Background: Dog with clinical leishmaniosis (ClinL), caused by Leishmania infantum, are commonly co-infected with other vector-borne pathogens (VBP). A recent PCR-based study found that dogs with ClinL are more likely to be co-infected with Ehrlichia canis. Further information on co-infections in ClinL cases with VBP, as assessed by serology, is required. We determined if dogs with ClinL are at greater risk of exposure to VBP than healthy control dogs using serology.
Methods: A prospective case–control study was performed on dogs with ClinL (qPCR and ELISA antibody for L. infantum positive on blood) and clinically healthy, preferably age-, breed-, and sex-matched, control dogs (qPCR and ELISA antibody for L. infantum negative on blood) from Cyprus, between April 2013 and March 2014. Demographic data were recorded and all dogs were tested for Anaplasma phagocytophilum/Anaplasma platys, Borrelia burgdorferi and E. canis/Ehrlichia ewingii antibodies, as well as antigen testing for Dirofilaria immitis (IDEXX Laboratories, Inc., Westbrook, Maine, USA). Logistic regression and structural equation modelling (SEM) were used to evaluate the possible risk of exposure to VBP between ClinL cases and controls.
Results: Of the 47 dogs with ClinL, antibodies for E. canis/E. ewingii were detected in 17 (36.2%), A. phagocytophilum/A. platys in 5 (10.6%) and antigen for D. immitis in 2 (4.3%). Of the 87 control dogs, antibodies for E. canis/E. ewingii were detected in 14 (16.1%) and A. phagocytophilum/A. platys in 2 (2.3%). No B. burgdorferi antibody tests were positive. There were no statistical differences between the ClinL and controls regarding lifestyle or ectoparasitic prevention use. The ClinL was significantly associated with 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 SEM.
Conclusions: There is an increased risk for E. canis/E. ewingii seropositivity in dogs with ClinL compared to clinically healthy dogs, despite similar lifestyles and ectoparasitic prevention use. Based on these findings we suggest testing dogs with ClinL not only for E. canis co-infection using PCR but also serologically for E. canis/E. ewingii.
Canine leishmaniosis (CanL) is a significant zoonotic disease in the Mediterranean basin and is caused by the parasite Leishmania infantum that istransmitted by phlebotomine sand fly vector . Often vector-borne pathogens (VBP) such as Ehrlichia canis, Anaplasma platys, Dirofilaria immitis, Babesia vogeli and Hepatozoon canis concurrently infect dogs with clinical leishmaniosis (ClinL) despite being transmitted by different vectors to L. infantum [2, 3, 4]. Such co-infections can result in an unexpected incubation period, atypical clinical outcome, more severe clinicopathological abnormalities and worse prognosis for the dogs with leishmaniosis, compared with dogs CanL alone [2, 3, 5]. Furthermore, a recent PCR-based case-control study found that it is 12 times more likely for dogs with ClinL to be co-infected with E. canis . Additional information on co-infections in ClinL cases with VBP, as assessed by serology in case-control studies, is required.
The aim of this study was to examine if dogs with ClinL are more likely to have been exposed to A. phagocytophilum/A. platys, B. burgdorferi and E. canis/Ehrlichia ewingii, or infected for D. immitis than clinically healthy controls.
Study design, site and populations
The samples we used for this serology study were collected under the frame of a previous case-control study design . All samples were collected from canine clinical cases presented to a small animal veterinary hospital in Paphos, Cyprus from April 2013 until March 2014. That area was selected since there are high numbers of CanL  and various canine VBP have been reported [8, 9].
The exact inclusion and exclusion criteria as well as the demographic characteristics recorded can be found in the previously published study . Briefly, the dogs with ClinL were naturally infected and matched with clinically healthy control dogs in-terms of breed, sex, age, living in the same geographical area as well as ideally lifestyle and the use of ectoparasitic prevention.
We used approximately 1-2 ml of surplus serum collected in plain tubes and stored at −20°C prior transport on dry ice to the Royal Veterinary College, University of London, UK.
A commercial in-clinic patient-side SNAP® 4Dx® Plus test kit (IDEXX Laboratories, Inc., Westbrook, Maine, USA) was used for the simultaneous detection of antibodies against E. canis/E. ewingii, A. phagocytophilum/A. platys, and B. burgdorferi, as well as antigens for D. immitis, following the manufacturer’s instructions. This ELISA kit utilises bi-directional flow of sample and automatic, sequential flow of wash solution and enzyme substrate. For E. canis it detects antibodies to the proteins p30 and p30-1, and for E. ewingii antibodies for p28 protein. For A. phagocytophilum/A. platys the assay detects antibodies against a peptide from the MSP2/p44 major surface protein and the C6 peptide is used for the detection of antibodies to a surface lipoprotein of B. burgdorferi. The assay detects antigens produced primarily from the uterus of female D. immitis (IDEXX Laboratories, Inc.).
Blood extracted DNA was submitted to IDEXX Laboratories, Ludwigsburg, Germany from all the cases that yielded positive antigens for D. immitis for further microfilaria specification using PCR specific assays for D. immitis, Dirofilaria repens, Acanthocheilonema reconditum and Acanthocheilonema dracunculoides. Additionally all samples underwent L. infantum serology , qPCRs for Leishmania spp. , Babesia spp. , “Candidatus Mycoplasma haematoparvum” and Mycoplasma haemocanis  as well as conventional PCR assays for Ehrlichia/Anaplasma spp.  and Hepatozoon spp.  under the framework of a previously published study .
The sample size was previously calculated  and analyses were performed using SPSS for Windows (version 25.0; SPSS Inc., Chicago IL, USA). A univariable analysis was initially performed to see how each of the explanatory variables was associated with ClinL using Pearson’s Chi-square test for categorical explanatory variables (breed, sex, lifestyle, use of ectoparasitic prevention, positivity for A. phagocytophilum/A. platys, positivity for B. burgdorferi, positivity for E. canis/E. ewingii, and positivity for D. immitis), and the two-sample t-test or Mann-Whitney U test for continuous variables (age). Any variables that showed a trend towards significant association with ClinL (P-value <0.1) were selected for entry into a multivariable logistic regression. A stepwise selection procedure was used to determine the final model (criteria for entry P-value ≤ 0.05 and for removal P-value >0.1).
Additionally, structural equation modelling (SEM) was performed reflecting the hypothesised mechanisms associated with ClinL and VBP exposure statuses in dogs: (a) causal effects of host characteristics, and (b) pathogen interrelationships, using a method previously described .
Serum was available in 47 dogs with ClinL and 87 dog controls that were included in this serology study. The age of these 134 dogs ranged from 1 to 12 years (median 4 years, interquartile range 3 years) and 98 (73%) were pedigree including Segugio Italiano, Cocker spaniel, Beagle, German Shepherd and other breeds.
In the ClinL group, antibodies for A. phagocytophilum/A. platys were detected in 5 (10.6%), E. canis/E. ewingii in 17 (36.2%) and antigen for D. immitis in 2 (4.3%) dogs. Of the 87 control dogs, antibodies A. phagocytophilum/A. platys for were detected in 2 (2.3%) and E. canis/E. ewingii in 14 (16.1%). No B. burgdorferi antibody tests were positive. (Figure 1). Table 1 summarizes the demographic characteristics and the serology findings. The two dogs with D. immitis antigens underwent microfilaria PCR specification which was positive for A. reconditum and negative for D. immitis for both cases.
A significant association between ClinL and the presence of E. canis/E. ewingii antibodies [odds ratio (OR) = 2.9, 95% confidence interval (CI): 1.3–6.7, P=0.010] was found, compared to healthy controls using multivariable logistic regression. The presence of A. phagocytophilum/A. platys antibodies was initially significantly associated with ClinL compared to controls using univariable analysis (OR = 5.1, 95% CI: 0.9–27.2, P=0.038) but this association was not maintained during multivariable logistic regression analysis. The numbers of D. immitis were very low hindering any further statistical analysis. Age, breed, sex, lifestyle, and use of ectoparasitic prevention were not statistically different between the ClinL and the control dogs.
Two associations were identified based on SEM (Figure 2, Table 2). Dogs with ClinL were more likely to be E. canis/E. ewingii seropositive and dogs seropositive for E. canis/E. ewingii are more likely to be infected with E. canis. A trend was identified between dogs with ClinL and A. phagocytophilum/A. platys seropositive.
The findings from this serology study are in agreement with previous studies [3, 16] and further support the findings from the initial PCR based study, using the same cohort of samples, in which it was demonstrated that dogs with ClinL are 12 times (CI: 1.5–106.0, P = 0.022) more likely to be co-infected with E. canis compared to healthy canine controls . A previous a 3-year longitudinal study, evaluating E. canis and L. infantum co-infection in naturally exposed dogs, found that E. canis infection preceded L. infantum infection in dogs with dual infections, thus suggesting that E. canis could contribute in the establishment of ClinL . Interestingly, a recent study by Baxarias et al.  from Catalonia (Spain) found that dogs with ClinL were four times more likely to be seropositive for Rickettsia conorii and 14 times more likely to be seropositive for A. phagocytophilum compared with healthy controls, but no association was found between ClinL and E. canis seroreactivity. This discrepancy probably reflects the different prevalence of these pathogens in Cyprus and other Mediterranean areas in comparison to Catalonia.
The seroprevalence of the various VBP in this specific studied population of 134 dogs from the area of Paphos in Cyprus, revealed a noticeably high seroreactivity to E. canis/E. ewingii (23%) and A. phagocytophilum/A. platys (13%) antibodies compared to other studies from Mediterranean countries using a similar in-house ELISA kit as the one utilised in this study [17, 18, 19]. If quantitative ELISA or IFAT with higher sensitivity, compared to the in-house kit, were used in this study, then seroprevalences of the VBP could have been even higher than these reported . The area of Paphos, Cyprus, may be Lyme disease free as no B. burgdorferi antibodies were detected in any of the dogs tested in this study, and the tick vectors can that transmit this pathogen, including Ixodes Ricinus, have not yet been identified in Cyprus . In two dogs (1%) antigens for D. immitis were detected but PCR failed to confirm this infection and instead an infection with A. reconditum was identified for both cases. These results may indicate that the dogs had dual infection with both D. immitis and A. reconditum, and the negative PCR for D. immitis was as a result of low level microfilaraemia. However, false positive D. immitis results cannot be ruled out entirely especially in light of a recent study from Cyprus in which, using a modified Knott’s testing for morphological identification of microfilariae in a total of 200 healthy dogs which did not receive any kind of heartworm prevention, only A. reconditum was identified in 9 dogs (4.5%) and no D. immitis was found .
We demonstrated that dogs with ClinL are three times more likely to be exposed to E. canis/E. ewingii than clinically healthy dogs in Paphos, Cyprus. Furthermore, dogs from this area have a high seroreactivity to E. canis/E. ewingii and A. phagocytophilum/A. platys while they are B. burgdorferi free.