J. Parasitol., 97(5), 2011, pp. 842–845 F American Society of Parasitologists 2011
ISOLATION OF VIABLE TOXOPLASMA GONDII FROM FERAL GUINEA FOWL (NUMIDA MELEAGRIS) AND DOMESTIC RABBITS (ORYCTOLAGUS CUNICULUS) FROM BRAZIL J. P. Dubey, L. M. F. Passos*, C. Rajendran, L. R. Ferreira, S. M. Gennari, and C. Su` U. S. Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Animal Parasitic Diseases Laboratory, Building 1001, Beltsville, Maryland 20705-2350. e-mail:
[email protected] ABSTRACT: Toxoplasma gondii was isolated from a feral guinea fowl (Numida meleagris) and domestic rabbits (Oryctologus cuniculus) from Brazil for the first time. Serum and brains from 10 guinea fowl and 21 rabbits from Brazil were examined for T. gondii infection. Antibodies to T. gondii were found in 2 of 10 fowl and 2 of 21 rabbits by the modified agglutination test (titer 1:25 or higher). Viable T. gondii (designated TgNmBr1) was isolated from 1 of the 2 seropositive fowl by bioassay in mice but not from the 8 seronegative fowl by bioassay in cat. Viable T. gondii was isolated from both seropositive rabbits (designated TgRabbitBr1, TgRabbitBr2) by bioassay in mice from 1 and by bioassay in cat from the other. The TgRabbitBr1 strain was highly virulent for out-bred mice; mice fed 1 infective oocyst died of acute toxoplasmosis. The remaining 2 isolates were relatively avirulent for mice; lethal dose for mice was 10,000 oocysts. All 3 isolates were grown in cell culture, and tachyzoite-derived DNA were genotyped using 10 PCR-restriction fragment length polymorphism markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). The TgNmBr1 was found to be clonal Type II, a rare finding in Brazil in any host. The rabbit isolates were atypical, similar to isolates from cats from Brazil (TgRabbitBr1 was identical to TgCatBr5, and TgRabbitBr2 was identical to TgCatBr1, a common genotype in Brazil denoted type BrII). This is the first genetic characterization of T. gondii isolates from the rabbits and guinea fowl in Brazil and the first host record for T. gondii in the guinea fowl.
Cats are considered key in the transmission of Toxoplasma gondii to humans and other animals because they are the only hosts that can excrete the environmentally resistant oocysts in their feces (Dubey, 2010). Small mammals and birds are considered important sources of infection for cats. We report isolation of viable T. gondii from feral guinea fowl and domestic rabbits for the first time from Brazil.
bicarbonate, centrifuged again, and mixed with antibiotics. The homogenate was inoculated subcutaneously into 5 Swiss Webster (SW) out-bred albino mice (Dubey, 2010). All inoculated mice were observed daily for illness. Impression smears from the lung of dead mice, or those killed when ill, were examined for T. gondii tachyzoites. Survivors were bled on day 41 post-inoculation (PI) from the retroorbital plexus, and a 1:25 dilution of serum from each mouse was tested for T. gondii antibodies with the MAT. Mice were killed 60 days PI and squash preparations of brains were examined for the presence of tissue cysts as previously described by Dubey (2010). Brains of all 8 seronegative (MAT ,1:5) fowl were pooled and fed to a T. gondii-free cat as described (Dubey, 1995). Brain tissue from 1 seropositive rabbit was fed to another cat. Feces of cats were examined for T. gondii oocysts starting 3 days PI as described previously (Dubey, 2010). Briefly, feces were floated in 33% sucrose solution, oocysts collected from the very top of the float, centrifuged, sediment suspended in 2% sulfuric acid, aerated in a shaker at room temperature for 1 wk, neutralized with 3.3% NAOH, and inoculated orally into 2–5 mice. The inoculated mice were observed for 2 mo and examined for T. gondii infection. Initially, all T. gondii isolates were cryopreserved in liquid nitrogen, in 2002–2003, using homogenates of tissues of infected SW mice. In 2010, these isolates were thawed and revived in 1–4 gamma interferon gene knock-out (KO) mice. The inoculated KO mice died of acute toxoplasmosis and homogenates of their lungs were seeded onto CV1 cells. Tachyzoites from cell cultures were cryopreserved in liquid nitrogen.
MATERIALS AND METHODS Naturally infected animals Samples (serum and brain tissue) were obtained from 10 feral guinea fowl (Numida meleagris) and 21 domestic rabbits (Oryctologus cuniculus) from the Metalurgica region of the State of Minas Gerais, Brazil (latitude 19u55900S, longitude 43u56900, and 770 m above sea level). Samples were transported with ice packs by air from Brazil to the Animal Parasitic Diseases Laboratory (APDL), Beltsville, Maryland. Samples were received in 2 batches (9 rabbits received 18 November 2002 and 10 guinea fowl and 2 rabbits received 5 February 2003). Serological examination Sera from animals were examined for T. gondii antibodies at 1:5 or 1:25 dilution using the modified agglutination test (MAT) as previously described by Dubey and Desmonts (1987).
Pathogenicity of oocysts of the 3 strains of T. gondii isolates in mice
Bioassay in mice and cats for T. gondii
Oocysts were obtained by feeding infected tissues of mice to cats. Oocysts were counted in a disposable hemocytometer and diluted 10-fold from 1021 to 1027 to reach an end point of $ oocyst. Aliquots (0.5 ml) from each dilution were orally inoculated into 5 SW mice (Table I). The last infective dilution was considered to have 1 viable organism for data presentation (in Table I).
Tissues of all guinea fowl and seropositive rabbits were bioassayed for T. gondii. Tissues were digested in pepsin and bioassayed in mice as previously described by Dubey (2010). Briefly, tissues were homogenized in saline (0.85% NaCl), mixed with acidic pepsin, and incubated in a shaker water bath for 60 min at 37 C. The homogenate was filtered through 2 layers of gauze, centrifuged, sediment neutralized with sodium
Genetic characterization for T. gondii Toxoplasma gondii DNA was extracted from cell–culture-derived tachyzoites and strain typing was performed using the 10 PCR-restriction fragment length polymorphism (RFLP) markers SAG1, SAG2, SAG3, BTUB, GRA6 c22-8, c29-2, L358, PK1, and Apico (Su et al., 2006; Su and Dubey, 2009; Su et al., 2010).
Received 16 December 2010; revised 21 March 2011; accepted 23 March 2011. * Escola de Veterina´ria, Universidade Federal de Minas Gerais, INCT – Informac¸a˜o Gene´tico-Sanita´ria da Pecua´ria Brasileira, Av. Antonio Carlos, 6627, 30123-970, Belo Horizonte, MG–Brazil. { Faculdade de Medicina Veterina´ria e Zootecnia, Universidade de Sa˜o Paulo (USP), Av. Prof. Dr. Orlando Marques de Paiva 87, Sa˜oPaulo, SP 05508-270, Brazil. { Department of Microbiology, The University of Tennessee, Knoxville, Tennessee 37996-0845. DOI: 10.1645/GE-2728.1
RESULTS Antibodies to T. gondii were found in 2 guinea fowls in titers of 1:10 in 1 and 1:20 in the other. Toxoplasma gondii was isolated 842
DUBEY ET AL.—T. GONDII IN GUINEA FOWL AND RABBITS
TABLE I. Infectivity and pathogenicity of oocysts from 3 isolates of Toxoplasma gondii from guinea fowl and rabbits from Brazil. Toxoplasma gondii strain Dose
TgNmBr1
TgRabbitBr1
TgRabbitBr2
100,000 10,000 1,000 100 10 1
5 (4,5,6,6,6)* 5 (6,6,7,7,10) 5 (7,10) 5 5 2
5 (5,6,6,6,6) 5 (6,6,6,6,6) 5 (8,8,8,8,8) 5 (9,9,9,10,10) 2 (11,12) 1 (12)
5 (4,5,5,5,5) 5 (7,11) 5 5 4 2
* No. of mice infected with T. gondii of 5 mice inoculated orally with oocyst aliquots from each dilution. The number in parentheses is day of death of each mouse. All TgRabbitBr1 infected mice died of acute toxoplasmosis.
from brain tissue of fowl with a MAT titer of 1:10; this isolate is designated here as TgNmBr1. The mice inoculated with brain tissue of fowl remained asymptomatic and antibodies to T. gondii were found in 1 of 5 mice when bled 60 days PI. Tissue cysts were found in the seropositive mice when killed 77 days PI; brain homogenate of this mouse was cryopreserved in liquid nitrogen and sub-inoculated into 2 SW mice that developed patent T. gondii infection. The cat that was fed brains of 8 seronegative fowl did not shed oocysts. In 2010, TgNmBr1 was revived in 4 KO mice. Two of the 4 KO mice inoculated with the thawed brain homogenate became ill and were killed 20 days PI; their lung homogenates were seeded onto CV1 cells for culture, and tissues were fed to a cat to obtain oocysts. The cat shed 800 million oocysts. Antibodies to T. gondii were found in 2 rabbits (MAT 1:100 in 1 and 1:40 in the other). Viable T. gondii was isolated from both rabbits. The cat fed brain of the rabbit with a MAT titer of 1:100 shed oocysts. Four of the 5 mice fed oocysts died of acute toxoplasmosis and tachyzoites were found in their mesenteric lymph nodes 7 days PI; tissue cysts were found in the fifth mouse when killed 104 days later. The homogenate of infected mesenteric lymph nodes was cryopreserved and inoculated into 2 SW mice; both inoculated mice became infected with T. gondii. This isolate is designated TgRabbitBr1. Four of the 5 mice inoculated with brain homogenate of rabbit with a MAT titer of 1:40 died of toxoplasmosis 14, 15, or 32 days PI; the other 2 mice remained seronegative and tissue cysts were not found in their brains 71 days PI. Lung homogenate from the mouse that died day 14 PI was cryopreserved in liquid nitrogen and inoculated into 2 SW mice; these mice died 14 days PI and their tissues were fed to a cat that shed oocysts. This isolate is designated TgRabbitBr2. Both the rabbit isolates were revived in 2010 in KO mice. The inoculated KO mice became ill and were killed 14 days PI. Tachyzoites were found in the lungs of these mice and these were seeded onto CV 1 cells and were also inoculated into SW mice. Toxoplasma gondii tachyzoites grew in inoculated cell cultures. Mice inoculated with rabbit strains also acquired T. gondii. Oocysts of both isolates were obtained by feeding infected mouse tissues to cats. Virulence of the 3 T. gondii isolates in mice varied (Table I). TheTgRabbitBr1 was the most virulent. All mice fed TgRabbitBr1oocysts died of acute toxoplasmosis between 4 and 12 days PI, depending on the dose; neither antibodies nor tissue cysts were
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found in mice that survived for 2 mo. The remaining 2 isolates were mildly virulent for mice (Table I). The T. gondii strain from the fowl was clonal Type I at all loci (Table II). The rabbit strains were atypical and matched 100% with TgCatBr1 and TgCatBr5, originally isolated from feral cats from Parana, Brazil (Dubey et al., 2004; Pena et al., 2008). DISCUSSION The isolation of T. gondii from rabbits is of historical and biologic significance. A T. gondii-like parasite was found in a rabbit from Sa˜o Paulo, Brazil by Splendore (1908) and in a rodent, Ctenodactylus gundi, from Tunisia by Nicolle and Manceaux (1908, 1909). This parasite was originally considered Leishmania sp. and subsequently named Toxoplasma gondii in 1909 by Nicolle and Manceaux. The rabbits were captive in the laboratory of Splendore and no clinical signs were observed before death (Splendore, 1908). Since then, very little is known of T. gondii infection in rabbits from Brazil. Ferreira et al. (2001) described mouse pathogenicity of a strain of T. gondii (N strain) from a rabbit reported to be from an outbreak of toxoplasmosis in rabbits in Sa˜o Paulo, Brazil 1952; all mice inoculated by Ferreira et al. (2001) with as few a 1–10 tachyzoites died of toxoplasmosis. Genetic characterization using 8 RFLP loci revealed that the N strain had a combination of Type I, II, and III loci (Ferreira et al., 2006). Although the RFLP markers used to type the N strain were not all the same as those in our present study, among the 4 shared markers (SAG1, 59+39SAG2, SAG3, and GRA6), the N strain has the same alleles with TgRabbit2 at loci SAG1, 59+39SAG2, and SAG3 but differs at locus GRA6 with a Type II allele. The N strain differs from TgRabbit1 at loci 59+39SAG2 and GRA6. Therefore, it is clear that these T. gondii strains from rabbits are not the same. The origin and source of N strain is not clear. A recent personal inquiry (J. P. Dubey, 28 October 2010 to R. W. A. Vitor) revealed that the N strain was brought to Belo Horizonte from Sa˜o Paulo in 1971 by Maria Paumgarten Deane (Deane and Nussenzweig, 1959). For the most part, the strain was maintained by twice weekly inoculation, intraperitoneal, in mice. Whether the strain changed biologically and genetically is unknown; prolonged mouse passage can alter T. gondii strain pathogenicity and, perhaps, geneticity (Dubey and Beattie, 1988; Dubey, 2010). Sogorb et al. (1972) reported Sabin Feldman dye test antibodies in 12 of 20 rabbits from Sa˜o Paulo and isolated T. gondii from 2 of 3 rabbits with titers of 1:4,096; no other details were given concerning the mouse pathogenicity of these isolates. In the present study, we fully characterized 2 rabbit isolates using 10 RFLP markers. The rabbits were from Minas Gerais, approximately 600 km from Sa˜o Paulo. The results indicate that these isolates from rabbits are widely spread in Brazil. Indeed, TgRabbitBr2 belongs to the common type BrII in Brazil, which was identified in cats and dogs in several states of this country (Pena et al., 2008). The isolation of T. gondii from rabbits is of public health importance because the demand for rabbit meat for human consumption is increasing (Dubey, 2010). There are no regulations for the slaughter of these animals commercially. In Brazil, rabbits are raised on small farms or by individual farmers in an environment frequented by cats; the meat is then sold in open markets. There is a need for a larger study on the prevalence of T. gondii infection in rabbits.
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THE JOURNAL OF PARASITOLOGY, VOL. 97, NO. 5, OCTOBER 2011
TABLE II. Genetic characterization of Toxoplasma gondii from guinea fowl and rabbits from Brazil. Genetic markers
Toxoplasma gondii isolate
SAG1
59+39 SAG2
alt.SAG2
SAG3
BTUB
GRA6
c22-8
c29-2
L358
PK1
Apico
RH88 PTG CTG TgCgCa1 MAS TgCatBr5 TgNmBr1* TgRabbitBr1{ TgRabbitBr2{
I II or III II or III I u-1 I II or III I I
I II III II I III II III I
I II III II II III II III II
I II III III III III II III III
I II III II III III II III III
I II III II III III II III III
I II III II u-1 I II I I
I II III u-1 I I II I III
I II III I I I II I I
I II III u-2 III u-1 II u-1 II
I II III I I I II I III
* Clonal Type II. { Identical to TbCatBr5 (Pena et al., 2008). { Identical to TgCatBr1 (Pena et al., 2008).
The isolation of T. gondii from 1 guinea fowl is also interesting because these birds are common in parks and zoos in Brazil, where they are an easy prey for cats. To our knowledge, there is no record of T. gondii prevalence in the guinea fowl. Humans become infected post-natally by ingesting tissue cysts from undercooked meat, consuming food or drink contaminated with oocysts, or by accidentally ingesting oocysts. However, only a small percentage of exposed adult humans or other animals develop clinical signs of disease. It is unknown whether the severity of toxoplasmosis in immunocompetent hosts is due to the parasite strain, host variability, or to other factors. Recently, attention has been focused on the genetic variability among T. gondii isolates from apparently healthy and sick hosts (Howe et al., 1997; Grigg et al., 2001). Severe cases of toxoplasmosis have been reported in immunocompetent patients considered to be due to infection with atypical T. gondii genotypes (Ajzenberg et al., 2004; Demar et al., 2007; Elbez-Rubinstein et al., 2009; Grigg and Sundar, 2009). Most T. gondii isolates from human and animal sources in North America and Europe have been grouped into 1 of 3 clonal lineages including Types I, II, and III (Darde´ et al., 1992; Howe and Sibley, 1995; Ajzenberg, Ban˜ uls et al., 2002; Ajzenberg, Cogne´ et al., 2002; Ajzenberg et al., 2004; Khan et al., 2007). When tachyzoites are used to infect out-bred mice, Type I strains are uniformly lethal. In contrast, Type II and III strains are significantly less virulent (Howe et al., 1996). In the present study, only 1 of the rabbit strains was highly mouse virulent, although both had atypical genotypes with Type I and Type III alleles at several markers (Table I). Toxoplasma gondii was considered to be clonal, with low genetic diversity (Howe and Sibley, 1995). However, we recently found that the isolates of T. gondii from Brazil are biologically and genetically different from those in North America and Europe (Dubey et al., 2002; Lehmann et al., 2006; Dubey and Su, 2009). Toxoplasma gondii isolates from asymptomatic chickens from mainland Brazil were, in general, more pathogenic to mice than were isolates from Europe or North America. Additionally, most isolates from chickens in Brazil were different from the major clonal lineages in North America and Europe, and the Type II strain was absent or rare (Dubey and Su, 2009). However, recently we reported that the Type II strain was frequent in chickens from the Fernando de Noronha, separated from
mainland Brazil by 354 km (Dubey et al., 2010). Because Fernando de Noronha was occupied by Europeans, it is likely that the Type II strain in Fernando de Noronha spread from Europe. More recently, da Silva et al. (2011) reported that 2 of 13 T. gondii isolates from sheep in Sa˜o Paulo were Type II at 9 loci but Type I at Apico. Since these sheep were of the Polwarth breed that was originated from Australia, the Type II strains isolated may be imported into Brazil. In the present study, the isolate from the guinea fowl was Type II at all 10 loci. Again, as guinea fowl is native to Africa and was introduced to the New World in recent history, the Type II T. gondii strain may also be introduced to Brazil from other regions of the world. To our knowledge, this is the first report of clonal Type II strain from mainland Brazil in any host. ACKNOWLEDGMENT We would like to thank Dr. R. W. A. Vitor, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil for providing the history of the N strain from rabbit.
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