. ectoparasitic prevention use. Methods Laboratory tests Study design, site and populations Approximately 1–2 ml of surplus serum collected in The samples used for this serology study were collected plain tubes and stored at − 20 °C until laboratory under the frame of a previous case-control study design processing at the Diagnostic Laboratories of the Royal [6]. All samples were collected from canine clinical cases Veterinary College, London, UK. Attipa et al. BMC Veterinary Research (2019) 15:331 Page 5 of 6 A commercial in-clinic patient-side SNAP® 4Dx® Plus Supplementary information test kit (IDEXX Laboratories, Inc., Westbrook, Maine, Supplementary information accompanies this paper at https://doi.org/10. 1186/s12917-019-2083-6. USA) was used for the simultaneous detection of anti- bodies against E. canis/E. ewingii, A. phagocytophilum/A. Additional file 1: Table S1. Raw datasets with signalment and test platys, and B. burgdorferi, as well as antigens for D. results per case enrolled. immitis, following manufacturer’s instructions. This ELISA kit utilises bi-directional flow of sample and Abbreviations automatic, sequential flow of wash solution and enzyme CI: Confidence interval; ClinL: Clinical leishmaniosis; ELISA: Enzyme-linked immunosorbent assay; IFAT: Immunofluorescence antibody test; OR: Odds substrate. For E. canis it detects antibodies to the pro- ratio; qPCR: Quantitative polymerase chain reaction; SEM: Structural equation teins p30 and p30–1, and for E. ewingii antibodies for modelling; VBP: Vector-borne pathogen p28 protein. For A. phagocytophilum/A. platys the assay Acknowledgements detects antibodies against a peptide from the MSP2/p44 Authors would like to thank the veterinarians Dr. Christos Yiapanis and Dr. major surface protein and the C6 peptide is used for the Kyriaki Neofytou from Cyvets Veterinary Centre, Paphos, Cyprus, for aiding in detection of antibodies to a surface lipoprotein of B. the collection of blood samples. burgdorferi. The assay detects antigens produced pri- Authors’ contributions marily from the uterus of female D. immitis (IDEXX CA, LSG, KP, JSB and ST conceived the study, and all participated in its Laboratories, Inc.). design and coordinated the experiments. CA, LSG and FS designed and Blood extracted DNA was submitted to IDEXX La- performed the collection of the samples. CA performed the serology analysis and the PCR analysis was performed by JB and CML. Statistical analysis was boratories, Ludwigsburg, Germany from all the cases performed by CA, and SC performed the SEM. CA and ST wrote the that yielded positive antigens for D. immitis for further manuscript with input from all of the authors. All authors reviewed and microfilaria specification using PCR specific assays for approved the final manuscript. D. immitis, Dirofilaria repens, Acanthocheilonema Funding reconditum and Acanthocheilonema dracunculoides. This clinical study has been sponsored by IDEXX Laboratories, Inc. by Additionally all samples underwent L. infantum serology providing the SNAP® 4Dx Plus® test kit as well as a clinical research grant from Langford Vets, University of Bristol which funded the PCR testing for [17], qPCRs for Leishmania spp. [18], Babesia spp. [19], Babesia spp., “Candidatus Mycoplasma haematoparvum”, Mycoplasma “Candidatus Mycoplasma haematoparvum” and Myco- haemocanis, Ehrlichia/Anaplasma spp. and Hepatozoon spp. Part of this work plasma haemocanis [20] as well as conventional PCR done by FS was supported by a Biotechnology and Biological Sciences Research Council Collaborative Awards in Science and Engineering assays for Ehrlichia/Anaplasma spp. [21] and Hepato- studentship (BB/1015655/1) in partnership with Zoetis and included zoon spp. [22] under the framework of a previously Leishmania spp. PCR and serology. CA is a Wellcome Trust Clinical Ph.D. published study [6]. Fellow (203919/Z/16/Z) and the open access publication fees have been sponsored by Wellcome Trust. Data analysis Availability of data and materials The sample size was previously calculated [6] and analyses The datasets supporting the conclusion of this article are included within the article and Additional file 1. were performed using SPSS for Windows (version 25.0; SPSS Inc., Chicago IL, USA). A univariable analysis was Ethics approval and consent to participate initially performed to see how each of the explanatory This study was ethically approved by the University of Bristol’s Animal Welfare and Ethical Review Board (Veterinary Investigation number: 15/022) variables was associated with ClinL using Pearson’s Chi- as well as the Royal Veterinary Collage’s Ethics and Welfare Committee square test for categorical explanatory variables (breed, (Veterinary Investigation number: 20141292). All procedures were performed sex, lifestyle, ectoparasitic prevention, positivity for A. in accordance with Cypriot legislation, which does not requires specific ethical approval from a local ethics committee [The Dogs LAW, N. 184 (I)/ phagocytophilum/A. platys, positivity for B. burgdorferi, 2002], following written and informed consent being obtained from all dog positivity for E. canis/E. ewingii, and positivity for D. owners. immitis), and the two-sample t-test or Mann-Whitney U test for continuous variables (age). Any variables that Consent for publication Not applicable. showed a trend towards significant association with ClinL (P-value < 0.1) were selected for entry into a multivariable Competing interests logistic regression. A stepwise selection procedure was CML, KP, JB and JSB work for IDEXX laboratories the manufacturer of SNAP® 4Dx Plus® test kit. used to determine the final model (criteria for entry P- value ≤0.05 and for removal P-value > 0.1). Author details 1 Additionally, structural equation modelling (SEM) was Molecular Diagnostic Unit, Diagnostic Laboratories, Bristol Veterinary School and Langford Vets, University of Bristol, Langford, UK. 2Department of performed reflecting the hypothesise mechanisms that Pathobiology and Population Sciences, The Royal Veterinary College, may be associated with ClinL and other VBP exposure University of London, Hatfield, Hertfordshire, UK. 3Cyvets Veterinary Center, statuses in dogs: (a) causal effects of host characteristics, Paphos, Cyprus. 4Department of Clinical Infection, Microbiology and Immunology, Institute of Infection and Global Health, University of Liverpool, and (b) pathogen interrelationships, using a method pre- Liverpool, UK. 5Departament de Medicina i Cirurgia Animals, Facultat de viously described [6]. Veterinària, Universitat Autònoma de Barcelona, Barcelona, Spain. 6IDEXX Attipa et al. BMC Veterinary Research (2019) 15:331 Page 6 of 6 Laboratories, Inc., West Sacramento, CA, USA. 7Present Address: IDEXX haemocanis and “Candidatus mycoplasma haematoparvum” in dogs. Vet Laboratories Ltd., Wetherby, UK. 8IDEXX GmbH, Ludwigsburg, Germany. Microbiol. 2010;140(1-2):167–70. 9 Department of Biological Sciences, University of Tasmania, Hobart, TAS, 21. Parola P, Roux V, Camicas J-L, Baradji I, Brouqui P, Raoult D. Detection of Australia. 10IDEXX Laboratories, Inc., Westbrook, ME, USA. 11Bristol Veterinary ehrlichiae in African ticks by polymerase chain reaction. Trans R Soc Trop School, University of Bristol, Langford, UK. 12The Linnaeus Group, Shirley, UK. Med Hyg. 2000;94(6):707–8. 22. Attipa C, Papasouliotis K, Solano-Gallego L, Baneth G, Nachum-Biala Y, Received: 9 February 2019 Accepted: 6 September 2019 Sarvani E, et al. Prevalence study and risk factor analysis of selected bacterial, protozoal and viral, including vector-borne, pathogens in cats from Cyprus. 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