Parasitological and serological diagnosis of Strongyloides stercoralis in domesticated dogs from southeastern Brazil

Veterinary Parasitology 136 (2006) 137–145 www.elsevier.com/locate/vetpar

Parasitological and serological diagnosis of Strongyloides stercoralis in domesticated dogs from southeastern Brazil A. Ferreira Ju´nior a, M.R.F. Gonc¸alves-Pires a, D.A.O. Silva b, A.L.R. Gonc¸alves a, J.M. Costa-Cruz a,* a

b

Laborato´rio de Parasitologia, Universidade Federal de Uberlaˆndia, Minas Gerais, Brazil Laborato´rio de Imunologia, Universidade Federal de Uberlaˆndia, Av. Para´ 1720, 38400-902 Uberlaˆndia, Minas Gerais, Brazil Received 27 May 2005; received in revised form 11 October 2005; accepted 13 October 2005

Abstract Canine strongyloidiasis is a parasitic infection caused by the nematode Strongyloides stercoralis and presents a great zoonotic potential. Its confirmation, using coproparasitological methods, is difficult. The detection of serum specific antibodies, however, may facilitate the diagnosis. The aims of this study were to determine the presence of S. stercoralis through the use of parasitological methods and to detect specific antibodies to the parasite in serum samples from domestic dogs by using the indirect fluorescent antibody test (IFAT) on slides and the enzyme-linked immunosorbent assay (ELISA). A total of 215 dogs of various breeds, from the cities of Uberlaˆndia, Araxa´ and Campo Belo in the State of Minas Gerais, were examined and distributed according to age into the following groups: (I) 19 males and 20 females of 1–2 months old; (II) 11 males and 20 females of 2-month- to 1-year-old and (III) 41 males and 104 females, from 1 to 7 years old. Coproparasitological results showed that 63/215 (29.3%) of the dogs presented some kind of parasite, with two (0.9%) dogs (one from Araxa´ and the other from Uberlaˆndia) passing S. stercoralis larvae in the feces. Serological results revealed antibodies to S. stercoralis in 45/215 (20.9%) of the dogs, with seropositivity rates of 0% (0/39) in Group I, 22.6% (7/31) in Group II, and 26.2% (38/145) in Group III. No serological cross-reactivity between S. stercoralis and hookworms or Ascaridae was found. Hookworm infections were seen in 31 dogs, but only one of these dogs (infected with both hookworm and Cystoisospora spp.) was S. stercoralis seropositive by IFAT. The present study demonstrated, for the first time, natural S. stercoralis infections in dogs diagnosed by coproparasitological and serological methods. It was concluded that the detection of specific antibodies to S. stercoralis by IFAT and ELISA may contribute to the diagnosis of canine strongyloidiasis. # 2005 Elsevier B.V. All rights reserved. Keywords: Strongyloides stercoralis; Enteroparasites; Dogs; Parasitological diagnosis; Serological diagnosis

1. Introduction * Corresponding author. Tel.: +55 34 3218 2187; fax: +55 34 3218 2333. E-mail address: [email protected] (J.M. Costa-Cruz).

In the recent times, there has been an increasing interest in the important role that pet animals play in

0304-4017/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2005.10.022

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societies throughout the world. In Brazil, especially in the large urban centers, dogs are the most frequent companion animals (Oliveira-Sequeira et al., 2002). A diverse range of infections and parasitic diseases can be transmitted to humans by domestic pets. Among them Strongyloides stercoralis, an intestinal nematode with worldwide distribution infects dogs, cats and non-human primates as well as humans (Grove, 1996; Ferreira, 2003). Although eggs and filariform larvae are rarely present in the fresh feces of dogs, canine strongyloidiasis could have zoonotic potential since it can be transmitted through filariform larvae that have developed in fecally contaminated soil from rhabditiform larvae, normally seen in dog feces (Robertson and Thompson, 2002). The major S. stercoralis infection routes in dogs are the percutaneous and via penetration of mucosal tissue. Although there are no reports on transplacental transmission of larvae, transmammary transmission can occur if the bitch acquires infection between the first and third day post-birth (Shoop et al., 2002). The pre-patent period ranges from 13 to 19 days and larval shedding in feces is interrupted between 52 and 68 days post-infection (Mansfield and Schad, 1992). Autoinfection can occur in dogs. It evolves from a hyper infection syndrome and can disseminate strongyloidiasis, depending on the immune status of the host (Malone et al., 1980; Schad et al., 1984). The infection can be clinically non-apparent or manifest dermatological, gastrointestinal and/or respiratory symptoms (Grove, 1996; Robertson and Thompson, 2002). The parasitological diagnosis of canine strongyloidiasis has been performed through fecal flotation and Baermann (1917) methods, as well as the direct smear of fresh feces to detect the presence of eggs, rhabditiform or filariform larvae of the parasite (Hendrix et al., 1987). Serological diagnosis has employed tests such as the indirect fluorescent antibody (IFA) test (Grove and Northern, 1982), an intradermal reaction test and radial immunodiffusion (Grove and Northern, 1982; Mansfield and Schad, 1992). In human strongyloidiasis, however, the most frequently used methods of diagnosis are the indirect fluorescent antibody test (IFAT) on slides (Costa-Cruz et al., 1997) and the enzyme-linked immunosorbent assay (ELISA) (Gam et al., 1987; Conway et al., 1993; Sato et al., 1995).

Since the detection of this helminth using parasitological methods in stool samples from dogs is difficult, the determination of specific antibodies in serum samples could contribute to the diagnosis. Thus, the aims of this study were to investigate the presence of S. stercoralis and other enteroparasites in stool samples from dogs using the Baermann (1917) and Lutz (1919) methods, and to detect specific antibodies to the parasite in serum samples from dogs using IFAT on slides and ELISA.

2. Materials and methods 2.1. Dogs, sera and fecal samples A total of 215 dogs, 71 males and 144 females, 167 being purebreds (PB) and 48 crossbreds (CB), aged from 1-month- to 7-year-old, was obtained from breeding kennels in the cities of Uberlaˆndia, Araxa´ and Campo Belo in the State of Minas Gerais, southeastern region of Brazil. The animals were distributed according to age as follows: Group I, 1–2 months old (19 males and 20 females; 8 PB and 31 CB); Group II, >2-month- to 1-year-old (11 males and 20 females, 30 PB and 1 CB); and Group III, >1–7 years old (41 males and 104 females, 129 PB and 16 CB). Three fresh fecal samples were collected on consecutive days, part without the addition of preservative (10% formalin solution) for parasitological examination by the Baermann (1917) method and another part with preservative for the Lutz (1919) method. Briefly, 5 g of fresh feces were maintained for 1 h in a Baermann apparatus containing water at 45 8C. The sediment was subsequently examined in a light microscope (Olympus, Japan) at 100 and 400 magnifications. For the Lutz method, 2 g of preserved feces were triturated in a beaker containing 25 ml of water, filtered through nylon mesh and four-foldgauze, and further diluted with water in a sedimentation cup to provide a sample of 200 ml. This suspension was left for 24 h at room temperature after which a drop of the sediment was examined in a light microscope. Blood samples without anticoagulant were obtained from the dogs’ radial veins and, after centrifugation, serum samples were stored at 20 8C for later

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examination. Positive control serum was obtained from a naturally infected, but not artificially immunosupressed, dog with S. stercoralis, shedding larvae in feces and infected with no other parasite. Negative control sera were obtained from healthy dogs after collection of three stool samples, consecutively negative by the Baermann (1917) and Lutz (1919) methods. All procedures were conducted according to the guidelines for animal experimentation (CIOMS, 1985) and the dogs’ owners were informed of the results.

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et al., 2004). Larvae were resuspended in PBS and subjected to a tissue homogenizer (Omnith International, US) using five cycles of 1 min each and subsequently eight ultra-sound cycles for 20 s at 40 kHz in an ice bath. After an overnight incubation at 4 8C under continuous agitation, the suspension was centrifuged at 12,400  g for 30 min at 4 8C and the supernatant (saline extract) was analyzed for protein content by the Lowry et al. (1951) method and stored at 20 8C. 2.3. Serological assays

2.2. Parasites and antigens S. stercoralis larvae were obtained from the feces of naturally infected humans and maintained in a male crossbred dog, aged 60 days, housed individually and receiving water and food ad libitum. The animal had been previously treated per os with the anthelmintics pyrantel (5 mg/kg body weight) and praziquantel (5 mg/kg body weight), and vaccinated according to the schedule routinely used in veterinary clinics. The animal was inoculated subcutaneously with 6000 L3 and received prednisone at 2 mg/kg/day, per os (Grove and Northern, 1982; Grove et al., 1983). Fifteen days after infection the feces were collected and mixed (v/ v) with an equal part of finely ground wood charcoal, moistened with water, spread in an uniform layer on Petri dishes and incubated at 25 8C and 70% humidity for 7 days (Malone et al., 1980). Larvae were harvested by the Baermann (1917) method, washed three times in 0.01 M phosphate-buffered saline (PBS) at 350  g for 5 min and the final sediment was stored at 20 8C. Particulate antigen was obtained from lyophilized 100,000 L3 of S. stercoralis (Costa-Cruz et al., 1997). Larvae were triturated in ethanol at 18 8C and centrifuged at 1110  g for 5 min at 4 8C. The pellet was washed twice with anhydride ether, resuspended in PBS containing 3% formalin and incubated for 24 h at 37 8C. After centrifugation, the material was washed in saline solution and resuspended in distilled water containing 0.5% goat normal serum. The particulate antigen was put on slides (26 mm  76 mm), incubated overnight at room temperature and stored at 20 8C. Saline soluble antigen was obtained from 150,000 L3 of S. stercoralis as previously described (Rodrigues

IFAT on the slides with particulate antigen and ELISA using saline antigen were employed to detect IgG antibodies to S. stercoralis in the 215 serum samples from dogs. Preliminary experiments were carried out in order to determine the optimal conditions for IFAT and ELISA through block titration of the reagents (antigens, control sera, and conjugates). IFAT was performed according to Machado et al. (2001). Slides containing the particulate antigen were incubated with dog sera at an optimal dilution of 1:40 for 30 min at 37 8C and then washed three times in PBS for 5 min each time. Next, the conjugate fluorescein isothiocyanate-labeled rabbit anti-dog IgG (Biomanguinhos, Brazil) at ideal dilution of 1:30 in PBS containing Evans blue at 3% was incubated for 30 min at 37 8C. After washing as explained above, slides were mounted with buffered glycerol (pH 8.5) and examined in an immunofluorescence microscope (Olympus BH2, Tokyo, Japan) at 400 magnifications. Positive sera (titer  40) were retested in serial two-fold dilutions up to the end point titer. ELISA was performed as previously described (Silva et al., 2003). Polystyrene microplates were coated and stored overnight at 4 8C with 5 mg/ml of S. stercoralis saline extract in 0.06 M carbonate–bicarbonate buffer (pH 9.6). Plates were washed three times with PBS containing 0.05% Tween 20 (PBS-T) and active sites were blocked with PBS-T plus 5% skim milk (PBS-TM) for 1 h at 37 8C. After washing, plates were incubated with serum samples diluted 1:100 for 45 min at 37 8C and subsequently with the secondary antibody consisting of peroxidase-labeled rabbit antidog IgG (prepared according to Wilson and Nakane,

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1978) at ideal dilution of 1:200 and left for 45 min at 37 8C. The reaction was revealed by adding the enzyme substrate (0.03% H2O2 and o-phenylenediamine [OPD] in 0.1 M citrate–phosphate buffer, pH 5.0) and incubating for 15 min at room temperature. The reaction was stopped by adding 2N H2SO4 and the optical density (OD) was determined at 490 nm in a plate reader (Metrolab-980, Argentina). Results were arbitrarily expressed as ELISA index (EI), previously reported in research on human strongyloidiasis (Rodrigues et al., 2004), according to the following formula: EI = OD sample/cut off, where the cut off was established as the mean OD of three negative control sera plus three standard deviations. Sera with EI > 1.0 were considered positive. 2.4. Statistical analysis Statistical analysis was performed using GraphPad Prism software, version 3.0 (Graph Pad Software, Inc.). Levels of IgG antibodies to S. stercoralis found in different groups of dogs were compared by the Student’s t-test and the positivity found for each parasite, as well seropositivity rates, were analyzed using the differences between two proportions, by Z statistics. Values of P < 0.05 were considered statistically significant.

3. Results Coproparasitological examinations were performed on a total of 1290 analyses (three fecal

samples for each of the 215 dogs) and evaluated by two observers. Table 1 shows results obtained from the parasitological examinations of the dogs according to age and breed. A significantly higher frequency of hookworm (Group I, Group II, Group III), Toxocara canis (Group I) and Cystoisospora spp. (Group III) was found in crossbred dogs as compared to purebred dogs (P < 0.05). Parasitological results demonstrated that 63/215 (29.3%) of the dogs showed some kind of intestinal parasite, with positivity rates of 82% (32/39) in Group I, 12.9% (4/31) in Group II, and 18.6% (27/ 145) in Group III. Two parasites were found in 24/ 215 (11.2%) of the total sample; including 19/39 (48.7%) of the dogs in Group I, none (0/31) in Group II, and 5/145 (3.4%) in Group III. S. stercoralis was detected in 2/215 (0.9%) of the dogs including one from Araxa´ (Group I) and one from Uberlaˆndia (Group III). Serological assays demonstrated that 45/215 (20.9%) of the dogs were seropositive to S. stercoralis and distributed as follows: no animals (0/39) in Group I, 7/31 (22.6%) in Group II, and 38/145 (26.2%) in Group III. IgG antibodies to S. stercoralis were detected using IFAT (Fig. 1) in 35 (16.3%) of the 215 dogs. None of the animals of Group I showed any reactivity, 4/31 (12.9%) of Group II were seropositive with titers ranging from 80 to 320, and 31/145 (21.4%) of the dogs in Group III showed reactivity with titers ranging from 40 to 160. From 35 seropositive samples, 25 (71.4%) demonstrated a titer of 80. IFAT seropositivity was significantly higher in Group III as compared to Groups I and II (P < 0.05).

Table 1 Parasitological diagnosis in 215 fecal samples from dogs, distributed according to age and breed in the following groups: Group I (1–2 months), Group II (>2-month to 1-year) and Group III (>1–7 years) Parasites

n+ (%) Group I

Hookworm Toxocara canis Giardia spp. Cystoisospora spp. S. stercoralis Trichuris vulpis

Group II

PB (n = 8)

CB (n = 31)

0 0 1 (12.5) 0 1 (12.5) 0

19 21 2 7 0 0

(61.3)* (67.7)* (6.5) (22.6)

Group III

Total (n = 215)

PB (n = 30)

CB (n = 1)

PB (n = 129)

CB (n = 16)

0 0 2 (6.7) 0 0 1 (3.3)

1 (100)* 0 0 0 0 0

4 4 10 0 1 0

7 (43.8)* 0 1 (6.3) 5 (31.3)* 0 0

PB: purebred; CB: crossbred; n+ (%): number and percentage of positive dogs. * P < 0.05 between PB and CB in each group.

(3.1) (3.1) (7.8) (0.8)

31 25 16 12 2 1

(14.4) (11.6) (7.4) (5.6) (0.9) (0.5)

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Fig. 1. Seropositivity determined by IFAT and ELISA for the detection of IgG antibodies to S. stercoralis in 215 serum samples from domesticated dogs, distributed according to age into the following groups: Group I (1–2 months), Group II (>2-month- to 1-year) and Group III (>1–7 years). Statistical significance was determined by the analysis of the difference between two proportions. *P < 0.05 (GI vs. GII); **P < 0.05 (GI vs. GIII).

Fig. 2. Levels of IgG antibodies to S. stercoralis, expressed by the ELISA index (EI), determined in 215 serum samples from domesticated dogs, distributed according to age in the following groups: Group I (1–2 months), Group II (>2-month- to 1-year) and Group III (>1–7 years). The horizontal line indicates the cut off value (IE = 1.0). Statistical significance was determined by the Student’s t-test. *P < 0.05.

When ELISA was used for the detection of IgG antibodies to S. stercoralis (Fig. 1), 23 (10.7%) of the dogs were found to be seropositive. While no animals of Group I showed any reactivity, 7/31 (22.6%) of Group II and 16/145 (11%) of Group III were seropositive. ELISA seropositivity was significantly higher in Group II than in Groups I and III (P < 0.05). Levels of IgG antibodies to S. stercoralis were significantly higher among the dogs of Group III than those of Groups I and II (P < 0.05) (Fig. 2). Table 2 shows concordant and discordant results obtained from IFAT and ELISA. In Group I, all results were negative, concordant for the two tests. In Group

II, 90.3% of the results were concordant (positive and negative) with only 9.7% of discordant results (IFAT /ELISA+). In Group III, concordant results were found in the majority of cases (80%), with 15.2% discordant results due to IFAT+/ELISA and only 4.8% to IFAT /ELISA+. Overall, a concordance of 85.1% (183/215 cases) was verified between IFAT and ELISA, with 22/215 (10.2%) discordant results attributed to IFAT+/ELISA and 10/215 (4.7%) to IFAT /ELISA+. One dog (male, 60 days of age, Shitzu, from Araxa´, Group I) that was shedding S. stercoralis larvae did not show positive serology. A second, copropositive dog

Table 2 Concordant and discordant results obtained from IFAT and ELISA for the detection of IgG antibodies to S. stercoralis in 215 serum samples from domesticated dogs, distributed according to age in the following groups: Group I (1–2 months), Group II (>2-month to 1-year) and Group III (>1–7 years) Tests

IFAT+/ELISA+ IFAT /ELISA IFAT+/ELISA IFAT /ELISA+

n+ (%) Group I (n = 39)

Group II (n = 31)

Group III (n = 145)

Total (n = 215)

– 39 (100)b,c – –

4 (12.9) 24 (77.4)b – 3 (9.7)

9 107 22 7

13 170 22 10

n+ (%) = number and percentage of positive sera. a P < 0.05 (S concordant vs. S discordant). b P < 0.05 (Group I vs. Group II). c P < 0.05 (Group II vs. Group III).

(6.2) (73.8)c (15.2) (4.8)

(6.0)a (79.1)a (10.2)a (4.7)a

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Table 3 Comparison between parasitological results and S. stercoralis serology (IFAT and ELISA) in dogs with positive coproparasitological diagnosis Parasites

n (%) IFAT+

Hookworm (n = 31) Toxocara canis (n = 25) Giardia spp. (n = 16) Cystoisospora spp. (n = 12) S. stercoralis (n = 2) Trichuris vulpis (n = 1) *

1 0 4 1 1 0

(3.2) (25) (8.3) (50)

IFAT 30 25 12 11 1 1

*

(96.8) (100)* (75) (91.7)* (50) (100)*

ELISA+

ELISA

0 0 1 (6.3) 0 1 (50) 0

31 25 15 12 1 1

(100)* (100)* (93.7)* (100)* (50) (100)*

P < 0.05.

(male, 2 years old, Pit Bull from Uberlaˆndia, Group III) was seropositive by both IFAT and ELISA, with an IFAT titer of 40 and ELISA index of 1.66. Fecal examination, however, showed that both dogs were infected only with S. stercoralis. A comparison between enteroparasite positivity and S. stercoralis serology is demonstrated in Table 3. A significantly higher percentage of sera from dogs with hookworm, T. canis; Cystoisospora spp. and Trichuris vulpis showed negative results in IFAT and ELISA for S. stercoralis as compared with positive results (P < 0.05), suggesting that hookworms seem not to interfere with the serology for S. stercoralis. No cross-reactivity was found between S. stercoralis seropositivity and hookworm or Ascaridae infections, however, one dog (female, 3 years old, crossbred) with hookworm and Cystoisospora spp. infections was seropositive in the IFAT with a titer of 160. Four dogs were copropositive to Giardia spp. and seropositive to S. stercoralis by IFAT with titers of 80 (25%), of these, one (male, 2 years old, Shitzu) was also seropositive by ELISA (EI = 1.16). The three remaining dogs were adult females (2, 3 and 4 years, respectively) and purebred (Maltes, Sharpei and Shitzu, respectively). Of the two dogs copropositive to S. stercoralis, only one was seropositive (both IFAT and ELISA positive).

4. Discussion Parasitological diagnosis of canine strongyloidiasis is based on the detection of parasite larvae in stool samples. However, parasitological methods have low sensitivity due to low parasite loads or fluctuations in larval excretion, particularly in chronic infections. Thus, necropsy survey results estimate the true

prevalence of S. stercoralis better than coproparasitological survey results, because necropsies demonstrate infections that cannot be easily diagnosed by fecal exams (Carneiro et al., 1973; Costa et al., 1990; Minnaar et al., 2002). Canine strongyloidiasis represents a problem in breeding kennels, particularly during periods of high temperature and humidity, affecting more severely the puppies (Hendrix et al., 1987). In India, 2–5% of dogs are infected with S. stercoralis (Traub et al., 2002). In the first of two coproparasitological surveys performed in Japan, S. stercoralis was found in 1.93% of 1505 dogs 1–6 months of age coming from breeding kennels and pet shops (Itoh et al., 2003). In the second survey, from 772 canine stool samples, 1.6% of animals were infected with S. stercoralis (Asano et al., 2004). In the present study, coproparasitological results concerning the parasite species detected were in agreement with other reports from Brazil (Oliveira et al., 1990; Oliveira-Sequeira et al., 2002; Blazius et al., 2005) except for the identification of S. stercoralis larvae in dogs from Araxa´ and Uberlaˆndia. The concomitant use of a specific method for detection of helminth larvae in feces (Baermann, 1917) associated with a concentration method of fecal material by spontaneous sedimentation (Lutz, 1919) enhanced the probability of a positive result. One of the cases of S. stercoralis was diagnosed only by the second method. Hookworm (14.4%), T. canis (11.6%), Giardia spp. (7.4%) and Cystoisospora spp. (5.6%) were the most common parasitic infections observed in the present research. In addition, crossbred dogs were observed to be more likely to have hookworms, T. canis and Cystoisospora spp. than purebred dogs.

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Coproparasitological results of the present study were similar to the findings of Oliveira et al. (1990), demonstrating that young dogs (<1-year-old) had higher frequencies of enteroparasites. Dogs are susceptible to hookworm and Ascaridae infections throughout their life, but as puppies the frequency of these parasites is higher because their immune systems are not completely mature (Oliveira-Sequeira et al., 2002). In addition, the larvae of these parasites remain in a state of latency in the muscular layer of bitches, and during pregnancy larvae are reactivated and capable of infecting the fetus (via transplacental) and puppies (via transmammary) (Burke and Roberson, 1985). S. stercoralis filariform larvae in human feces can infect dogs (Hendrix et al., 1987; Robertson and Thompson, 2002), particularly in areas where sanitary conditions are inadequate (Traub et al., 2002). In contrast, a case report in New York demonstrated the occurrence of human strongyloidiasis from contact with canine feces contaminated with S. stercoralis filariform larvae (Georgi and Sprinkle, 1974). The hypothesis that dogs would present S. stercoralis copropositive results similar to those reported from humans in Uberlaˆndia by Machado and Costa-Cruz (1998) could not be confirmed. However, it is worth noting that dogs in this region are hosts for S. stercoralis as well as other zoonotic parasites such as the hookworm, Giardia and T. canis seen in this study. Serological methods have been found to be helpful for evaluation of the host immune response in asymptomatic cases and valuable for clinical diagnosis, in addition to their frequent employment in seroepidemiological surveys (Costa-Cruz, 2000). The present study was innovative in the employment of IFAT on slides (homologous particulate antigen) and ELISA (homologous saline extract) for the detection of antibodies to S. stercoralis in serum samples from dogs. Serological assays demonstrated that 20.9% of the dogs were seropositive for S. stercoralis, particularly according to IFAT, with the majority of seropositive samples showing titers of 80. Among the dogs of Group I, none was seroreactive, including the animal that was copropositive for S. stercoralis. A probable explanation for this finding is that this dog had been recently infected and had no detectable seroconversion. Levels of IgG to S. stercoralis are usually

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detected in the serum from dogs only after 2 weeks of infection. However, a positive serology does not always indicate that the dog was actually infected with S. stercoralis since parasite-specific IgG titers remain elevated for a period of time after infection and may reappear if a dog is reinfected with larvae, even if no adult worms develop, thus reflecting a spontaneous self-cure and resistance to challenge infection (Grove and Northern, 1982). In this context, many of the dogs examined in the present study may have been serologically positive because of an exposure to larvae that did not result in adult worms developing, in according with the findings of Mansfield et al. (1996) who showed no evidence that the populations of parenteral larvae re-established patent, adult worm infections in mature, experimentally infected female dogs. Therefore, a seropositive dog could be exposed to but not necessarily infected with S. stercoralis. A general concordance of 85.1% between IFAT and ELISA for S. stercoralis-specific antibodies, in the present study, indicates that these complementary serological tests may be useful for the detection of canine strongyloidiasis. Among the discordant results (14.9%), the IFAT showed higher positivity than ELISA, suggesting that infective larvae surface antigens, rather than soluble antigens, may be recognized with more facility by the host immune system. A comparison between parasitological and serological results in copropositive dogs demonstrated no cross-reactivity between S. stercoralis seropositivity and hookworm or Ascaridae, except for one dog that harbored two parasites (hookworm and Cystoisospora spp.) and that was seropositive only in IFAT. The detection of specific serum antibodies to S. stercoralis in dogs copropositive to Giardia spp. but copronegative to S. stercoralis suggests the existence of either a chronic occult infection with larval excretion too low to be detected or exposure with self-cure, leaving an antibody level high enough to still be detected. The present study demonstrated, for the first time, natural S. stercoralis infection in dogs that had been diagnosed by coproparasitological (Baermann, 1917; Lutz, 1919) and serological (IFAT and ELISA) methods. Results suggest that the detection of specific antibodies to S. stercoralis by IFAT and ELISA may contribute to the diagnosis of canine strongyloidiasis, even though the detection does not differentiate between past and present infections. Thus the use of

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complementary serological tests, combined with parasitological methods, may be useful in the determination of whether dogs have been exposed or infected.

Acknowledgements We would like to thank Maria das Grac¸as Marc¸al, Rosaˆngela Maria Rodrigues, Camila Siqueira Silva and Mariana Nascimento Manhani for their help in the laboratorial assays. We are also grateful to the Fundac¸a˜o Ezequiel Dias for providing the conjugate for IFAT, the Associac¸a˜o de Protec¸a˜o Animal de Uberlaˆndia and all the dog owners for permitting the participation of their animals in the study. This work was supported by grants from the Brazilian Research Council (CAPES).

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