Low Seroprevalence of Leishmania infantum and Toxoplasma gondii in the Horse Population in Israel

Abstract

A cross-sectional investigation was done on the seroprevalence of Leishmania infantum and Toxoplasma gondii infection among apparently healthy horses in Israel. This survey included 383 horses distributed in 22 farms throughout Israel during the years 2011–2013. Serum samples were tested for the presence of immunoglobulin G (IgG) antibodies using the direct agglutination test (DAT) specific to Leishmania and by the modified agglutination test (MAT) for the presence of IgG antibodies to T. gondii. Low seroprevalences were detected for both L. infantum and T. gondii in the horse population in Israel; of the 338 horses tested, 6 (1.4%) were found to be seropositive for L. infantum and 11 (2.5%) for T. gondii, with no significant association between seroprevalence and demographic/environmental factors. An ongoing geographical expansion of L. infantum, previously reported in humans and dogs in Israel, was also supported by our results in horses. Here we present evidence of exposure of horses to L. infantum and T. gondii in Israel. Continuous seroprevalence surveillance in horses, such as the one performed in this study, might further elucidate the eco-epidemiology of these two important zoonotic parasites in this country.

Introduction

Leishmaniosis and toxoplasmosis are two protozoal zoonotic diseases that are prevalent worldwide. Leishmaniosis preferentially occurs in tropical and subtropical countries, including the Mediterranean area (Müller et al. 2009). Depending upon the infecting species of Leishmania, the disease can present as cutaneous, mucocutaneous, or visceral leishmaniosis (Murray et al. 2005). Cutaneous leishmaniosis (CL) is the most common and least fatal form of the disease, identified by ulcerative skin lesions. Mucocutaneous leishmaniosis results in the complete or partial destruction of mucous membranes of the nose and mouth. The most life-threatening form of the disease is visceral leishmaniosis (VL), in which the pathogen disseminates to internal organs like the liver, spleen, and bone marrow (Salam et al. 2014).

Both the cutaneous and visceral diseases are endemic in Israel (Greenblatt et al. 1985), where, similarly to other parts of the eastern Mediterranean basin, Leishmania major and Leishmania tropica cause CL and Leishmania infantum causes VL (Jaffe et al. 2004). Dogs are the main reservoir of L. infantum, and the parasite is transmitted to animals and humans by the bite of female phelobotomine sand flies. Canine leishmaniosis is usually manifested as a chronic viscerocutaneous disease, whereas in humans infections with L. infantum can manifest either in a severe visceral or a mild cutaneous form. In horses, clinical manifestations are usually characterized by single or multiple nodular or papular lesions on the head, limbs, or axillary and inguinal regions (Gama et al. 2014). Cases of CL in horses have been reported sporadically in various European countries, including Spain (Solano-Gallego et al. 2003), Portugal (Rolão et al. 2005), Germany (Koehler et al. 2002), and Switzerland (Muller et al. 2009) and recently also from the Americas (Soares et al. 2013).

Toxoplasma gondii is a zoonotic intracellular protozoan parasite, with wild and domestic felids being its definitive hosts. Virtually all warm-blooded species (including humans) can be intermediate hosts and become infected by ingestion of food or water contaminated with sporulated oocysts, by consumption of tissue cysts in infected animal tissues, or congenitally (Dubey 2010). Toxoplasmosis is endemic in Israel and, recently, a high age-adjusted seroprevalence rate (60.4%) was found in Arabs (non-Bedouins) compared to the rate in Jews (19.9%) and Bedouins (27.5%) (Markovich et al. 2014). In horses, infection with T. gondii is usually subclinical, but clinical signs may include fever, ataxia, retinal degeneration, encephalomyelitis, and abortion or stillbirth in pregnant equids (Miao et al. 2013). Cases of human toxoplasmosis were reported following consumption of raw horsemeat (Pomares et al. 2011). Hence, the role of horses in the transmission of parasitic zoonoses, either as a source of infection to vectors or through contamination of raw horse meet, is gaining importance worldwide. Although Israel is considered endemic to both zoonotic parasites, this is the first study that aims to characterize the seroprevalence of T. gondii and L. infantum among the horse population in the country.

Materials and Methods

Blood samples, serum preparation, and detection of L. infantum and T. gondii immunoglobulin G antibodies

During the years 2011–2013, blood samples were collected from 338 clinically healthy horses from 22 farms distributed in various regions of Israel. The study was approved by the ethics committee of the Koret School of Veterinary Medicine, The Hebrew University, and blood collection was performed under owners' consent. Blood samples were collected from the jugular vein of each horse into sterile vacuum tubes without any anticoagulant agent. Sera were obtained from clotted blood samples by centrifugation (3000 × g for 8 min) and stored at −80°C until use. Serum samples were tested for the presence of immunogobulin G (IgG) antibodies using the direct agglutination test (DAT) specific to Leishmania, as previously described by Lopes et al. (2013), and by the modified agglutination test (MAT) commercial kit (Toxo-Screen DA^®, bioMérieux, Lyon, France) for the presence of IgG antibodies to T. gondii (Lopes et al. 2013).

Collection of demographic and environmental data

During blood collection in sentinel farms, information was recorded on the age, sex, color, and breed of the horses, as well as on the geographical location and surrounding human population density (rural/urban). Climate, location, and human density data were collected for all 338 horses, whereas other variables were only recorded partially (breed, 257/338 horses; age, 313/338 horses; gender, 319/338 horses; color, 148/338 horses). The average age of sampled horses was 9 years, ranging from 3 months to 30 years. Horses were divided by their sex to males (46%) and females (54%). For geographical and environmental analyses, Israel was divided either by latitude into north/center/south or by its climatic zones (from the wettest to driest region)—Mediterranean, semi-arid, steppe arid, desert arid, and extreme arid.

Statistical analysis

Statistical analyses were performed using SPSS 22.0^® software. Seroprevalence was calculated for each category in the demographic and environmental variables with 95% confidence intervals (CI). Differences in seroprevalence among the various groups were assessed by the two-sided chi-squared test or Fisher exact test. A p value <0.05 was regarded as statistically significant.

Results

Detection of antibodies to L. infantum and T. gondii and demographic and environmental risk factors

Of the 338 horses tested, 6 (1.4%) were found to be seropositive for L. infantum and 11 (2.5%) for T. gondii. Horses seropositive for either agent were found to be distributed throughout Israel (Fig. 1). For L. infantum, seropositive horses had titers of 200 (n = 2), 400 (n = 2), 3200 (n = 1), and 12,800 (n = 1). Fourteen horses were found seronegative for L. infantum (titer <200). However, seven of the horses seropositive for T. gondii (cutoff, 20) had a titer of 20 and the other four horses had a titer of 40, 640, 5120, and 40,960 each. None of the horses was seropositive for both parasites.

FIG. 1.

Seroprevalences of Leishmania infantum (A) and Toxoplasma gondii (B) in horses throughout Israel. All circles represent farms tested for the two parasites. Farms with seropositive horses (red circles); farms with no seropositive horses (green circles). Color images available online at www.liebertpub.com/vbz

None of the demographic variables (age, breed, gender, color) was found to be significantly associated with seroprevalence to neither L. infantum nor T. gondii by univariate analysis. In addition, none of the environmental factors (i.e., climate, location, and human population density [rural/urban]) were found to be associated with seroprevalence to neither of the parasites (Tables 1 and 2).

Table 1.

Univariate Analysis of the Association Between Demographic (Age, Breed, Gender, and Color) and Environmental (Climatic Zone, Location, and Urban/Rural) Risk Factors and Leishmania infantum Seroprevalence in Horses in Israel

Variable	Category	Positive/total (%)	OR	95% CI	p
Climate zone	Mediterranean	1/159 (0.6)	0.16	0.0–1.67	0.085
	Semi arid	4/106 (3.8)	REF
	Steppe arid	0/38 (0)	0.00	0.0–4.24	0.573
	Extreme arid	1/35 (2.9)	0.75	0.01–7.94	1.0
Location	North	2/126 (1.6)	0.66	0.05–5.84	0.684
	Center	3/125 (2.4)	REF
	South	1/87 (1.1)	0.47	0.01–6.02	0.646
Age	0–2	0/17 (0)	0.00	0.0–12.99	1.0
	3–5	1/81 (1.2)	0.24	0.0–4.88	0.261
	6–8	2/88 (2.3)	0.45	0.03–6.51	0.591
	9–11	2/41 (4.9)	REF
	12–14	0/29 (0)	0.00	0.0–7.53	0.508
	15+	1/57 (1.8)	0.35	0.01–6.98	0.570
Breed	Cross/LO	2/129 (1.6)	0.44	0.03–6.25	0.589
	QH	2/58 (3.4)	REF
	AR	0/23 (0)	0.00	0.0–13.54	1.0
	PO	0/19 (0)	0.00	0.0–16.46	1.0
	WB	0/7 (0)	0.00	0.0–46.58	1.0
	AP	0/19 (0)	0.00	0.0–10.71	1.0
	TB	0/1 (0)	NR
Gender	Male	2/169 (1.2)	0.44	0.04–3.11	0.425
	Female	4/150 (2.7)	REF
Color	Grey	0/39 (0)	0.00	0.0–10.82	1.0
	Palomino	0/15 (0)	0.00	0.0–28.65	1.0
	Brown	2/79 (2.5)	REF
	Dark brown/black	0/7 (0)	0.00	0.0–63.61	1.00
	Paint	0/8 (0)	0.00	0.0–55.19	1.00
Urban/rural	Urban	2/88 (2.3)	REF
	Rural	4/250 (1.6)	0.70	0.10–7.87	0.653

OR, odds ratio; CI, confidence interval; REF; reference value; LO, local; QH, quarterhorse; AR, Arabian; PO, pony; WB, warmblood; AP, Appaloosa; TB, thoroughbred.

Table 2.

Univariate Analysis of the Association Between Demographic (Age, Breed, Gender, and Color) and Environmental (Climatic Zone, Location, and Urban/Rural) Risk Factors and Toxoplasma gondii Seroprevalence in Horses in Israel

Variable	Category	Positive/total (%)	OR	95% CI	p
Climate zone	Mediterranean	8/159 (5.0)	REF
	Semi-arid	2/106 (1.9)	0.36	0.04–1.88	0.324
	Steppe arid	1/38 (2.6)	0.51	0.01–4.02	1.0
	Extreme arid	0/35 (0)	0.0	0.0–2.66	0.355
Location	North	3/126 (2.4)	0.48	0.08–2.33	0.334
	Center	6/125 (4.8)	REF
	South	2/87 (2.3)	0.47	0.05–2.70	0.476
Age	0–2	1/17 (5.9)	0.79	0.01–10.79	1.0
	3–5	0/81 (0)	0.0	0.0–1.20	0.036
	6–8	3/88 (3.4)	0.45	0.06–3.52	0.381
	9–11	3/41 (7.3)	REF
	12–14	1/29 (3.4)	0.45	0.01–6.04	0.637
	15+	3/57 (5.3)	0.70	0.09–5.56	0.692
Breed	Cross/LO	6/129 (4.7)	0.88	0.10–42.60	1.0
	QH	1/58 (1.7)	0.32	0.0–26.15	0.435
	AR	1/23 (4.3)	0.82	0.01–67.75	1.0
	PO	1/19 (5.3)	REF
	WB	0/7 (0)	0.0	0.0–105.86	1.0
	AP	1/19 (5.3)	1.0	0.01–82.80	1.0
	TB	0/1 (0)	NR
Gender	Male	7/169 (4.1)	1.58	0.39–7.49	0.550
	Female	4/150 (2.7)	REF
Color	Grey	1/39 (2.6)	0.37	0.0–30.97	0.482
	Palomino	1/15 (6.7)	REF
	Brown	0/79 (0)	0.0	0.0–7.41	0.160
	Dark brown/black	0/7 (0)	0.0	0.0–83.57	1.0
	Paint	0/8 (0)	0.0	0.0–73.13	1.0
Urban/rural	Urban	4/88 (4.5)	OR
	Rural	7/250 (2.8)	0.60	0.15–2.89	0.486

OR, odds ratio; CI, confidence interval; REF; reference value; LO, local; QH, quarterhorse; AR, Arabian; PO, pony; WB, warmblood; AP, Appaloosa; TB, thoroughbred.

Discussion

L. infantum and T. gondii are two well-studied zoonotic parasites that are endemic in Israel. However, this is the first study investigating the seroprevalences of these two parasites in the horse population in Israel and assessing demographic and environmental risk factors associated with such seroprevalence values. We hereby report low seroprevalence (1.4%) for L. infantum in horses in Israel. Similarly, low seroprevalences were also detected in horses in the north of Portugal (4.0%) (Lopes et al. 2013) and in Greece (0.3%) (Kouam et al. 2010). Risk factor analyses did not yield any significant associations between demographic/environmental factors and seroprevalence to L. infantum. This is perhaps due to the very low seroprevalence observed for this parasite. In the past, both CL and VL in humans and dogs were thought to be restricted to geographically defined areas in Israel, with CL occurring primarily in the Negev Desert, Jordan, and Arava Rift Valleys, and VL limited to the Galilee region of northern Israel. However, in the last decade, these diseases seem to have expanded geographically, and VL has spread throughout the entire central region of Israel (Jacobson et al. 2003, Jaffe et al. 2004).

Interestingly, despite the low seroprevalence detected in this study, horses seropositive for L. infantum were detected not only in the northern and central parts of the country but also in the southern region, suggesting further geographical expansion of the parasite. Hence, although only a few horses were found seropositive to this parasite, these results indicate that leishmaniosis should be added to the differential diagnosis list in horses with typical lesions in Israel. Furthermore, horses may have a role in the ecology of L. infantum because they are not only exposed to phlebotomines sand flies, but are also exposed to Culicoides spp., which are prevalent in their environment and were recently demonstrated to be a potential vector for leishmaniosis (Slama et al. 2014).

A low seroprevalence value was also found for T. gondii (2.5%). Comparable low prevalences were also detected in horses in Greece (1.8%) (Kouam et al. 2010) and in Korea (2.9%) (Lee et al. 2014), whereas higher seroprevalences were detected in horses in Yunnan Province, southwestern China (30.5%) (Miao et al. 2013), in Tunisia (17.7%) (Boughattas et al. 2011), and in the north of Portugal (13.3%) (Lopes et al. 2013). Similar to the risk factor analysis performed for L. infantum, no significant associations were detected between demographic/environmental factors and seroprevalence to T. gondii. This is in accordance with two studies in Portugal (Lopes et al. 2013) and Korea (Lee et al. 2014) that reported no significant influence of gender and age on the seropositivity to T. gondii. On the other hand, older horses, males, and Arab horses were more likely to be seropositive to T. gondii in a study from Tunisia (Boughattas et al. 2011), and significant differences in seropositivity were found between breeds and regions in Korea (Lee et al. 2014). Those authors suggested that the differences in prevalence might be explained by the different management types, proposing that horses that are allowed to graze freely are more likely to acquire parasites from the natural environment (Koehler et al. 2002). Variations in hygiene and climate conditions were also suggested to partially explain the differences between studies in the prevalence of T. gondii in cats (Miao et al. 2013). In Israel, grazing areas are limited, which might partially explain the low seroprevalence that was found in this study. Compared to the low number of T. gondii–seropositive horses that were found in this study, higher seroprevalences were found in cattle (15%), sheep (25%) (Shkap et al. 1992), and dogs (35%) in Israel (Baneth et al. 1996).

Recently, very high T. gondii seroprevalence was found in scavenger birds that feed on animal carcasses, 52 of 122 (42.6%) crows (Corvus cornix, Corvus monedula, Corvus splendens) and 40 of 101 (39.6%) Griffon vultures (Gyps fulvus) were found to be seropositive (Salant et al. 2013). High T. gondii seroprevalence (46.6%) was also found in free-range chickens (Gallus domesticus) from a commercial farm located in the north of Israel (Dubey et al. 2004). In contrast, low T. gondii seroprevalence (4%) was found in wild pigeons (Columba livia) captured from various locations in Israel (Salant et al. 2009). None of the horses sampled in the extreme arid climatic zone were found to be seropositive to T. gondii. This might be attributed to the dry and hot climate conditions in this geographical region. This finding is in accordance with the low seroprevalence found in Bedouins in Israel (residing in this region), despite their poor living conditions and close contact with livestock (Markovich et al. 2014). The low seroprevalence observed for T. gondii suggests that in Israel the risk of infection from equids to people is very low, especially as consumption of horsemeat is uncommon in this country. Surprisingly this is in contrast to the reported situation in Egypt, a neighboring country of Israel, where 79 (52.6%) out of 150 horse meat samples were found to be infected with T. gondii (Shaapan and Ghazy 2007).

Conclusion

Low seroprevalences were detected for both L. infantum and T. gondii in the horse population in Israel. Still, an ongoing geographical expansion of L. infantum previously reported in humans and dogs in Israel is also supported by our results in horses. Because horses may serve as hosts for both L. infantum and T. gondii, continuous seroprevalence surveillance in horses, such as the one performed in this study, might further elucidate the eco-epidemiology of these two important zoonotic parasites in this country.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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