Abstract: This study was undertaken to evaluate the viability and infectivity of the tachyzoites in different temperatures and times of milk by bioassay methods. Tachyzoites of RH strain were transferred into sterile milk and kept in different temperatures (4, 20 and 37°C) for different time intervals (10 and 30 min) and their viability was evaluated by vital stain (methylene blue) and the infectivity by intra-peritoneal and oral inoculation to different groups of inbred BALB/C mice (2-4 mice in each group obtained from Pasteur Institute, Tehran, Iran). Tachyzoites in saline were intra-peritoneally inoculated to inbred BALB/C mice as positive controls. All animals were kept in Laboratory Animal Center of Shiraz University of Medical Sciences in Shiraz, southern Iran. The touch smear of spleen and liver of dead mice were stained by Giemsa and were parasitologically evaluated. Eighty percent of tachyzoites were stained by methylene blue indicating identical parasite viability. All mice died after intra-preritoneal inoculation of tachyzoites present in sterile milk. Only one out of eight mice which was orally inoculated remained alive. All mice fed by tachyzoites in saline died. Duration of mice vitality was more in oral route than peritoneal inoculation (n1 = 11, Mean1 = 9.55 days and n2 = 17, Mean2 = 6.65 days, p = 0.001). Tachyzoites in drinks and foods undergoing heating up to 37°C for 30 min such as milk can be a possible route of transmission of toxoplasmosis to human. Therefore strict public health measures should be carried out to prevent transmission of disease in high risk areas.
INTRODUCTION
Toxoplasma gondii is an obligate intracellular protozoan that infects humans and a wide range of warm-blooded animals (Smith and Reduck, 2000). The parasite is known to cause congenital disease and abortion both in humans and livestock (Dubey, 2004; Sharif et al., 2007). Maternal toxoplasmosis during early pregnancy of human may leads to death of fetus or cause chorioretinitis, hydrocephaly, microcephaly and jaundice in neonates (Higa et al., 2010; Joynson and Wreghitt, 2001). However, acquired toxoplasmosis has mild flu like symptoms in immunocompetent humans, but the disease is severe in immunocompromised persons, for example 23% of HIV-positive patients will develop toxoplasmic encephalitis (Alvarado-Esquivel et al., 2010; Davarpanah et al., 2007; Oksenhendler et al., 1994).
The prevalence of toxoplasmosis in Fars Province, Southern Iran was reported 20.24% in bovine (Asgari et al., 2010) 26.5% in ovine, (Asgari et al., 2009) 14.02% in caprine, (Asgari et al., 2007) and 36.1% in chicken (Asgari et al., 2006). Human seropositivity in North and South of Iran using indirect fluorescent antibody method was shown to be 55 and 29%, respectively and a seroprevalence of 51.8% was reported in all regions of the Country (Ghorbani et al., 1978; Sedaghat et al., 1978; Assmar et al., 1997). Infections in man occurs because of ingestion of oocysts from the feces of contaminated cats and/or dogs (Clementino et al., 2007; Jittapalapong et al., 2007) and by ingesting raw or under-cooked products containing tissue cysts (Garcia et al., 2006; Gilot-Fromont et al., 2009). Milk may serve as a potential source for human toxoplasmosis (Ertug et al., 2005; Jones et al., 2009; Riemann et al., 1975; Skinner et al., 1990). Additionally, tachyzoites of T. gondii were found in the milk of several species, including sheep, goats, cows, mice and cats (Dubey, 1998; Inpankaew et al., 2010; Powell et al., 2001; Tenter et al., 2000).
Since bovine milk as a dietary food may be contaminated by Toxoplasma gondii tachyzoites, this study was undertaken to evaluate the viability and infectivity of tachyzoites in milk by bioassay methods.
MATERIALS AND METHODS
Inbred BALB/C mice were obtained from Pasteur Institute, Tehran, Iran at 10-14 weeks of age. Animals were kept at temperature of 22°C and 40-50% relative humidity in Laboratory Animal Center of Shiraz University of Medical Sciences in Shiraz, southern Iran. The protocol of all procedures and sacrifice were identical for all animals. During the experiments from June to December 2008, the animals were housed in cage and maintained under controlled environmental conditions (21±2°C, 65-70% RH and a balanced diet with free access to food and water). All experiments and the sacrifice procedure were adhered to the same guidelines under supervision of Animal Care Committee of Iran Veterinary Organization.
Toxoplasma gondii tachyzoites of the RH strain were obtained from Tehran University of Medical Sciences, Iran. Dye Test was based on the fact that live Toxoplasma gondii tachyzoites can actively absorb methylene blue dye from the culture medium and the dead ones remain colorless. Tachyzoites were obtained by mouse inoculation. T. gondii tachyzoites were injected intraperitoneally in Balb/C mice and the animals were killed 48 h after injection. Tachyzoites were collected after repeated flushing of the peritoneal cavity with phosphate-buffered saline (PBS; pH 7.4) and adjusted to a concentration of 25x106 mL-1. Fifty micro litre of the sample (1.25x106 parasites) was transferred into eppendorf tubes, which already contained 150 μL of sterile milk or saline solutions. Three series of these tubes incubated in different temperatures (4, 20 and 37°C) for different times (10 and 30 min). After this incubation, 200 μL of methylene blue solution was added to a serial of the tubes and were incubated for 30 min. The viability of tachyzoites was evaluated by the percent of the stained tachyzoites under an invert microscope at x400 magnification.
The infectivity was compared by intra-peritoneal and oral inoculation of the tachyzoites transferred in milk to different groups of inbred Balb/C mice (2-4 mice in each group and 50 μL of milk containing 3x105 parasites for each mouse). All mice died and their touch smears of spleen and liver were stained by Giemsa and observed under light microscopy (X400) for detection of parasite. Tachyzoites in saline were also intra-peritoneally inoculated to mice as positive controls. Intra-peritoneal and oral inoculation of 2 mL of uncontaminated milk was considered as negative control. The data were analyzed using SPSS software (version 11.5, Chicago, IL, USA) by non-parametric test of Mann-Whitney. A p<0.05 was considered statistically significance.
RESULTS AND DISCUSSION
82.5 to 88% of tachyzoites in all media were stained by methylene blue whereas 93.5% of them in control group absorbed the dye and no significant difference was observed between the two groups indicating to the same rate of viable parasite.
All mice (n = 10) that were intra-preritoneally inoculated by tachyzoites with sterile milk died. Only one out of eight which was orally inoculated with milk was alive. Moreover, all of mice which were fed by tachyzoites in saline died (Table 1). Duration of the mice vitality in oral inoculation with Mean = 9.55 days was more than it in peritoneal inoculation with Mean = 6.64 days (n1 = 11, n2 = 17 and p = 0.001 by using Mann-Whitney test).
In this study, any significant differences between applied temperatures and times were not seen. The animals which were intraperitoneally and orally inoculated by bovine milk were not shown clinical signs.
| Table 1: | Infectivity of Toxoplasma gondii inoculations in different temperatures and times |
| Values in parenthesis are no of groups, + = Alive, - = Died | |
The sources of infection for humans, worldwide, vary greatly with culture, ethnic, geographical location and food habits differences. Food animals such as pigs, sheep and goats have a highest frequency of tissue cysts in comparison with other animals. The many factors such as management and hygienic standards in breeding livestock, density of cats and environmental conditions are effective on the acquisition of T. gondii oocysts (Tenter et al., 2000). The association between infection and unpasteurised milk or milk products was unexpected (Cook et al., 2000). However, the milk of goat may serve as a potential source for human toxoplasmosis (Chiari and Neves, 1984; Ertug et al., 2005; Jones et al., 2009; Sacks et al., 1982; Skinner et al., 1990). T. gondii tachyzoites have been isolated from goats' milk and cows' colostrums (Dubey, 1988; Hiramoto et al., 2001; Jones et al., 2009).
Toxoplasma gondii was detected in the milk of five of six experimentally infected cats by either bioassay or PCR (Powell et al., 2001). Generally, Tachyzoites are not considered an important source of oral transmission of T. gondii because they are rapidly killed outside the host and because they are considered sensitive to proteolytic enzymes (Powell et al., 2001).
Conversely, tachyzoites were recently shown to survive up to 2 h in pepsin solutions and adult cats become infected when high numbers of tachyzoites are given orally (Dubey, 1998).
Pettersen, (1984) suggested that when the milk from lactating mother mice was exposed to HCl, that some of the parasites present were "acid-resistant" cystozoites. Furthermore, infectivity of bradyzoite in bovine milk was maintained even after storage for 20 days at refrigerator temperatures (Hiramoto et al., 2001). Moreover, Walsh et al. (1999) reported tachyzoites of RH strain survived for 3-7 days in goat milk at 4°C while this strain survived for up to 14 days in Hank’s Balanced Salt Solution (HBSS) at this temperature.
Hence there is an obvious risk of acquiring toxoplasmosis by consuming milk from infected donors. Alternatively, infection by tachyzoites might occur by penetration of the oropharyngeal mucosa (Sacks et al., 1982). Fusco et al. (2007) demonstrated that contamination of milk should not be underestimated since it can represent a critical point in food safety. Unhygienic water supplies were other sources of infection which was also reported Ertug et al. (2005).
This study showed that tachyzoites in all media were stained by methylene blue which indicates the parasite viability. Considering the our mentioned results, tachyzoites are not only resistant to milk media and conserve their infectivity for up to 30 min but were infective due to oral transmission. Moreover, the higher duration of the mice vitality in parasite oral inoculation showed this route has less virulent but is however infective.
Tachyzoites in drinks and foods undergoing heating up to 37°C for 30 min such as milk can be a possible route of transmission of toxoplasmosis to human. Therefore, strict public health measures should be carried out to prevent transmission of disease in high risk areas.
ACKNOWLEDGMENT
The authors would like to thank the Office of Vice-Chancellor for Research of Shiraz University of Medical Sciences, Shiraz University for financial support of this project.