Can fleas from dogs infected with canine visceral leishmaniasis transfer the infection to other mammals?

Veterinary Parasitology 147 (2007) 320–325 www.elsevier.com/locate/vetpar

Short communication

Can fleas from dogs infected with canine visceral leishmaniasis transfer the infection to other mammals? Maria Teresa Zanatta Coutinho a, Pedro Marcos Linardi b,* a

Centro de Controle de Zoonoses, Prefeitura Municipal de Contagem, Av. Coronel David Sarnoff, 3113 Contagem, Minas Gerais, Brazil Departamento de Parasitologia, Instituto de Cieˆncias Biolo´gicas, Universidade Federal de Minas Gerais (UFMG), Caixa Postal 486, Avenida Antoˆnio Carlos 6627, Campus UFMG, CEP 31270-901, Belo Horizonte, Minas Gerais, Brazil

b

Received 16 January 2007; received in revised form 12 April 2007; accepted 12 April 2007

Abstract In order to investigate the possible role of dog fleas in the transmission of trypanosomatids, ectoparasites were removed from 59 dogs testing positive for canine zoonotic visceral leishmaniasis according to the indirect fluorescent antibody test (IFAT). Of the fleas collected, 4/207 (1.9%) showed the presence of promastigotes in smears stained by Giemsa, whilst 43/144 (29.9%) exhibited positive polymerase chain reaction (PCR) amplification assays for Leishmania DNA. Fleas (409) from 9 Leishmania chagasiinfected dogs, each hosting more than 20 fleas per animal, were macerated and administered by peritoneal injection or orally to 36 hamsters. After 6 months, the 30 surviving hamsters were sacrificed and liver and spleen fragments were removed for PCA assay and to produce imprint smears, whilst blood samples were subjected to IFAT assay. Sixteen hamsters tested positive for Leishmania infection, 14 on the basis of PCR amplification and four by IFAT assay (two animals testing positive in both assays). Of the infected hamsters, 11/16 (68.7%) had been infected peritoneally and 5/16 (31.2%) orally. The imprint smears for all animals were, however, negative. Since both PCR and IFAT could present cross-reactivity for Leishmania and Leptomonas, the possibility of oral transmission of L. chagasi by fleas cannot be proven unambiguously even though the hamsters developed infection. # 2007 Elsevier B.V. All rights reserved. Keywords: Canine visceral leishmaniasis; Ctenocephalides felis felis; Fleas; Leishmania chagasi; Leptomonas; Monoxenous trypanosomatids

1. Introduction Zoonotic visceral leishmaniasis (ZVL) is a disease caused by Leishmania chagasi (syn. L. infantum), a protozoan that is widely distributed in South and Central America. The infective forms of the parasite (the metacyclic promastigotes) develop in the intestines of the phlebotomine sand fly Lutzomyia longipalpis, the main vector of ZVL (Lainson and Shaw, 1988; Desjeux, 1991). Whilst sand flies are abundant in areas that are endemic for ZVL, the rates of infection of these insects * Corresponding author. Tel.: +55 31 34992874; fax: +55 31 34992970. E-mail address: [email protected] (P.M. Linardi). 0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.04.008

with L. chagasi have been found to be generally low in a number of South American countries. Thus, a natural infection rate of 0.28% has been reported in Venezuela (Feliciangeli et al., 1999), whilst that in Colombia is between 0.29 and 0.9% (Corredor et al., 1989; Ferro et al., 1995). In Brazil, published infection rates range from 0.2 to 0.5% (Ryan et al., 1984; Sherlock, 1996), although a rate of 7.1% was claimed for Santare´m in the State of Para´ (Lainson et al., 1985). Although it is accepted that the natural transmission of canine ZVL mainly occurs through the bite of an infected sand fly, other potential mechanisms cannot be dismissed. In this context, Coutinho et al. (2005) have recently described the oral transmission of L. chagasi by the tick Rhipicephalus sanguineus in Brazil.

M.T.Z. Coutinho, P.M. Linardi / Veterinary Parasitology 147 (2007) 320–325

Among the vertebrate hosts incriminated in the epidemiology of ZVL, the dog is the principal source of infection for sand flies and is also the main domestic reservoir of L. chagasi. On the other hand, the crabeating fox, Cerdocyon thous, found in Amazonian and north-eastern Brazil, is the main sylvatic reservoir of the disease (Lainson et al., 1990; Couternay et al., 1996), although various other mammals including opossums may also be involved in this role (Sherlock et al., 1984). All known ZVL reservoirs are parasitised by fleas, most commonly by Ctenocephalides felis felis (Linardi and Nagem, 1973; Linardi and Guimara˜es, 2000). Fleas collected from C. thous in the ZVL-endemic area of Jacobina (Bahia, Brazil) also belonged to the genus Ctenocephalides (Cerqueira et al., 2000). Owing to the close association with their hosts, fleas affect the health and well-being of these animals. Certain characteristics of flea biology and behaviour, such as their remarkable capacity for exchange between hosts (Rust, 1994) together with their voracity, feeding frequency and vectorial capacity, make them susceptible to infection by diverse pathogens (Bibikova, 1977) including trypanosomatids (Wallace, 1966; Beard et al., 1990). Additionally, the feeding behaviour of C. felis felis (Dryden and Gaafar, 1991) prompts dogs to carry out grooming (Hinkle et al., 1998) in an attempt to relieve themselves of discomfort by removing the fleas with their teeth. Such actions, together with the common submissive gestures of licking and fondling around the muzzles of other dogs, facilitate the ingestion of flea gut contents by the animal, and this might act as a mode of transmission in canine ZVL. Since infection of an HIV-positive patient with a species of Leptomonas (a monoxenous trypanosome) isolated from the dog flea Pulex simulans has already been reported (Pacheco et al., 1998), we have investigated the role of dog fleas in relation to the possible oral transmission of trypanosomatids. 2. Materials and methods All experiments were performed in accordance with international guidelines for biomedical research involving animals and the recommendations of the Cole´gio Brasileiro de Experimentac¸a˜o Animal (COBEA). 2.1. Collection of dog fleas and preparation of assay samples and smear slides In the State of Minas Gerais, Brazil, dogs that are found to be positive for L. chagasi on the basis of the indirect fluorescent antibody test (IFAT) are transported

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to the laboratories of the Centro de Controle de Zoonoses (CCZ) in Montes Claros, Sabara´ or Belo Horizonte (Minas Gerais, Brazil) as part of the standard ZVL control policy. Fifty-nine L. chagasi-infected dogs, presenting diverse symptomologies, were immobilised, subjected to a clinical ectoscopic examination and then sedated so that the ectoparasites could be removed from the body surface by prising them off manually or through the use of forceps. Fleas were stored individually in glass tubes fitted with rubber stoppers until required for identification and subsequent dissection in 0.9% saline under a stereomicroscope. Part of the material, including the exoskeleton of the flea, was homogenised in ca. 80 ml of saline in an Eppendorf tube, mixed with an equal volume of Tris–EDTA buffer and frozen at 20 8C for later analysis by polymerase chain reaction (PCR) amplification. The remaining material was used to prepare a smear slide that was fixed in methanol, stained with Giemsa and examined for promastigotes under an oil-immersion lens. The ectoparasites of a selected sample of 9 infected dogs, each of which had been host to more than 20 fleas in total over the entire body, were separately identified, counted and then collectively macerated in a tissue grinder. Each macerate was mixed separately with 2 ml of 0.9% saline containing 1 ml/l of penicillin G solution (5,000,000 U dissolved in 10 ml water) and 1 ml/l streptomycin (1 g dissolved in 5 ml water), and the supernatant removed using a hypodermic syringe. Blood samples were taken from the dogs in order to confirm the serological diagnoses based on IFAT and TRALd (rapid test for Leishmania donovani) (Camargo and Robonato, 1969; Burns et al., 1993). 2.2. PCR analysis The detection of DNA characteristic of species of Leishmania was performed by PCR amplification. Flea samples (ca. 80 ml) were thawed, re-homogenised in a disposable plastic pestle, mixed with 12 ml of proteinase K (final concentration 100 mg/ml) and made up to a final volume of 100 ml by the addition of ca. 10 ml of Milli-Q water (prepared using a Millipore Milli-Q1 purification system). The mixture was incubated for 3 h at 56 8C with further homogenisation every 30 min in order to digest all of the proteinaceous material. Remaining proteinase K was deactivated by boiling for 15 min, the mixture was centrifuged for 5 min at 14,000 rpm and the supernatant transferred to another Eppendorf tube. DNA was extracted from each sample and amplified by PCR using primers 150 [50 GGG(G/T)AGGGGCGTTCT(G/C)CG AA-30 ] and 152

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[50 -(G/C)(G/C)(G/C)(A/T)CTAT(A/T)TTACACCAACCCC-30 ], which correspond to the conserved region of the minicircles of kDNA of Leishmania, according to published methods (Degrave et al., 1994). PCR amplicons were separated on polyacrylamide gels (6%) according to the method of Marques et al. (2001): the gels were subsequently placed in fixing solution (15 ml ethanol, 750 ml acetic acid, 150 ml Milli-Q water) for 10 min and then stained with 0.2% silver (Santos et al., 1993).

specimens, one was male and four were females, giving a natural infection rate of 2.4 and 1.8%, respectively. PCR analysis was carried out on 144 flea homogenate samples. A single DNA band of ca. 118 bp, corresponding to the expected amplicon from the conserved region of the minicircles of kDNA of Leishmania, was observed in 43 samples (29.9%) deriving from 31 female (72%) and 12 male (28%) fleas. There were no significant differences between the infection rates in males and females ( p < 0.05).

2.3. Infectivity of trypanosomatids found in dog fleas

3.2. Infectivity of the dog flea homogenates

The infectivity of the flagellates encountered in the fleas collected from nine infected dogs was determined by infection experiments (carried out between May and August 2001) involving hamsters that had been bred and maintained under appropriately controlled conditions in the animal house at the Universidade Federal de Minas Gerais. Thirty-six hamsters were randomly assigned to two sets of nine cages with one pair of animals per cage. The supernatant of the flea homogenate obtained from an infected dog was transferred to a syringe and 0.5 ml was injected peritoneally into each of a pair of caged hamsters and 0.5 ml was administered orally (directly from the syringe with the needle removed) to each of a second pair of caged animals. After approximately 6 months the hamsters were sacrificed and subjected to necropsy (December 2001/January 2002). Hamsters blood samples were collected for IFAT assay, and portions of the viscera (spleen and liver) were used to prepare smear slides and samples for PCR analysis as described above. 2.4. Statistical analysis The x2-test with the Yates correction was applied in order to evaluate the degree of association between rates of infection of fleas and gender of ectoparasite, and between routes of infection of hamsters by flea homogenates. 3. Results 3.1. Analysis of smear slides and PCR assays A total of 207 flea specimens (166 females and 41 males) were collected from the 59 L. chagasi-infected dogs studied. All fleas were identified as C. felis felis. Following dissection and staining, four fleas (1.9%) were found to contain trypanosomatid promastigotes. Of these

All of the nine specially selected L. chagasi-infected dogs presented positive TRALd and IFAT assays with titre values from the later ranging from 1:40 to 1:40,960 (Table 1). A total of 409 fleas were collected from these dogs but none were positive for trypanosomatids as determined from the smear slides. Homogenates were prepared from the total complement of fleas collected separately from each dog, a value that ranged between 20 and 86 ectoparasites per animal (Table 1). Of the 36 hamsters treated with the flea homogenates, six died before the end of the experiment, five animals having received treatment by peritoneal injection and one by oral administration. The results of the serological and PCR amplification assays conducted on the remaining animals (n = 30) are presented in Table 1. According to the IFAT assay, four animals were positive for L. chagasi, 11.8% (2/17) having received the flea homogenate by oral administration whilst 15.4% (2/ 13) had been treated by peritoneal injection. The difference in infection rate between the two routes was not significant ( p > 0.05). According to the PCR amplification assay, 24.1% (14/58) of the hamsters were found to be positive for Leishmania DNA, 40.0% (10/25) of which had been infected peritoneally and 12.1% (4/33) orally (Table 1). Peritoneal infection was clearly more effective and the difference between the two routes was significant (x2 = 4.61; p < 0.05). On the basis of the IFAT assay, 13.3% (4/30) of the hamsters were infected by flea macerates, two via the oral route and two by peritoneal injection. The difference between the routes of infection was not significant in this case ( p > 0.05). When the results from PCR and IFAT assay were pooled, 16 (53.3%) of the 30 surviving animals were found to be infected. PCR and IFAT were both positive in two hamsters (12.5%), whilst PCR alone revealed 12 positive animals (75%) and IFAT alone only 2 (12.5%). Of these infected hamsters, 68.7% (11/16) had been infected peritoneally and 31.2% (5/16) orally.

IFAT titre of dogs

Number of fleas in the homogenate

Number of treated hamsters showing positive PCR assays of viscera fragments Spleen

Liver b

Peritoneal (n = 13) 1:40 1:160 1:2560 1:20,480 1:20,480 1:20,480 1:40,960 1:40,960 1:40,960 Total Percentage of tests showing positive results a b c

c

Oral (n = 16)

Peritonealb (n = 12)

Number of treated hamsters showing positive IFAT assays

Total number of hamsters infected as determined by PCR/IFAT assays

Total number of hamsters completing each experimenta

Oralc (n = 17)

20 33 20 59 31 86 21 61 78

1 1 – 1 1 – 1 – 1

– – – – – – – – –

– 1 2 – – – – – 1

– – – – 1 1 – 1 1

1 – 1 – – – – – 2

2 2 3 1 2 1 1 1 3

3 4 3 3 4 3 4 2 4

409

6

–

4

4

4

16

30

46.1

0

33.3

23.5

13.3

Thirty-six hamsters were initially treated, 18 by peritoneal injection and 18 by oral administration. Number of animals surviving peritoneal injection of flea homogenate = 13. Number of animals surviving oral administration of flea homogenate = 17.

53.3

M.T.Z. Coutinho, P.M. Linardi / Veterinary Parasitology 147 (2007) 320–325

Table 1 Relationship between IFAT titres from nine L. chagasi-infected dogs and the number of hamsters testing positive for the presence of the parasite, as determined by PCR amplification and IFAT assays, following administration of the homogenate by peritoneal injection or orally to the experimental animals

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4. Discussion A number of factors suggest that fleas might represent efficient agents for the natural transfer of infective pathogens, and these include the mode of obtaining blood, the duration of blood feeding, digestion and contact with the host, and frequency of host exchange. A female flea lays on average 40–50 eggs per day and must engorge with blood each time it oviposits. Thus females of C. felis felis consume up to 13.6 ml of blood per day and are able to reach a mean weight of 0.95 mg (Dryden and Gaafar, 1991). Fleas deposit 0.77 mg of faeces per day on the fur of their host, and these faeces contain partially digested blood, which is consumed by the flea larvae that live in the nests of the hosts. In this manner, the feeding behaviour of the adult flea assists larval development and also ensures that the intestine of an adult flea always contains fresh blood mixed with blood from previous meals that is partially digested. In the present study it is noteworthy that the rate of infection of C. felis felis by trypanosomatids was determined to be 1.9% according to smear slides and 29.9% according to PCR amplification assays. In Florida, the prevalence of trypanosomatids in fleas obtained from dogs and cats was 4.5% (Beard et al., 1990). Recently, de Avelar et al. (2007) found a prevalence of 3.1% of these flagellates in C. felis felis from dogs of Brazil. In contrast, the typical natural infection rate of sand flies by L. chagasi has been estimated as being less than 1% (Deane, 1958; Mayrink et al., 1979; Ryan et al., 1984; Corredor et al., 1989; Ferro et al., 1995; Sherlock, 1996; Feliciangeli et al., 1999). The reason for this disparity may be related to the fact that sand flies feed only once on their host and at a site that may or may not harbour Leishmania parasites, whilst fleas feed over the entire body of the host on several occasions over a period of many days. All of the flea homogenates obtained from L. chagasi-infected dogs gave rise to infection in hamsters as determined by PCR or IFAT analysis independent of the titres of the dogs’ blood: the infection rates varied from 25% (dog’s blood titre value 1:40,960) to 100% (dog’s blood titre value 1:2560) (Table 1). However, in spite of a light or moderate abdominal swelling observed in 26 animals (72.2%), none of the organs from hamsters treated with flea homogenates tested positive for the presence of amastigotes of L. chagasi according to imprint smears, in spite of a light or moderate abdominal swelling observed in 26 animals (72.2%). Although hamsters that had been inoculated

intraperitoneally with macerates of sand flies containing promastigotes of L. chagasi, have been followed for up to 11 months (Montoya-Lerma et al., 2003), it is known that some L. chagasi strains induce a fulminating form of ZVL that invariably leads to the death of the experimental animal (Hommel et al., 1995). Since dogs are very likely to ingest flea gut contents when licking their own fur or that of other dogs, the presently recorded infection rate of 23.5% (4/17) following oral administration could suggest a serious danger of ZVL infection occasioned by the habitual ingestion of these arthropods and their contents by their hosts (Hinkle et al., 1998). In sand flies, development into metacyclic forms after taking a sugar meal, and the presence of a peritrophic membrane, constitute fundamental steps in biological transmission (Schlein, 1993). However, the ingestion of flea contents could represent an alternative route for the transmission of L. chagasi between dogs, and might well explain the occurrence of the disease in regions where the traditional vector is absent. However, since species of Leishmania and Leptomonas present cross-reactivity in both PCR and IFAT tests (Garin et al., 2001; Anders, 2003), and the visceral smear slides proved negative, the possibility of oral transmission of L. chagasi by fleas is not unambiguously proven by our experiments even though the hamsters developed infection. Consequently, the raised question remains unanswerable. Acknowledgements The authors are grateful to Lilian Lacerda Bueno, Annelise Sterzik and members of the Centro de Controle de Zoonoses (CCZ) of Belo Horizonte and Sabara´ for their cooperation in this study. The study described in this paper forms part of the Ph.D. thesis of M.T.Z.C. within the Parasitology/ Programa de Po´s-graduac¸a˜o em Parasitologia/Instituto de Cieˆncias Biolo´gicas/Universidade Federal de Minas Gerais and was partially supported by the Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES/Brasil) and the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq/Brasil). P.M.L. is a research fellow from CNPq/Brasil. References Anders, 2003. PCR diagnosis of leishmaniasis in Israel and the West Bank, Ph.D. Thesis, Humboldt-Universita¨t zu Berlin, Berlin, Deutschland. Beard, C.B., Butler, J.F., Hall, D.W., 1990. Prevalence and biology of endosymbionts of fleas (Siphonaptera: Pulicidae) from dogs and cats in Alachua County, Florida. J. Med. Entomol. 27, 1050–1061.

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