Antibodies to Selected Canine Pathogens and Infestation with Intestinal Helminths in Golden Jackals (Canis aureus) in Israel

The Veterinary Journal 2001, 162, 66–72 doi: 10.1053/tvjl.2001.0572, available online at http://www.idealibrary.com on

Antibodies to Selected Canine Pathogens and Infestation with Intestinal Helminths in Golden Jackals (Canis aureus) in Israel M. SHAMIR*, B. YAKOBSON†, G. BANETH*, R. KING‡, S. DAR-VERKER*, A. MARKOVICS† and I. AROCH* *Koret School of Veterinary Medicine, Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100 Israel, †Kimron Veterinary Institute, P.O. Box 12 Beit Dagan, Israel, ‡Nature Reserve Authority, P.O. Box 667, Eilat, Israel

SUMMARY Blood and faecal samples, collected from 46 healthy adult free-ranging golden jackals captured in two different locations in Israel, were examined. A serological survey was conducted to investigate the prevalence of circulating antibodies reacting with four common canine pathogens: canine distemper virus (CDV), canine parvovirus (CPV), Ehrlichia canis and Leishmania infantum. Faecal floatation and haematological tests were also performed. The seroloprevalence of CPV, E. canis, CDV, and L. infantum were 72.3% (34/47), 54.3% (25/46), 52.2% (24/46), and 6.5% (3/46) respectively. Faecal floatation tests revealed a high prevalence of Ancylostoma caninum (13/17, 76%) and a low prevalence (1/17) of Dipilidium caninum infestation. Examination of blood smears revealed Hepatozoon canis gamonts in one jackal. Golden jackals are among the most common free-ranging carnivores in Israel and neighbouring countries. Their habitats are in proximity to densely populated areas and they bear close phylogenetic relation to the domestic dog. These facts, combined with the high prevalence of the jackals’ exposure to the major canine pathogens demonstrated in this study, suggest that they may serve as a reservoir for the transmission of certain diseases to domestic dogs. © 2001 Harcourt Publishers Ltd KEYWORDS: Jackal; Canine distemper virus; parvovirus; Ehrlichia canis; Leishmania infantum; Ancylostoma; Hepatozoon.

INTRODUCTION The golden jackal (Canis aureus syriacus) is one of the most prevalent wild canids in Israel and neighbouring countries and has been a natural inhabitant of the Middle East for many years (Mendelson & Yom-Tov, 1987). Jackals are known to be omnivores and scavengers, which prey on small mammals, chickens, carcasses and insects. They also feed on vegetables, fruit and garbage. Thus they adapt to a wide range of habitats (McKenzie, 1993) and tend to infringe on rural and urban areas (Mendelson & Yom-Tov, 1987). This pattern of behaviour results in increased contact between Correspondence to: M. Shamir. Tel: 972 3 9688558; Fax: 972 3 9604079; E-mail: [email protected] 1090-0233/01/010066 + 01 $35.00/0

jackals and domestic dogs (Canis familiaris) and the phylogenetic relationship between the two species is considered to be very close. These facts may contribute to the transmission of infectious diseases between them (Mendelson & Yom-Tov, 1987; Alexander et al., 1994). Jackals have been reported to host a large spectrum of pathogens also found in the domestic dog including rabies (Yakobson et al., 1998), canine parvovirus, canine distemper virus (Alexander et al., 1994), Ehrlichia canis (Waner et al., 1999), Leishmania donovani, Toxoplasma gondii (van der Merwe, 1953), Ancylostoma caninum (Macchioni, 1995) and Echinococcus granulosus (Macpherson et al., 1983). A recent study demonstrated a high incidence of seropositivity for E. canis (35.8%) and other rickettsiae in Israeli golden jackals (Waner et al., 1999). © 2001 Harcourt Publishers Ltd

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A similar seroprevalence of 30% E. canis was found in Israeli domestic dogs (Baneth et al., 1996). An outbreak of visceral leishmaniasis in man and domestic dogs has been described recently as an emerging problem in Israel and a number of seropositive jackals were found in the disease focus (Baneth et al., 1998). It has been suggested that wild canids, including the golden jackal, may have a role in the transmission of this disease to nonendemic areas in the country (Baneth et al., 1998). Canine distemper virus and canine parvovirus are among the most common viral infections in both unvaccinated and partially vaccinated dogs in Israel, in spite of extensive vaccination throughout the country during the last 15 years. Analysis of epidemiological data on the diseases of the golden jackal may aid in understanding the role of wild canids in the transmission of canine infectious diseases and certain zoonoses. This paper describes a survey of certain selected important canine pathogens including CDV, CPV, E. canis, L. infantum and intestinal helminths in healthy free ranging golden jackals held in captivity. MATERIAL AND METHODS

Animals Forty-six adult golden jackals were trapped in central and northern Israel by foot and bait traps, as a part of an experiment on oral rabies vaccination conducted in the summer of 1998 by the Nature Reserves Authority and the Israeli Veterinary Services. The animals were sedated with xylasine hydrochloride (Rompun, Bayer; 1.1 mg/kg i.m.) and ketamine hydrochloride (Ketaset, Fort-Dodge; 5 mg/kg i.m.) and transported to an isolation unit where they were allowed to acclimatize for a period of seven days. During this period, the jackals were closely observed for clinical abnormalities, and were found to be clinically normal. The animals were anaesthetized, using ketamine hydrochloride (Ketaset, Fort-Dodge; 10 mg/kg i.m.), underwent physical examination and were bled from the cephalic or the jugular vein.

Samples Blood samples placed in calcium-EDTA tubes were submitted for haematological studies within one hour and peripheral blood smears were stained in May-Grünwald-Giemsa. Blood samples for serological tests were collected in plain tubes, allowed to clot, and separated by centrifugation. Sera for serological

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tests were stored at –20˚C pending analysis. Faecal samples were obtained rectally and sent for analysis.

Haematological and faecal tests A complete blood count (CBC) was performed on 31 jackals using an automated analyser calibrated for canine blood (Minos ST). Blood smears were examined microscopically for the presence of blood parasites. Faecal samples collected from 17 jackals were floated in Sheather’s solution (specific gravity 1.23), and examined microscopically for eggs. Egg numbers were counted in 1 g of faeces.

Serological tests Dot-blot ELISA tests for CDV, CPV and E. canis IgG antibodies were performed, as previously described (Waner et al., 1996; Waner et al., 1998; Waner et al., 2000) using commercially available test kits (Immunocomb, Biogal) containing plastic combs sensitised with CDV, CPV and E. canis antigens (CDV-Rockborn strain, CPV-2 strain C-780916 and E. canis Israel No. 611 strain). Sera were diluted (1:36 for CDV and for E. canis, and 1:20 for CPV) in buffer and incubated with antigen spots for five minutes. After washing to displace unbound antibodies, the combs were allowed to react with purified whole molecule goat anti-dog IgG alkaline phosphatase conjugate ( Jackson Immunosearch Laboratories Inc.). After two successive washing steps, bound antibody was detected with a precipitating chromogen, 5-bromo-4-chloro-3-indolyl phosphate and nitro-blue tetrazolium (Biosynth International). The concentration of the antibodies for each sample was measured utilizing a colourcoded scale provided in the test kits. By this method, translation of the colour reaction to the IgG antibody units was achieved by comparison of each spot with the colour reaction of a spot from a known positive pre-titrated serum sample. The results were expressed in ‘S’ units on a scale of 0 to 6. For CDV, CPV and E. canis, three ‘S’ units were assigned the positive control serum titre. ‘S’ unit 3 is equivalent to a viral neutralization (VN) titre of 1:80 for CDV, haemagglutination inhibition (HI) titre of 1:80 for CPV and to immunofluorescent (IF) titre of 1:160 for E. canis. The method has been validated and calibrated with the above golden standard tests previously (Waner et al., 1996; Waner et al., 1998; Waner et al., 2000). The ‘S’ values reading for CDV and CPV were as follows: ‘S’ units 4, 5 and 6 are equivalent to 1:160, 1:480 and 1:1280 respectively in their respective serological

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test. The ‘S’ values reading of 4 and 5 for E. canis were 1:320 and 1:960. Anti-leishmanial antibodies were tested by ELISA using crude leishmanial antigen, as previously described (Baneth et al., 1998). Briefly, the jackal sera were diluted (1:100), and incubated with antigen coated plates for one hour at 37˚C. The plates were then washed with 0.1% Tween 20 in PBS, pH 7.2, and incubated with whole molecule rabbit antidog IgG conjugated to horseradish peroxidase (1:10 000 dilution, Jackson Laboratories Inc.) for one hour at 37˚C. Excess conjugate was removed by extensive washing in PBS-Tween and the plates were developed by addition of the substrate 2,2′azino-di-3-ethylbenzthiazoline sulfonate (ABTS) (Boehringer Mannheim). Each plate was read when the absorbency (405 nm) of the positive canine serum reached a value between 0.95 and 1.0. A titration of positive and negative reference dog sera was included on each plate to monitor

interassay variation. A control group consisting of six jackal sera originating from an area considered to be non-endemic for leishmaniasis was used to determine a cut-off for distinguishing between positive and negative sera. A sample was considered positive if the optical density was higher than the mean control plus 2.6 times the standard deviation of the control group (99% confidence interval). RESULTS Thirty-three jackals (72.3%) were serologically positive for CPV (‘S’ unit > 3) with ‘S’ units equivalent to HI titre levels of 1:160–1:1280 and 13 (27.7%) were negative (‘S’ unit ≤ 3) (Table I). Twenty-five (54.3%) jackals were serologically positive for E. canis (‘S’ units ≥ 3) with ‘S’ units equivalent to IF titre levels of 1:160–1:960 and 21 (45.7%) were negative (‘S’ units < 3) (Table I). Twenty-four jackals (52.3%) were serologically positive for CDV

Table I Prevalence of IgG antibodies for E. canis, canine distemper virus and canine parvovirus, given in ‘S’ units, from 46 Israeli golden jackals Ehrlichia canis ‘S’ unit IF titre equivalent of n (%) Positive/negative Total

<3 <1:160 21 (45.7) Negative 21 (45.7%)

3 1:160 4 (8.7)

4 1:320 9 (19.6) Positive 25 (54.3%)

5 1:960 12 (26)

≤3 ≤1:80 22 (47.8) Negative 22 (47.8%)

4 1:160 15 (32.6)

5 1:480 9 (16.6) Positive 24 (49.2%)

6 1:1280 0 (0)

≤3 ≤1:80 13 (28.3) Negative 13 (28.3%)

4 1:160 5 (10.8)

5 1:480 9 (19.6) Positive 33 (71.7%)

6 1:1280 19 (41.3)

Canine distemper virus ‘S’ unit VN titre equivalent of n (%) Positive/negative Total

Canine parvovirus ‘S’ unit HI titre equivalent of n (%) Positive/negative Total

CANINE PATHOGENS IN JACKALS IN ISRAEL

(‘S’ unit > 3) with ‘S’ units equivalent to titre VN levels of 1:160–1:480, and 22 (47.8%) were negative (‘S’ unit ≤ 3), (Table I). Three (6.4%) jackals were serologically positive to L. infantum (optical density higher than the mean control plus 2.6 times the standard deviation of the control group). Fifteen jackals were seropositive to one pathogen only, 19 were seropositive to two and 11 to three pathogens (Table II). Complete blood counts showed thrombocytopenia (platelets < 200 × 109/L) in three jackals and anaemia (haematocrit < 0.3) in four jackals. Examination of blood smears revealed Hepatozoon canis gamonts in 1.5% of the neutrophils in one jackal. No other blood parasites were detected. Faecal floatation studies revealed Ancylostoma caninum eggs in 13/17 jackals (76%). Egg numbers range was from 50–800/g (mean 220/g, standard deviation 230/g). In addition, one jackal was found to be infested with Dipylidium caninum. DISCUSSION In this study, the seroprevalence of IgG antibodies against E. canis in golden jackals in Israel was found to be 54.3%. This prevalence is higher than that reported in a previous study, which demonstrated a seroreactivity of 35.8% in 53 Israeli golden jackals (Waner et al., 1999). However, similar findings were reported in the silver-backed jackal (Canis mesomelas) in Kenya, where 50% of the jackals in a study, performed by cell culture tests, were infected (Price & Harstad, 1980). A survey conducted on clinically healthy dogs in Israel found an overall seropositive incidence of 30%, with 23.9% in pet dogs and 37.5% in stray dogs (Baneth et al., 1996). Our findings suggest that golden jackals are frequently exposed to E. canis, as no other canine infective agent, cross-reacting with E. canis is known in Israel (Waner et al., 1999). The high seropositivity in golden jackals correlates well to the higher seropositivity described in stray dogs comparing to pet dogs in Israel. A similar relation has been previously reported in silver-backed jackals compared to domestic dogs in Kenya (Price & Harstad, 1980). The higher rate of seropositivity in jackals in comparison to dogs may be attributed to higher exposure of jackals to the tick vector Rhipicephalus sanguineus. Despite this high seropositivity for E. canis, all jackals included in this study were found to be clinically normal, suggesting that jackals may be sub-clinically infected with E. canis, as has been

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described in dogs (Ristic & Holland, 1993; Harrus et al., 1998), or recovered spontaneously from a clinical disease. Thrombocytopenia has been described as a consistent haematological finding in canine monocytic ehrlichiosis (CME) (Harrus et al., 1997). In this study, only 3/31 (9.7%) jackals had low platelet numbers (platelets < 200 × 109/L) and, of these, two were seropositive for rickettsia. This may imply that the two jackals were subclinically infected. Of four jackals with low haematocrits (< 0.3), three were found positive for E. canis. This finding may suggest that anaemia in jackals may also be associated with previous exposure to the rickettsia. One jackal had H. canis parasitaemia, diagnosed through identification of the gamonts of the parasite in the blood smear. To the best of our knowledge, this is the first reported case of H. canis in a golden jackal. Infection with H. canis was previously described in other wild canids, such as the crabeating fox (Cerdocyon thous) (Alencar et al., 1997) and the red fox (Vulpes vulpes) (Conceicao et al., 1998). This jackal was also found to be strongly positive for E. canis. Co-infection with both parasites has been reported to occur in dogs in Israel (Baneth & Weigler, 1997). Canine distemper virus has been previously reported in wild carnivores, including jackals, from different countries (Gese et al., 1991; Alexander et al., 1994; Spencer et al., 1999; Frolich et al., 2000). The seroprevalence for CDV in our study was 52.2%. Similar studies in Kenya demonstrated prevalence of 7.3% and 0% in Canis mesomelas and Canis aureus respectively, and in Zimbabwe 50% and 63.6% in Canis adustus and Canis mesomelas, respectively. A survey conducted in Germany reported a significantly higher prevalence of CDV in foxes from urban and suburban areas compared to foxes from rural areas, suggesting transmission of the virus from domestic dogs to the wild carnivores (Frolich et al., 2000). Outbreaks of distemper in unvaccinated and partially vaccinated dogs are common in Israel, and have also been previously reported in different European countries (van Moll et al., 1995; Ek-Kommonen et al., 1997). The high prevalence for CDV in clinically healthy jackals found in this study indicates that jackals are frequently exposed to CDV, and survive the viraemic stage of the disease. The question of whether wild carnivores serve as a reservoir of CDV for dogs and other species was previously addressed by Allinger et al. (1993). A study that investigated the expression of five CDV-specific proteins and their epitopes

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Table II The distribution of seropositivity for selected canine pathogens in 30 jackals positive for more than one pathogen

Number of Jackals

Distemper virus and parvovirus

Ehrlichia canis and parvovirus

8

6

Ehrlichia Leishmania canis and infantum distemper virus and parvovirus

4

in the CNS of dogs and mustelids in Germany showed that the distemper virus demonstrated in these species of wild carnivores exhibits antigenic characteristics of CDV (Alldinger et al., 1993). It remains to be determined if Israeli golden jackals carry a new strain of CDV, and whether any such jackal-distemper virus is virulent for dogs, and hence making the golden jackal a potential source of CDV infection for domestic dogs or the opposite. The seropositivity for CPV in our study was 72.3%; of these, 55% were strongly positive (‘S’ unit 6, equivalent to HI titre of 1:1280). A lower prevalence of CPV seropositivity was reported in Kenya in golden jackals (56%), side-stripped jackals (Canis adustus) (33.3%) and black-backed jackals (Canis mesomelas) (25%) (Alexander et al., 1994). Another study, from Zimbabwe, reported a seroprevalence of 18.2% and 12.5% in Canis mesomelas and Canis adustus respectively (Spencer et al., 1999). Our findings may differ from those reported in African countries due to the high population density in Israel, which results in closer contact between golden jackals and domestic dogs. These data support the concept of transmission of CPV between the two species. Visceral leishmaniasis has been described in a golden jackal previously (Hervas et al., 1996). The emergence of visceral leishmaniasis in a new location in central Israel was reported in both man and domestic dogs (Baneth et al., 1998). This previous study, conducted around two village foci in central Israel, reported 7.6% and 5% seropositivity for L. infantum in golden jackals and red foxes, respectively. In our study, jackals from different locations in Israel were trapped and examined, and a similar rate of seropositivity (6.4%) was found, thus indicating that L. infantum infection in jackals is probably prevalent in many locations. Similar overall seroprevalence (6.8%) for L. infantum has been

1

Ehrlichia canis, parvovirus and distemper virus

Leishmania infantum, parvovirus and distemper virus

9

2

also reported in domestic dogs in central Israel (Baneth et al., 1998). A higher seroprevalence to L. infantum (11.5%) was found in domestic dogs examined in two neighbouring village foci, while only 1% seropositivity was found in dogs living in nearby settlements. This may indicate that sandflies, the vectors of the disease, are not the sole factor in the spread of the disease to new distant locations. This is especially so, as Phlebotomus flies are weak fliers (Soulsby, 1982). Other ecological factors, such as infected wild canids, may have an important role in the transmission of leishmaniasis from endemic to non-endemic locations. Thirty jackals were seropositive for more than one canine pathogen. This finding may therefore make the exposure of domestic dogs to jackals a serious risk, as one jackal may transmit several diseases. Acylostoma caninum and Dipylidium caninum have been found in the domestic dog and many other wild canids, including jackals, and have a worldwide distribution (Soulsby, 1982; Maccionni, 1995). The very high rate of infection (76%) by Ancylostoma caninum in Israeli golden jackals found in this study suggests that golden jackals may also serve as a reservoir for the parasite, which may lead to an increased risk of infection of dogs in suburban and rural areas. Since ancylostomiasis has zoonotic implications, people in these areas may also be affected. Wild canid populations in Israel came close to extinction in the 1950s and 1960s as a result of a rabies control program that included the elimination of potential reservoir hosts (Yakobson et al., 1998). However, recent annual counts of wild canids in central Israel have shown a dramatic increase in the jackal population, and a progressive incursion into southern Israel (Eli Shay, Nature Reserves Authority, unpublished data). This trend, coupled with the close contact between the jackal

CANINE PATHOGENS IN JACKALS IN ISRAEL

population and densely populated areas in Israel, and the close phylogenetic relation between jackals and dogs, may lead to an increase in the risk of exposure of dogs to certain diseases. Our study has shown that the prevalence of canine pathogens in golden jackals in Israel is high, and that individual jackals are exposed to multiple canine infectious agents, suggesting that jackals may serve as an important reservoir and a potentially efficient transmitter of certain canine pathogens. The data presented in this study, limited to a group of 46 golden jackals, warrant further investigations into the specific pathogen strains found in domestic dogs and golden jackals before definite conclusions regarding the role of the golden jackal as a reservoir can be drawn. ACKNOWLEDGEMENTS The authors would like to thank Ms Shlomit Mazar from Biogal laboratories and Dr Dalit Strauss for their technical assistance. This research was partially funded by grant number S0 220/5-1 of the Deutsche Forschungsgemeinschaft (DFG) for the Palestinian-German-Israeli Cooperative Project on leishmaniasis in Palestine and Israel. REFERENCES ALENCAR, N. X., KOHAYAGAWA, A. & V. A. SANTAREM (1997). Hepatozoon canis infection of wild carnivores in Brazil. Veterinary Parasitology 70, 279–82. ALEXANDER, K. A., KAT, P. W., WAYNE, R. K. & FULLER, T. K. (1994). Serologic survey of selected canine pathogens among free-ranging jackals in Kenya. Journal of Wildlife Diseases 30, 486–91. ALLDINGER, S., BAUMGÄRTNER, W., van MOLL, P. & Örvell, C. (1993). In vivo and in vitro expression of canine distemper viral proteins in dogs and non-domestic carnivores. Archives of Virology 132, 421–8. BANETH, G., WANER, T., KOPLAH, S., WEINSTEIN, A. & KEYSARY, A. (1996). Survey of Ehrlichia canis antibodies among dogs in Israel. Veterinary Record 138, 257–9. BANETH, G. & WEIGLER, B. (1997). Retrospective casecontrol study of hepatozoonosis in dogs in Israel. Journal of Veterinary Internal Medicine 11, 365–70. BANETH, G., DANK, G., KEREN-KORNBLATT, E., SEKELES, E., ADINI, I., EISENBERGER, C. L., SCHNUR, L. F. & JAFFE, C. L. (1998). The emergence of visceral leishmaniasis in central Israel. American Journal of Tropical Medicine and Hygiene 59, 722–5. CONCEICAO, F. M., ABRANCHES, P., SILVA-PEREIRA, M. C. D. & JANZ, J. G. (1998). Hepatozoonosis in foxes from Portugal. Journal of Wildlife Diseases 24, 344–7. EK-KOMMONEN, C., SIHVONEN, L., PEKANEN, K., RIKULA, U. & NUOTIO, L. (1997). Outbreak of canine distemper

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