Toxoplasma gondii: Evidence for the transmission by semen in dogs

Experimental Parasitology 123 (2009) 190–194

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Toxoplasma gondii: Evidence for the transmission by semen in dogs Tiago Pereira Arantes, Welber Daniel Zanetti Lopes *, Roberta Machado Ferreira, Juliana S. Pinto Pieroni, Vanessa M.R. Pinto, Claudio A. Sakamoto, Alvimar José da Costa CPPAR – Animal Health Research Center – Faculdade de Ciências Agrárias e Veterinárias, UNESP, Via de acesso prof. Paulo Donatto Castellani, s/n CEP:14884-900, Jaboticabal, São Paulo, Brazil

a r t i c l e

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Article history: Received 11 January 2009 Received in revised form 14 July 2009 Accepted 15 July 2009 Available online 19 July 2009 Keywords: Dogs PCR Semen Toxoplasma gondii Transmission by semen

a b s t r a c t Ten male dogs were distributed into three experimental groups for infection with Toxoplasma gondii: GI – three dogs inoculated with 2.0  105 P strais oocysts, GII – three dogs infected with 1.0  106 RH strain tachyzoites, and GIII – four controls dogs. Several clinical parameters were evaluated. IFAT was performed to detect anti-T. gondii antibodies. Presence of the parasite in semen was evaluated by PCR and bioassay techniques. Tissue parasitism was examined using bioassays and immunohistochemistry in testicle and epididymis fragments collected after orchiectomy. In semen samples collected from these two groups, the presence of T. gondii was verified by bioassays and PCR. T. gondii was detected by immunohistochemistry in tissues (testicle and epididymis fragments) of all six experimentally infected dogs. The T. gondii-positive seminal samples were used in the artificial insemination (AI) of four female dogs free of toxoplasmic infection. Seven days after AI, all of the female dogs presented serologic conversion (IFAT). Fetal reabsorption occurred in two of the dogs, while the others sustained full-term gestation. Several T. gondii cysts were detected in the brains of four offspring. These results suggest that T. gondii can be sexually transmitted in domestic dogs. Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction Toxoplasmosis is a geographically widespread zoonosis caused by Toxoplasma gondii (Nicolle and Manceaux, 1909), an obligatory intracellular protozoan parasite that can infect a great variety of cells in most vertebrate hosts. This coccidium usually causes an asymptomatic infection (Yap and Sher, 1999). The T. gondii life cycle includes three main forms: (a) oocysts, which are the final product of sexual reproduction, only occur in the digestive tract of felids (their permanent hosts) and are eliminated in the environment together with the feces of these animals, after which they become infectious following sporulation; (b) rapidly multiplying tachyzoites, which are found in the acute phase of infection; and (c) bradyzoites, which are located in tissue cysts and are found in chronic infection (Dubey and Beatite, 1988). Toxoplasmosis in dogs was described for the first time by Mello (1910) in Italy. Clinical and subclinical toxoplasmosis in dogs has since been reported in many countries (Ehrensperger and Pospischil, 1989). Toxoplasmic infection in the canine species is an extremely important disease, as it can cause serious harm. Ocular lesions were the main abnormalities detected in the study of ca-

* Corresponding author. E-mail addresses: [email protected] (T.P. Arantes), [email protected] (W.D.Z. Lopes). 0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2009.07.003

nine toxoplasmosis carried out by Wend and Townsend (2008). Hecley (1963) verified that experimental toxoplasmic infection in female dogs (regardless of gestational stage) can cause the death of offspring from 4 to 75 days after birth. The correlation of toxoplasmosis with immunosuppressive diseases or with canine distemper has been described by several authors (Dubey et al., 1989; Pimenta et al., 1993). Kavinski (1980) observed chorioretinitis in dogs with an indication of distemper and titers up to 1:1024 against T. gondii were found in serum analyses (Sabin and Feldman). Several works in the literature have reported the high prevalence of T. gondii in dogs. Souza Pinto et al. (2004) concluded that dogs’ access to the street is a significantly important factor in the occurrence of infection. Ragozo et al. (2004) added risk factors such as age (higher prevalence in animals over 12 months old) and the presence of cats in the household. Although the isolation of T. gondii in seminal samples from sheep (Spence et al., 1978), goats (Dubey and Sharma, 1980), swine (Moura et al., 2007) and cattle (Scarpelli et al., 2009) has already been carried out successfully, there are no references in the literature regarding either the viability of transmission of this coccidium via semen in dogs, or even its isolation in semen from this species. Therefore, this study was motivated by the high rate of dogs naturally infected with T. gondii, the promiscuity of these animals and the lack of evidence reported in the literature on the transmission of toxoplasmosis by semen in this animal species.

T.P. Arantes et al. / Experimental Parasitology 123 (2009) 190–194

2. Materials and methods The P (Jamara and Vieira, 1991) and RH strains of T. gondii (Sabin, 1941) were used in this study; they were maintained in the Animal Health Research Center (Centro de Pesquisas em Sanidade Animal, CPPAR) of the Faculty of Agrarian and Veterinary Sciences (Faculdade de Ciências Agrárias e Veterinárias, FCAV) of São Paulo State University (UNESP). The inoculates were obtained through periodic inoculation of brain cysts (P strain) and/or tachyzoites (RH strain) in albino mice. T. gondii oocysts were obtained using a technique similar to that described by Dubey et al. (1972). Ten male mixed-breed dogs, 18–19 months old, were caged in individual boxes and given only commercially canned and dry food and water ad libitum. The kennel was closed and had a solarium area. Dogs presenting good clinical and reproductive conditions and that were serologically negative for T. gondii were selected, identified, randomized and inoculated according to the experimental outline described in Table 1. Serological exams to detect antibodies against other infectious diseases that could provoke reproductive disorders (brucellosis, neosporosis and leptospirosis) were conducted on all of the experimental dogs, before and after inoculation. The presence of antibodies against T. gondii was evaluated using IFAT (Camargo, 1964) on serum obtained from all of the animals 2 days prior to inoculation, on the 7th day post-inoculation (DPI) and weekly thereafter until the 70th DPI. Concurrently, seminal samples from the 10 dogs were obtained by means of ‘‘penian massage” (Núñez-Martinez et al. 2006). Detection of T. gondii in the semen was conducted using a bioassay (Teale et al., 1982) and Polymerase Chain Reaction (PCR) (Fuents et al., 1996). In the bioassay, an aliquot of approximately 0.5 mL of semen (from each dog) was inoculated into five mice, which were observed and examined according to the methodology adopted by Costa et al. (1977). The mouse was considered positive if anti-T. gondii antibodies was detectable at a dilution of >1:64 by IFAT and if it presented brain cysts. DNA from the semen samples and from the positive control (RH strain) was extracted (Sambrook and Russell, 2001) to detect the presence of T. gondii. For this assay, the T. gondii gene fragment B1 (194 bp) was amplified using the primers 50 -GGAACTGCATCCGTTCATGAG-30 (B11) and 50 -TCTTTAAA GCGTTCGTGGTC-30 (B12), according to the method described by Fuents et al. (1996). PCR was conducted with 500 ng of genomic DNA in a reaction medium containing 2 mM MgCl2, 50 mM KCl, 10 mM Tris–HCl, pH 9, 0.01% Triton X-100, 0.2 mM dNTPs, and 10 pmol of initiator and 5.0 units of Taq DNA polymerase. Restriction fragments of the amplified products were analyzed by electrophoresis in 2% agarose gel. The gel was stained for 20 min in a 0.5 lg/mL solution of ethidium bromide in water and observed on a UV transilluminator. After the experimental period of semen collection, all of the male dogs (control and inoculated groups) were orchiectomized; samples of testicle and epididymis tissue were collected for bioas-

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says (Dubey, 1998) and immunohistochemistry (Guesdon et al., 1979) to assess tissue parasitism. Female dogs of reproductive age and serologically negative for toxoplasmosis, neosporosis, brucellosis and leptospirosis were used in the second phase. Due to the impossibility of inducing fertile and synchronized estrus in canine species, several female dogs held at the CPPAR/FCAV/UNESP were observed daily through serial vaginal cytology (Allen, 1995), and four were selected. The selected reproductive females were artificially inseminated (AI) twice with samples of ejaculate containing 1  106 tachyzoites of RH strain T. gondii (added at the moment of artificial insemination). Thirty days after the last AI, ultrasound scans were taken to diagnose gestation. Clinical and serological exams were carried out on the four female dogs as well as on the males. Tissue parasitism was assessed using a bioassay (Dubey, 1998) in the resulting ‘‘products” of reproductive disorders (macerated aborted fetuses, stillborn offspring and offspring that died up to 30 days after birth). The inoculated mice that survived until the 42nd DPI were euthanized to search for T. gondii cysts.

3. Results The experimental infection of the dogs with T. gondii was confirmed by the seroconversion of all the inoculated males (Table 2). Although the dogs carried acute toxoplasmic infection, relevant clinical signs that could be attributed to T. gondii were not observed in any of them; in other words, the six inoculated dogs presented, in general, asymptomatic cases of toxoplasmosis. The titers of antibodies against T. gondii (Table 2) showed high levels of antibodies (IgG) on the 14th and 21st DPI in dogs inoculated with tachyzoites. The increase in titers was less pronounced in the dogs inoculated with oocysts; the titers in these dogs reached maximum serologic levels of 1:1024 between the 21st and the 35th DPI. In this study, T. gondii was isolated using a bioassay on four seminal samples from the dogs inoculated with tachyzoites, which were ejaculated on the 7th, 21st and 35th DPI. The presence of the parasite was detected in the 7th DPI seminal sample of one of the dogs inoculated with oocysts (Table 3). Out of the positive samples in the bioassay (Table 3), only one from dog inoculated with T. gondii oocysts was not confirmed by PCR (Fig. 1). Another important result was obtained by immunohistochemistry (Fig. 2), which detected the presence of T. gondii in the testicle and epididymis samples of all the (male) dogs inoculated either with oocysts or tachyzoites. Having proven that dogs carrying acute toxoplasmic infection could eliminate the coccidium in ejaculation samples, the viability of such semen (containing T. gondii tachyzoites) as a source of toxoplasmic infection was verified using female dogs free from antibodies against this protozoan. All the female dogs artificially inseminated with semen containing T. gondii tachyzoites serocon-

Table 1 Experimental outline of dogs inoculated with Toxoplasma gondii. Dog number

Group

Toxoplasma gondii oocysts

Toxoplasma gondii tachyzoites

Inoculation route

1 15 24 14 31 71 5 21 22 75

I

2  105 2  105 2  105 – – – Placebo Placebo Placebo Placebo

– – – 1  106 1  106 1  106 Placebo Placebo Placebo Placebo

Oral Oral Oral Subcutaneous Subcutaneous Subcutaneous

II

III

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T.P. Arantes et al. / Experimental Parasitology 123 (2009) 190–194

Table 2 Results of serological titers obtained by indirect immunofluorescence (IIF) in dogs inoculated with Toxoplasma gondii (2.5  105 oocysts, 1.0  106 tachyzoites or non-inoculated [control]). Days post-inoculation

Serological titer results Dog number (control)

2 7 14 21 28 35 42 49 56 63 70

Dog number (oocysts)

Dog number (tachyzoites)

5

21

22

75

1

15

24

14

31

71

– – – – – – – – – – –

– – – – – – – – – – –

– – – – – – – – – – –

– – – – – – – – – – –

– – 16 64 16 64 64 64 16 16 64

– –

– –

–

–

16 16 1024 16 16 16 16 16 16

64 256 64 16 16 16 16 16 16

– – 1024 4096 256 64 64 64 16 16 16

16 4096 4096 16 64 64 16 16 16 16

16 256 4096 256 64 64 64 16 16 16

– = negative serology.

Table 3 Presence of Toxoplasma gondii in seminal samples of non-inoculated (control) and T. gondii oocyst-inoculated (2.0  105) or tachyzoite-inoculated (1.0  106) dogs. Dog #

Inoculate

Days post-inoculation 2

7

14

21

28

35

42

49

56

63

70

Total

5 21 22 75 Total

Control

– – – – 0

– – – – 0

– – – – 0

– – – – 0

– – – – 0

– – – – 0

– – – – 0

– – – – 0

– – – – 0

– – – – 0

– – – – 0

0 0 0 0 0

1 15 24 Total

Tachyzoites

– – – 0

Positive* – – 1

– – – 0

Positive* – – 1

– – – 0

Positive* – Positive* 2

– – – 0

– – – 0

– – – 0

– – – 0

– – – 0

3 0 1 4

14 31 71 Total

Oocysts

– – – 0

– Positive – 1

– – – 0

– – – 0

– – – 0

– – – 0

– – – 0

– – – 0

– – – 0

– – – 0

– – – 0

0 1 0 1

Positive = IFAT for T. gondii (P1:64) and presence of brain cysts in inoculated mice. Positive* = presence of brain cysts in inoculated mice and positive in seminal samples by PCR. – = negative serology (mice).

verted on the 7th DPI (Table 4), with antibody titers reaching their peak between the 14th and the 21st DPI, indicating acute infection. This was the first time that a toxoplasmic infection transmitted by

Fig. 1. Electrophoresis in 2% agarose gel of PCR product extract from seminal samples of dogs experimentally infected with Toxoplasma gondii. (1) Molecular weight DNA Ladder (100 bp) marker. (2) Dog 01 (DPI 21). (3) Dog 01 (DPI 35). (4) Dog 01 (DPI 7). (7) Dog 24 (DPI 35). (8) Dog 31 (DPI 7). (10) Positive control. (11) Negative control. (12) Molecular weight DNA Ladder (100 bp) marker.

semen was reported not only in dogs but in any other animal species. Out of the four inseminated female dogs, fetal reabsorption occurred at the beginning of gestation in two of them (number 1 and 2). As for dogs number 3 and 4, the diagnosis of pregnancy was confirmed by ultrasound scan and the gestations came to term. Female dog number 3 had two offspring, one of which died during

Fig. 2. Toxoplasma gondii immunoreactivity in epididymis of canines inoculated with 1  106 T. gondii tachyzoites. Mag. 40.

T.P. Arantes et al. / Experimental Parasitology 123 (2009) 190–194 Table 4 Results of serological titers obtained by indirect immunofluorescence (IIF) in female dogs artificially inseminated with seminal samples positive for Toxoplasma gondii (1.0  106 tachyzoites). Days post-insemination

30 14 2 7 14 21 28 35 42 49 56

Serological titer results for female dogs 1

2

3

4

– – –

– – –

– – –

– – –

64 1024 64 64 64 64 64 64

64 64 1024 256 64 64 64 16

64 1024 64 64 64 64 64 64

64 1024 64 64 64 64 16 16

– = negative serology.

delivery, while the other survived for 18 days. The fourth inseminated female dog (number 4) had only one offspring that survived for 7 days. Pyrexia was the symptom observed in this offspring. The only noteworthy discovery in female dog number 3’s stillborn offspring was the liquefaction of the encephalic mass. The other offspring of dog number 3, which survived for 18 days, presented anatomopathologic alterations related to the positioning of the pelvic members, which were turned inward and therefore useless for locomotion. This offspring was directed to the Section of Orthopedics of the Veterinary Hospital of FCAVJ/UNESP, where it was diagnosed as having a 4th degree dislocation of the palette. Female dog number 4’s offspring, which survived for 7 days, presented asymmetry in the cranium and signs of imperfect epitheliogenesis with subsequent tissue necrosis. In the necropsy, this offspring also presented liquefaction of the encephalic mass and pulmonary lesions. Several cerebral cysts containing T. gondii bradyzoites were isolated (by bioassay) in the four offspring of the artificially inseminated female dogs.

4. Discussion The results of the clinical parameters obtained in this research are similar to those found by Kuhn et al. (1972) and Dubey (1985). In contrast, Dubey and Beatite (1988) described ocular lesions as the main alteration found in dogs carrying toxoplasmic infection. Oppermann (1971) related the following alterations in experimentally infected dogs: hyperthermia, lymphadenopathy and respiratory and digestive disturbances. Precocity of the humoral immune response was also detected by Silva et al. (2002), although these authors used ELISA tests to evaluate humoral immune response in dogs infected with the T. gondii RH strain. Studying the kinetics of the acute immunological response (IgM), these same authors reported that the peak in IgM titers occurred on average between the 7th and 21st DPI. The results found in this study are consistent with the antigenic characteristics of each strain of T. gondii in hosts (Dressen, 1990). The P strain has chronic features, causing, for example, the formation of oocysts in cats (Dubey 1995), whereas infection with the RH strain, which was isolated from humans, is characterized by a more acute response (Scarpelli et al., 2009). This study reports the first description of the isolation of T. gondii from the semen of experimentally infected dogs (bioassay and PCR). In similar reports related to different strains, the results regarding the recovery of this protozoan from canine semen seem to agree with the results found by Spence et al. (1978) in sheep, by

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Dubey and Sharma (1980) in caprines and by Moura et al. (2007) in swine. The PCR results reinforce the results of the bioassay and confirm this technique as an important auxiliary tool in the diagnosis of toxoplasmic infection; the B1 gene of T. gondii was used as the amplification template because it was found to be highly conserved in several isolates and present in at least 35 loci of T. gondii (Fuents et al., 1996). These inferences are reinforced by the results obtained by Hitt and Filice (1992), who emphasized the superiority of the bioassay in relation to PCR for the isolation of T. gondii. Some authors (Aquizerate et al., 1993; Dubey and Thulliez, 1993) do not discard the possibility of the parasitic agent being present in samples diagnosed as negative by PCR; they suggest that part of the genomic DNA could be lost due to the DNA extraction technique that is used, and that 500 ng of genomic DNA (host + parasite) per reaction could contain an insufficient quantity of parasite DNA to visualize the 194 bp fragment on 2% agarose gel. The successful detection of T. gondii in tissue fragments from the reproductive system of experimentally infected males was also achieved by Moura et al. (2007) in swine. The conclusion that fetal reabsorption occurred in two female dogs (numbers 1 and 2) is based on the fact that these dogs showed signs of pregnancy, such as an increase of the mammary glands, in the first 20 days after the artificial insemination; these initial signs were followed by a gradual regression of the mammary glands’ volume soon afterwards, and the dogs subsequently presented negative results for pregnancy in the ultrasound scan performed on the 32nd day after artificial insemination. Pyrexia was the symptom observed by Kuhn et al. (1972) and Bresciani et al. (1999) in offspring and in primo-infected pregnant female canines, respectively. The main anatomopathologic alterations observed in the offspring studied in this research (pulmonary lesions) are in line with the observations of Bresciani et al. (2009). Rhyan and Dubey (1992) mention lungs and livers as the organs usually harmed by this protozoan. Jubb et al. (1993) related that pulmonary lesions represent the most common change found in canine toxoplasmosis. The results of this work show that male dogs infected with T. gondii can eliminate viable tachyzoites from the semen during the acute phase. Moreover, the addition of this protozoan to the semen resulted in transmission to artificially inseminated females and, consequently, in vertical transmission to their offspring. These results suggest the sexual transmission of T. gondii in domestic dogs.

Acknowledgments The authors would like to thank FAPESP (Research Support Foundation of the State of São Paulo) and CPPAR (Centro de Pesquisas em Sanidade Animal) at the Faculdade de Ciências Agrárias e Veterinárias, UNESP-Jaboticabal, SP, Brazil, for their financial and infrastructural support.

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