Cross-Sectional Serosurvey and Factors Associated with Exposure of Dogs to Vector-Borne Pathogens in Greece

Abstract

Blood samples from 2620 dogs living in 7 different areas were used for the detection of antibodies against Ehrlichia canis and Borrelia burgdorferi and Dirofilaria immitis antigen. Previously published seropositivity data against Leishmania infantum from the same samples were also used to identify multiple-pathogen seropositivity. The associations between seropositivity against each pathogen and gender, age, utility, hair length, and region of residence were evaluated by multivariate logistic regression analyses. The prevalence of seropositivity against E. canis, B. burgdorferi, and D. immitis was 12.25%, 2.23%, and 5.96%, respectively. Double- and triple-pathogen seropositivity was detected in seven different combinations. Age and region of residence were strongly associated with seropositivity against all pathogens. The association between seropositivity and the area of residence highlights the need for regular testing of dogs for vector-borne pathogens in areas with similar conditions to define control measures.

Introduction

Canine vector-borne diseases (CVBD) seem to be of increasing significance in Greece as in other countries where factors influencing vector distribution and abundance exist. Arthropod vectors are dependent on climate conditions and habitat suitability for survival and reproduction (Rogers and Randolph 2006). Therefore, epidemiological data regarding vector-borne infections are useful for risk identification of animal and zoonotic transmission in different areas, with dogs serving as potential sentinels for human infection (De Tommasi et al. 2013).

Vectors and vector-borne pathogens such as Leishmania infantum, Dirofilaria immitis, Ehrlichia canis, and Borrelia burgdorferi alone or in combination can result in a variety of infection states from asymptomatic carriage to severe clinical disease (Day 2011a). Apart from representing a diagnostic and therapeutic challenge for the veterinarians, Leishmania and B. burgdorferi are also of medical importance because of their zoonotic potential (Day 2011b). Moreover, although very rarely, D. immitis can also cause pulmonary dirofilariosis in humans (Irwin and Jefferies 2004, Simon et al. 2005). Contrary to other rickettsiae causing life-threatening disease to humans, such as Ehrlichia chaffeensis, dogs can only serve as sentinels of rickettsial infection (Walker 2005).

So far, numerous serological and/or molecular surveys have demonstrated that canine leishmaniasis is constantly endemic in Greece (Leontides et al. 2002, Athanasiou et al. 2012). Although there are many studies on the clinical and clinicopathological findings of canine ehlrichiosis in Greece (Mylonakis et al. 2004a, 2011, Gianopoulos et al. 2016), there is paucity of epidemiological data within the country. Similarly, no data have been available regarding the occurrence of Lyme borreliosis in dogs in Greece whereas there is only one recent study (Diakou et al. 2016) on dirofilarial infections in different areas of the country.

Asymptomatic seropositive infections are of both veterinary and medical importance as clinically healthy infected dogs pose a hidden risk of future clinically patent disease (Baneth et al. 2012) whereas infectivity of seropositive dogs to vectors has been proven by xenodiagnoses, at least for Leishmania (Michalsky et al. 2007). Further, co-infections by two of more vector-borne pathogens have been previously reported (Cardoso et al. 2012, De Tommasi et al. 2013, Farkas et al. 2014, Pantchev et al. 2015a) and they are also important to consider.

The aims of this study were to: (a) determine seropositivity of CVBD pathogens in dogs in seven different areas in Greece, (b) identify factors associated with the presence of antibodies or antigen against these pathogens, and (c) identify double-, triple-, or multiple-pathogen seropositivity.

Materials and Methods

Blood samples that had been collected from 2620 dogs living in 7 different areas for a previous study on the seroprevalence of canine leishmaniasis (Athanasiou et al. 2012) and their demographic data were used. Antibodies against E. canis and B. burgdorferi were detected by using commercial indirect immunofluorescent assays (Agrolabo SPA, Italy) and D. immitis antigen by the serological commercial kit Pet Check^® (IDEXX). Previously published results on the seropositivity against Leishmania were also used to identify seropositivity against multiple pathogens.

The associations between seropositivity and gender, age, utility, hair length, and region of residence against all pathogens were evaluated by multivariate logistic regression analyses.

The associations between seropositivity and demographic characteristics (gender, age, utility, hair length, and region of residence) as well as among seropositivity against different vector-borne pathogens were evaluated through multivariate backward logistic regression analyses, using statistical software R.

The dependent variables were a positive or not serological result against Leishmania, E. canis, D. immitis, and B. burgdorferi. Independent variables were gender (female, male), hair length (short, long), utility (hunting, guard, pet), age (young adult, young, adult, geriatric), and region of residence. To assess the relationship between seropositivity against Leishmania and against other vector-borne pathogens, the presence or not of coexisting seropositivity in at least one of the other vector-borne pathogens in dogs found seropositive to Leishmania was also included as an independent variable. Univariate analysis was performed, and variables with a significance level of 0.1–0.2 were included in the final multivariate model. The final model contained the selected independent variables and their interactions. Variables and/or interactions that were found to be statistically insignificant (p > 0.05) were removed unless confounding occurred (a change of at least 25% of the parameter estimates when compared with the full model).

Results

The percentage of seropositivity against E. canis, D. immitis, and B. burgdorferi in the total population of dogs sampled was 12.25% (286/2334), 5.96% (153/2565), and 2.23% (55/2467), respectively, and the corresponding value for each pathogen per region is presented in Fig. 1.

FIG. 1.

Depiction of seroprevalence against Ehrlichia canis, Borrelia burgdorferi, and Dirofilaria immitis in dogs living in seven different areas on the map of Greece.

Double- and triple-pathogen seropositivity that was detected in seven different combinations and the statistically significant differences among them are presented in Table 1.

Table 1.

Frequency (%) of Double- and Triple-Pathogen Seropositivity in 10 Different Combinations Based on 4 Pathogens (Leishmania, Dirofilaria, Ehrlichia, Borrelia)

Combination of pathogens	Seropositivity (N)	Seropositivity (%)
Leishmania+Dirofilaria	14/2620	0.53^ad
Leishmania+Ehrlichia	36/2620	1.37^b
Leishmania+Borrelia	6/2620	0.23^a
Dirofilaria+Ehrlichia	7/2620	0.27^a
Dirofilaria+Borrelia	2/2620	0.076^c
Ehrlichia+Borrelia	17/2620	0.65^d
Leishmania+Dirofilaria+ Ehrlichia	1/2620	0.04^c
Leishmania+Dirofilaria+Borrelia	0/2620	0
Leishmania+Ehrlichia+Borrelia	8/2620	0.3^a
Dirofilaria+Ehrlichia+Borrelia	1/2620	0.04^c

abcd

Combinations with different superscripts differ significantly (p < 0.05).

Predictor variables hair length and gender were found to be statistically insignificant in all models. Dogs' utility was only significantly associated with the possibility of becoming or not seropositive against D. immitis. More specifically, guard dogs and pets have significantly lower odds to be found seropositive against D. immitis compared with hunting dogs. The dogs' age and region of residence were strongly associated with seropositivity against all vector-borne pathogens (Table 2).

Table 2.

Factors Associated with Seropositivity Against Dirofilaria

		Dirofilaria
Factors	Tested (n)	Positive (n)	OR (95% CI)	p
Age, years
<1	221	16	2.8 (1.46–5.21)	0.001
1–3	1009	33	—	—
4–8	1075	91	5.85 (3.59–9.69)	<0.0001
>9	315	13	6.22 (2.37–15.92)	0.0001
Region
Attiki	1006	32	—	—
Evia	140	7	2.07 (0.8–4.76)	0.1
Evritania	200	28	6.45 (3.68–11.03)	<0.0001
Evros	416	28	1.24 (0.69–2.21)	0.5
Florina	146	9	1.71 (0.73–3.64)	0.2
Ioannina	233	17	2.13 (1.11–3.97)	0.02
Serres	479	32	2.33 (1.38–3.94)	0.001
Utility
Hunting	1641	105	—	—
Guard	814	44	0.25 (0.15–0.42)	<0.0001
Pet	165	4	0.12 (0.03–0.37)	<0.0001
Gender
Male	1279	69	0.85 (0.61–1.18)	0.2
Female	1341	84	—	—
Hair length
Long	1098	74	—	—
Short	1522	79	0.76 (0.55–1.05)	0.1

CI, confidence interval; n, no. of samples; OR, odds ratio.

Adult dogs versus young adult dogs are more likely to become seropositive against Leishmania, D. immitis, or E. canis and less likely against B. burgdorferi. The odds for young and geriatric dogs to become seropositive against Leishmania are lower but higher against D. immitis compared with young adult dogs. In addition, always in comparison with young adult dogs, there is a two-fold increase in the odds of a young dog being seropositive against B. burgdorferi; on the other hand, there is a significant reduction in the odds of geriatric dogs being seropositive against B. burgdorferi. Further, young dogs are two times more likely to be seropositive to E. canis than young adult dogs (Table 3).

Table 3.

Factors Associated with Seropositivity Against Ehrlichia and Borrelia

		Ehrlichia			Borrelia
Demographic data	Tested (n)	Positive (n)	OR (95% CI)	p	Positive (n)	OR (95% CI)	p
Age, years
<1	221	32	2.04 (1.29–3.16)	0.002	12	2.31 (1.09–4.63)	0.02
1–3	1009	77	—	—	26	—
4–8	1075	134	1.59 (1.18–2.16)	0.002	16	0.49 (0.25–0.93)	0.03
>9	315	43	1.5 (0.98–2.28)	0.06	1	0.13 (0.01–0.63)	0.05
Region
Attiki	1006	85	—	—	21	—	—
Evia	140	13	1.003 (0.52–1.81)	0.99	8	4.47 (1.75–10.5)	0.001
Evritania	200	13	0.77 (0.4–1.38)	0.4	7	2.14 (0.82–4.95)	0.1
Evros	416	53	1.51 (1.02–2.21)	0.04	4	0.81 (0.23–2.23)	0.7
Florina	146	9	0.7 (0.32–1.36)	0.3	3	1.52 (0.35–4.59)	0.5
Ioannina	233	37	1.88 (1.21–2.88)	0.004	4	1.19 (0.34–3.2)	0.8
Serres	479	76	1.96 (1.39–2.75)	0.0001	8	1.11 (0.45–2.46)	0.8
Utility^a
Hunting	1641	183	1.1 (0.84–1.46)	0.5	44	1.74 (5.28–5.41)	0.3
Guard	814	83	—	—	11	—	—
Pet	165	20	1.21 (0.71–2.01)	0.5	0	0	0.9
Gender^a
Male	1279	137	1.16 (0.91–1.48)	0.2	41	2.03 (0.85–4.98)	0.1
Female	1341	149	—	—	14	—	—
Hair length^a
Long	1098	127	—	—	10	—	—
`Short	1522	159	0.89 (0.7–1.14)	0.2	45	0.89 (0.49–1.8)	0.1

ORs adjusted for age and region.

Dogs living in Attiki region compared with all other regions are more likely to be found seropositive against Leishmania than against D. immitis. The probability to find a seropositive dog against E. canis increases by twofold when the regions of residence are Evros, Serres, and Ioannina, compared with Attiki region. Moreover, dogs residing in the island of Evia have a fourfold increase in the odds of being seropositive against B. burgdorferi compared with dogs residing in Attiki.

The associations between seropositivity against Leishmania and against other vector-borne pathogens found were evaluated. Dogs that are seropositive against at least one of the other vector-borne diseases have significantly lower odds of becoming Leishmania seropositive than seronegative dogs. An interaction between dogs' age and seropositivity against B. burgdorferi was present, specifically between adult and young adult dogs. In detail, adult dogs that are seropositive against B. burgdorferi (OR: 0.14, 95% CI: 0.02–0.75, p = 0.03) are less likely to be Leishmania seropositive compared with young adult dogs. No associations between the other age categories and young adult dogs were statistically significant.

Discussion

Diseases caused by vector-borne pathogens in dogs seem to be quite frequent in Greece as in other countries in the area sharing similar environmental conditions (Pantchev et al. 2015b, Hamel et al. 2016, Vascellari et al. 2016, Attipa et al. 2017, Guven et al. 2017). Awareness of the extent of infection in a specific geographic area facilitates diagnostic, therapeutic, and preventive interventions.

This is the first serosurvey on antibodies against E. canis in Greece. The overall seroprevalence in Greece as well as in northeastern areas is approximately half than that found in neighboring areas of Turkey (27.25%) (Cetinkaya et al. 2016). This could be attributed to differences in sampled populations; owned animals in Greece versus animals in shelters in Turkey, and presumed differences in preventive care. The seroprevalence in different countries in Europe has been reported to range from 0.16% in Hungary (Farkas et al. 2014) to 46% in Italy (Pennisi et al. 2012).

The presence of antibodies against E. canis denotes either current or past infection since antibody titers may persist for months or years (Gaunt et al. 2010). After an incubation period of 8–20 days, three phases have been documented in the progression of ehrlichiosis: acute, subclinical, and chronic. In an experimental study (Waner et al. 1997), infected dogs remained asymptomatic throughout the 6-month period of monitoring with high IFAT titers, indicative of a prolonged infection period and chronic antigenic stimulation. Therefore, seropositivity of dogs in this study represents either a subclinical phase of the infection or a resolved infection with a persistent antibody reaction. Cross-seroreactivity between E. canis and other Ehrlichia species such as E. chaffeensis, Ehrlichia ewingii, and Ehrlichia ruminantium has also been reported (Neer et al. 2002, Little 2010, Harrus and Waner 2011). However, this is not likely to occur in this study since neither these organisms nor their vectors (Amblyomma americanum, Amblyomma hebraeum, Dermacentor variabilis) have been reported in Greece (Papadopoulos et al. 1996, Papazahariadou et al. 2003, Siarkou et al. 2007). The presence of Anaplasma (former Ehrlichia) platys and Anaplasma phagocytophilum has been reported to exist in Greece (Kontos et al. 1991, Mylonakis et al. 2004b). However, antibodies against E. canis cross-react only with the latter (Waner et al. 1998).

This is also the first serosurvey on antibodies against B. burgdorferi in dogs living in Greece. A positive serologic result is considered indicative of previous exposure instead of active infection (Greene 1990, Littman et al. 2006). In an experimental study, antibodies were detected at 4 weeks and remained present for at least 11 weeks postinfection; whereas clinical signs did not coincide with antibody detection (Cerri et al. 1994).

Seropositivity against E. canis and B. burgdorferi was the second in frequency of double seropositivity in this study attributed to the shared arthropod vectors and/or concurrent exposure to multiple pathogen-carrying vectors.

Dirofilaria was the third pathogen investigated in this study. In a previous study (Diakou et al. 2016), the overall seropositivity was slightly lower, 4.1% compared with 5.96% in this study. In both studies, the lowest seropositivity was observed in Attiki. In this study, dogs from different regions of Greece were sampled, most of them in the northern and eastern parts of Greece. The finding of the previous study (Diakou et al. 2016) that the prevalence of dirofilariosis is higher in northern regions of Greece compared with Athens has been also shown in this study.

Differences in the seroprevalence of all pathogens determined in this study could be attributed to climate and environmental conditions favoring the survival and abundance of vectors. However, this seems not to be the case in dirofilariasis in Athens, an area with a history of mosquito-borne West Nile outbreaks related to high mosquito densities (Fotakis et al. 2017). A more efficient protection against vectors in dogs living in Athens compared with in other regions could be an explanation. However, since the highest seropositivity against Leishmania was recorded in Athens (Athanasiou et al. 2012), this assumption can only be made for mosquitos and not for sandflies.

The double and triple seropositivity identified in this study could be attributed to the exposure of dogs to different vectors and to the immunomodulation caused by the first invading pathogen that probably facilitates a sequential infection (Day 2011b).

Despite the presence of double and triple seropositivity in this study, dogs seropositive against at least one of the other vector-borne diseases were found to be less likely to become Leishmania seropositive than seronegative dogs. The fact that dogs sampled in this study were asymptomatic could explain this finding; the co-infections are known to enhance pathogenicity and aggravate the clinical picture (De Tommasi et al. 2013). Dogs with clinical leishmaniosis were found more likely to be infected by E. canis than clinically healthy controls (Attipa et al. 2018).

Apart from the region of residence, the age of the dog was associated with seropositivity. Most of the seropositive dogs against all pathogens were either young adults or adults. These two age groups are exposed for a prolonged period to these pathogens compared with young dogs. On the contrary, geriatric dogs would have developed clinical disease and/or died because of concurrent clinical conditions.

Conclusions

Antibodies against E. canis and B. burgdorferi and antigen of D. immitis are prevalent in dogs living in Greece.

Exposure to multiple vector-borne pathogens is quite common in the country. The region of residency and age are the main factors associated with seroprevalence. Therefore, regular testing is required in dogs in the age and residency of elevated risk of exposure to permit earlier diagnosis and improve treatment approaches in dogs, identify human infection risk, and define preventive measures.

Footnotes

Author Disclosure Statement

No conflicting financial interests exist.

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