Isolation and characterization of Toxoplasma gondii from small ruminants (sheep and goats) in Chennai City, South India

Abstract

The present study aimed for the isolation and genotyping of Toxoplasma gondii from small ruminants (sheep and goats). 14 out of 193 tissue samples (either brain and heart) tested positive by MDAT for anti-T. gondii antibodies, were selected and bioassayed, which resulted 4 samples positive for T. gondii after 40 days of post inoculation. Four samples consisting of 3 numbers of sheep and 1 number of goat tissues out of 14 samples detected by B1 PCR, were genotyped at SAG3 locus by nested polymerase chain reaction–restriction fragment length polymorphism technique (nPCR–RFLP). The results of the present study revealed that the four isolates designated as TgShIn19, TgShIn76, TgShIn77 and TgGtIn27 were circulating in small ruminants, were belonged to genotypes of type II (TgShIn19) and type III (TgShIn76, TgShIn77 and TgGtIn27) which are in concordance with the previously reported genotypes from other animal species and further this presumptive results indicating that the genotype II and III could be the predominant in different animal species including birds and humans in India.

Introduction

Toxoplasma gondii is a single celled, intracellular protozoan parasite which infects all warm blooded animals including human (Dubey 2008). Felids are the definitive hosts, in which the parasites can undergo sexual replication to generate oocysts. A large numbers of unsporulated oocysts are shed in the cat’s feces, however, oocyst shedding occurs for only 1–2 weeks. Within 1–5 days, oocysts in the environment sporulate and become infective. Infection with T. gondii is generally asymptomatic in immunocompetent individuals (Montoya and Liesenfeld 2004) but some individuals suffer from severe acquired forms of chorioretinitis (Burnett et al. 1998), or pulmonary forms of toxoplasmosis (Carme et al. 2002), which can be a life threatening when acquired congenitally (Wong and Remington 1994) or a consequence of reactivation in the immunocompromised patients (Luft and Remington 1992). Further, it can cause abortion and destructive inflammation that targets multiple organs including the brain and eyes (Wong and Remington 1994).

Sheep and goat rearing plays a major role in the economy of the farming community and fetal loss is a serious problem among them (Buxton and Rodger 2008; Dubey and Adams 1990; Mainar et al. 1996). Due to heavy numbers of population of stray cats in India, which may seed the environment with millions of oocysts of T. gondii (Dubey and Beattie 1988). According to 19th livestock census 2012, the total sheep populations are 65.06 million whereas the goat is 135.17 million all over India and most of them are being reared in free range or in semi-intensive system. So the possibility of ingestion of oocysts by these grazing ruminants are expected to be so high. The ingestion of undercooked infected lamb is considered to be one of the important source of infection to humans, which was identified as a high risk factor for T. gondii infection in pregnant women in Europe (Cook et al. 2000). Secondly, mutton and chevon are the very important sources of protein in India, thus acts as a potential mode of transmission to humans (Sacks et al. 1982) and recent findings revealed transmission through goat milk as another important risk factor (Dubey et al. 2011).

Early studies classified T. gondii into type I, II and III based on mouse virulence (Darde et al. 1992). A considerable number of studies from North America and Europe revealed three distinct lineages of type I, II and III in addition to recent type 12 (fourth clonal lineage) (Dubey et al. 2011; Khan et al. 2011). Limited studies from the African continent (Velmurugan et al. 2008; Al-Kappany et al. 2010), the Middle East and the Arabic peninsula, revealed mostly of type II and III (Salant et al. 2009; Dubey et al. 2010). In the Asian continent, various studies specially in China from animals (Yan et al. 2014; Tian et al. 2014; Li et al. 2015) and humans (Wang et al. 2013, 2015) revealed the predominance of Chinese I (ToxoDB#9) genotype with the limited numbers of archetypal lineages I, II and III (Tian et al. 2014; Wang et al. 2013, 2015; Zhou et al. 2009); in India, limited genotyping revealed types II and III (Sreekumar et al. 2003; Biradar et al. 2014), type I (Sudan et al. 2015). On the contrary, isolates from South America are highly diverse (Dubey et al. 2002, 2007; Lehmann et al. 2006; Rajendran et al. 2012; Shwab et al. 2014) and grouped them into different classification (Pena et al. 2008). Therefore, the present study has been focused on the isolation and genotyping of T. gondii circulating in small ruminants (sheep and goats) in and around Chennai metropolitan City.

Materials and methods

Samples and sampling area

14 number titre (136 numbers of sheep and 57 numbers of goats) of samples showed positivity by MDAT at 1:20 titre by MDAT out of 193 samples (either of lung, brain and heart) used for our previous study (Satbige et al. 2016) were utilized for the isolation and genotyping studies in T. gondii free BALB/c mice (Table 1).

Table 1.

The results of the bioassay for the isolation of T. gondii from different tissue samples of small ruminants with seropositivity at 1:100 in MDAT

Sample no.	Sheep or goat tissues	Tissue to be bioassayed	Mouse inoculation after 35 dpia	B1 PCR	Genotypes
S19	Sheep	Heart	No symptoms	Positive	TgShIn19
S76	Sheep	Heart	No symptoms	Positive	TgShIn76
S77	Sheep	Heart and brain	No symptoms	Positive	TgShIn77
G27	Goat	Heart	No symptoms	Positive	TgGtIn27

^aDays of post inoculation

Mouse bioassay studies

The tissue samples were prepared for the bioassay as per the method described (Dubey 1998). The experimental grouping was made into 14 groups with three animals per group that is free from T. gondii infection, Swiss albino mice, approximately 30 days old, weighing 20–25 g. 1 ml of tissue digest was mixed with two broad spectrum antibiotic solutions (50 µg of streptomycin and 100 IU of benzyl penicillin per ml of fluid), were injected subcutaneously for further observation. All mice were not showed any visible symptoms of T. gondii infection but all subjected to MDAT after 40 dpi and the four groups of seropositive mice were examined for the presence of tissue cysts of T. gondii (Dubey et al. 1998). Ethical clearance was obtained from the institutional animal ethical committee (IAEC) to carry out the mouse bioassay experiment. The brain samples were collected in sterile vials and subjected to extraction of total genomic DNA using DNeasy Tissue Kit as per the manufacturer’s instructions (Qiagen, Germany).

B1 PCR and SAG3 genotyping of T. gondii strains

The PCR targeting 194 bp of B1 gene was used for the detection of T. gondii (Angel et al. 1997) from the seropositive mice. Further, it was subjected to genotyping with SAG3 marker along with standard positive control such as GT1 (type I), PTG (type II), CTG (type III), TgCgCa1, MAS, TgCtBr5, TgCtBr64 and TgRsCr1 using polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) according to the method described (Su et al. 2006).

Results

Fourteen positive samples (10 from sheep and 4 from goats, 14/193) at 1:100 titre were subjected to mouse bioassay for the isolation and genotyping studies (Table 1). Acute toxoplasmosis was not observed during this study. All mice were tested positive at 1:100 by MDAT at 40 dpi. There was no evidence of either cysts in the brain or tachyzoites in the lung, when the impression smear taken from all the mouse. The tissues collected from these four groups subjected to B1 PCR (194 bp) showed positivity. These were grouped into four isolates of T. gondii (TgShIn19, TgShIn76, TgShIn77 and TgGtIn27) based on SAG3 genotyping with PCR–RFLP which revealed one isolate of type II (TgShIn19) and three isolates of type III (TgShIn76, TgShIn77 and TgShIn27).

Discussion and conclusions

The sheep and goats so called “small ruminants” are mostly reared in free range rearing or semi intensive system in India. Therefore, the possibility of expected ingestion of oocysts of T. gondii from the environment could be high. A considerable number of seroprevalence studies in India revealed 5–100% from various animal species (Devada et al. 1998; Selvaraj et al. 2007; Vijaya Bharathi et al. 2003, 2011; Hannah et al. 2013) and 8.8–37.3% in immunocompetent humans (Dhumne et al. 2007; Singh et al. 2014) in addition to a major outbreak of T. gondii in humans through water contamination (Manikandan et al. 2006). In the intensively managed sheep had lower antibodies titre than the semi-intensively managed sheep (Savio and Nieto 1995; Abu Samra et al. 2007; Romanelli et al. 2007; Ragozo et al. 2008) indicating that the seroprevalence is directly reflected on the management in the western countries. T. gondii is considered to be one of the important abortifacient in sheep and goats worldwide but little is known about this protozoa induced abortion in Indian livestock (Sharma et al. 2008). Some workers have reported presumptive T. gondii serological evidence of abortion in goats (Sharma and Gautam 1972; Srivastava et al. 1984).

In this study, higher numbers of sheep were infected with T. gondii as compared to goats, which may be associated with the presence of cats in the farm area, and the probability that a cat might shed oocysts on a sheep farm and farm premises (Weiland and Dalchow 1970; Dubey 1994). Excreted toxoplasma oocysts from the infected cats would remain infectious for a long time (Ghorbani et al. 1983). Since the information regarding genotypes circulating in small ruminants is obscure in India, this present study focussed mainly on the prevailing genotypes in sheep and goats. The four strains of T. gondii from 4 animals (3 no. of sheep and 1 no. of goat) out of 14 tissue samples (TgShIn19, TgShIn76, TgShIn77 and TgGtIn27) were mouse bioassayed. The resultant positive samples were genotyped on SAG3 marker revealed one strain belonged to type II (TgShIn19) and three strain belonged to type III (TgShIn76, TgShIn77 and TgGtIn27). Majority of the sheep harboured T. gondii as compared to goat in this limited study. The chosen genotyping marker, SAG3 is the most sensitive marker within 10 common genotyping set, thus it is the easiest to detect when parasite DNA concentration is limited (Khan et al. 2005). Our study is in agreement with the previously reported studies. Some of the studies showed the genotypes of T. gondii circulating among backyard chicken species in India had revealed two type II and 5 type III genotypes based on SAG2 genotyping (Sreekumar et al. 2003), type I based on SAG3 (Sudan et al. 2015) and type III based on GRA6 (Biradar et al. 2014); Further, a recent study in the HIV (human immune deficiency virus) infected human patients, who succumbed to severe cerebral toxoplasmosis revealed recombinant genotypes of type II/III and type I/III (Vijaykumar et al. 2016). Further detailed studies are needed to accurately assess the genotypes circulating among small ruminants.

However, it is concluded that this study exhibited type II and III are the genotypes mainly circulating among small ruminants. Other studies from various other hosts such as chicken and human from India had revealed a similar findings. Therefore, it is presumptively concluded that the type II and III could be the predominant genotypes circulating among different hosts such as animals and birds including human.