Zoonotic Chicken Toxoplasmosis in Some Egyptians Governorates

MATERIALS AND METHODS

Experimental animals: This research study was conducted from 5-2012 to 12-2012. Swiss albino laboratory breed mice about 1 month-old were used for bioassay of the suspected infected chicken tissues and maintenance of isolated T. gondii local strain in the lab. All mice were kept in clean cages, fed on pelleted food and clean water supply with multivitamins.

Chicken tissue samples: Meat and tissue samples were obtained from slaughtered chicken at different regions, in Egypt. Pooled tissue sample, about 25-50 g, from heart, brain and pectoral muscles were prepared as described by Shaapan and Ghazy, 2007, the tissues and organs was cut into small cubes about 5x5x5 cm and stored at 4°C for few hours until used for bioassay in mice.

Isolation of local T. gondii chicken strain: Tissue samples were digested by pepsin according to method of Sharma and Dubey (1981), followed by microscopically examined at low and high powers for the presence of T. gondii tissue cysts containing the bradyzoites. Bioassay of the digested positive tissues in mice according method mentioned by Dubey (2010a), in order to obtain the tachyzoites stage which maintained in the laboratory through repeated intra peritoneal mouse inoculation in mice (Johnson et al., 1979).

Chicken blood collection and sera preparation: Blood samples were collected in dry, sterile screw capped tubes of 15 cm from 439 apparently healthy chickens (free range and commercial farms) from six governorates include El Gharabia, Kafr El-shiekh, El Sharkia, Sohag Quena and Cairo. Samples were centrifuged at 3000 rpm for 15 min and stored at -20°C until used in serological investigation.

Serological survey for detection of T. gondii antibodies: The collected chicken sera were examined serologically for detection of T. gondii antibodies using the Enzyme Linked Immuno-sorbant Assay (ELISA), the whole soluble tachyzoites antigens were prepared as described by Waltman et al. (1984) and the procedures adopted according to Lind et al. (1997).

Polymerase chain reaction (PCR): In this study, PCR was adopted for confirmation that tachyzoites as previously isolated from chicken tissues and used in serological diagnosis (as local strain) belongs to T. gondii strain. Genomic DNA was extracted from tested T. gondii local and RH (positive control) strains using a commercially available kit (Dneasy blood and Tissue kit, Qiagen Co., Cat. No. 69504) with modifications to the manufacturer’s protocols (Howe et al., 1997). Primers used for PCR were targeting the repetitive 35-fold B1 gene. The amplified product was analyzed on 1% agarose gel and stained with Ethidium bromide. Every PCR run included controls (Burg et al., 1989).

RESULTS

Morphological studies of the isolated T. gondii developmental stages: Two developmental infective stages of T. gondii were isolated; trachyzoites and bradyzoites.

Tachyzoites: Tachyzoites were obtained from the peritoneal exudates of previously inoculated mice with infected digested chickens’ tissues 6-8 post inoculation. Tachyzoites were found inside leucocytes (lymphocytes and macrophages) or free in the peritoneal exudates after rupture of leucocytes. The Giemsa stained isolated tachyzoites were often crescent in shape or banana shape pointed at one end and rounded or blunt at the other one, their size were 2x7 μm. It showed pale blue cytoplasm with reddish purple nucleus which centrally located or near the blunt end. The leucocytes appear as dark blue color (Fig. 1).

Tissue cysts containing bradyzoites: The tissue cysts of isolated T. gondii strain were noticed in the fresh smears after digestion of heart and brain tissues.

Fig. 1:	Giemsa stained T. gondii tachyzoites (X400)

The tissue cyst is the resting stage of parasite within the host, they were usually sub-spherical to spherical in shape and its cyst wall was thin elastic and will defined enclosing up several hundreds of crescent shaped Bradyzoites (Fig. 2).

Confirmatory PCR results: As number of base pairs (94 pb) of the isolated T. gondii strain obtained from chicken tissues (lane, 3 and 4) was the same of compared positive control RH strain (lane, 2) with the presence of negative control sample (lane,1) and matched with standard marker (lane, M) (Fig. 3). So the PCR results confirm that tachyzoites which previously isolated is belong to T. gondii.

Fig. 2:	Giemsa stained T. gondii tissue cysts (X1000)

Fig. 3:	Detection of T. gondii by PCR. Lane 1: Negative control, Lane 2: Positive control (RH stain), M: MW marker (100 bp ladder) and Lanes 3 and 4: Positive PCR samples of isolated chicken strain at 94 bp

Table 1:	Seroprevalence of T. gondii in six governorates
C: Commercial forms, FR: Free range

Serological investigation of T. gondii antibodies in chicken sera: Examination of the total 439 serum samples of chickens by ELISA revealed that 302 sample (68.8%) had antibodies against T. gondii which was considered the percentage of infection. The obtained results indicate that prevalence rate was slightly higher, (69.5%) in free range chicken (FR) than that (68.5%) in chicken from commercial farms (C) in the most of 6 examined governorate and also results refer to that the prevalence rates was less in Upper Egypt than governorates than that of Lower Egypt and Cairo (Table 1).

DISCUSSION

The high prevalence rate of chicken toxoplasmosis (68.8%) in the present study which comprised 6 examined Egyptian governorates; refer to the importance of chicken’s meat as important source of infection among Egyptian consumers which agreed with that previously detected in Egypt by El-Massey et al. (1990), who reported that sheep, goat and chicken are important intermediate hosts for T. gondii and their meat are main source of infection to man if un-hygienically consumed.

However, present survey showed that seroprevalence rates was higher in Free-Range (FR) (69.5%) than commercial farm Chicken (C) (68.5%) indicated that the distribution of T. gondii in chicken might be related to the cats that disseminated the oocysts in the soil (Asgari et al., 2006). On the other hand, cats could be infected from eating chicken meat, wild birds and rodents infected with T. gondii (Hassanain et al., 2008). Therefore, T. gondii infection in chickens is epidemiologically significant and people have the habit of eating under-cooked chicken meat, should be aware of the risk of encountering T. gondii infection.

Little is known concerning the validity of the serologic tests for the detection of T. gondii antibodies in avian sera. Viable T. gondii was isolated from up to 100% of free-range chickens bio-assayed in mice (Dubey et al., 2007b). The present study, ELISA test used for seroprevalence of 439 chicken blood samples collected from six Egyptian governorates. The ELISA test has the advantage that it specific and of great sensitivity, objective, quantitative, low coast and can be automated and is convenient for large-scale surveys of toxoplasmosis in human and animals but it need a refinement in the procedures and standardization (Shaapan et al., 2008).

Many surveys detecting T. gondii infection in FR chickens have been conducted in a number of countries and the prevalence rates in most of these studies ranged between 17.9% and 85% (Deyab and Hassanein, 2005; Sreekumar et al., 2003; Dubey et al., 2007a; Dubey et al., 2006; Yan et al., 2009; El-Massey et al., 2000; Dubey et al., 2003). The seroprevalence of T. gondii in domestic birds varies within countries due to the testing methods, the number of examined animals, type and hygiene of animal breeding Dubey (2010a). The total seroprevalence T. gondii among chicken in our study (68.7%) was lower than some results other results (Sreekumar et al., 2003; Dubey et al., 2007a; Yan et al., 2009; El-Massey et al., 2000) similar to that (64%) in FR chicken in Thailand (Chumpolbanchorn et al., 2009), While lower than 85% obtained in Nicaragua (Dubey et al., 2006). Reasons for this variability could be many, including the age of the chicken, number examined, type of serological test used and the tissues bio-assayed. Overall, prevalence of viable T. gondii in chickens raised indoors was low (Dubey et al., 2007a).

Concerning PCR results, the matched sequence of base pairs with the stander for T. gondii confirm that PCR assay is a specific, speedy, sensitive and cost-effective method for detecting T. gondii DNA in chickens. The choice of using the fragment of 94 bp from the B1 gene as target to PCR amplification was based on the observations made by other studies (Howe et al., 1997; Dubey et al., 2005).

CONCLUSION

In considering the above-mentioned findings, it could be concluded that T. gondii infection was spread widely in Egyptian poultry, so it give aware of the public health danger of poultry as an intermediate host which transmit the infection to man. The significantly higher infection rate than previous studies indicate that ELISA is more sensitive method for the diagnosis of toxoplasmosis. Moreover, this study is the first was used antigen from locally isolated T. gondii chicken strain for the diagnosis of chicken toxoplasmosis.