Abstract: Cryptosporidium spp. and Toxoplasma gondii are public health important protozoan parasites. The presence of Cryptosporidium spp. oocysts and anti-Toxoplasma gondii antibodies in fecal and serum samples from native quails in different farms in Giza, Egypt has been carried out, respectively for the first time in Egypt., through the parasitological and serological examination. The extent of Cryptosporidium oocysts invasion was found to be 31.9% (lower extent 30.8% in bobwhite and higher 33.3% in brown quails). Three Cryptosporidium spp. oocysts were detected varied from small (Cryptosporidium meleagridis), medium (Cryptosporidium baileyi) and large size (Cryptosporidium galli). The T. gondii antibodies prevalence was 29.8 and 25.5%, using Modified Agglutination Test (MAT) and Latex Agglutination Test (LAT), respectively (lower prevalence 25.5 and 22.4% in bobwhite while higher prevalence 34.4 and 28.8% in brown quails). The obtained results indicated that Egyptian quails are reservoirs for both zoonotic Cryptosporidium spp. and Toxoplasma gondii. So that, it is imperative for human to avoid consuming insufficiently cooked quail meat. Also fecal contamination from quail should be controlled.
INTRODUCTION
Cryptosporidia and Toxoplasma are emerging coccidian parasite zoonoses that transmitted to humans either by ingesting environmentally robust transmissive resistant stage oocysts through contact with contaminated faeces or ingestion of contaminated water and food in case of both Cryptosporidia and Toxoplasma or by eating raw or undercooked meat containing infective tissue stages in case of Toxoplasma (Nissapatorn, 2007; Dorny et al., 2009; Smith and Nichols, 2010).
Cryptosporidiosis is one of the most prevalent parasitic infections in domesticated, caged and wild birds (Ryan, 2010). Cryptosporidium species have been reported in more than 30 avian species worldwide (Sreter and Varga, 2000; Ng et al., 2006; Seva Ada et al., 2011). The infection caused by several genotypically diverse Cryptosporidium species, has been dynamically changing over the past decade from that of a rare, largely asymptomatic infection to an acute enteric disease of animals and humans (Ramirez et al., 2004; Moghaddam, 2007; Fayer and Xiao, 2008).
There have been extensive studies conducted on avian cryptosporidiosis; however, the prevalence data for the determination of Cryptosporidium infection in quails in the different localities of the world is much less known. In USA, examined intestinal content from different quail species for Cryptosporidium oocysts, only 18 (14.9%) of 121 birds had Cryptosporidium oocysts in their feces at time of collection (Duszynski and Gutierrez, 1981). In Azerbaijan, the invasion extensities of Cryptosporidium oocysts invasion in chickens, pheasants, peacocks and quails were 50.6% (135 out of 269), 7.9% (16 out of 203), 12.9% (8 out of 62) and 21.7% (26 out of 120), respectively (Musaev et al., 1998).
However, many avian species are intermediate hosts of Toxoplasma gondii, the prevalence of T. gondii infection in quail species is little known, experimental toxoplasmosis in bobwhite and Japanese quails through oral inoculation with ME 49 strain T. gondii oocysts had been done by Dubey et al. (1993) and Dubey et al. (1994), respectively, the infective stages were isolated from the brains, hearts and skeletal muscles of all quail by bioassays in mice and quails that survived develop severe protozoal pneumonia, myocarditis or meningo-encephalitis. The T. gondii antibodies were found in quail sera 63 Days After Infection (DAI) using MAT and LAT. Also, Albuquerque et al. (2001), observed T. gondii tachyzoites from 7th DAI, in the liver, lung and spleen and the T. gondii tissue cysts observed in 70th DAI in brain and heart of Japanese quails experimentally inoculated with tachyzoites of 'P' strain while no parasites were observed in any animals after inoculation with tachyzoites of SERO-47 strain of T. gondii.
Since largely unknown concerning Cryptosporidium and T. gondii infection prevalence in Egyptian native quails, so the objectives of the study reported here were to investigate the native quails parasitological and serological to Cryptosporidium and T. gondii and refer to their zoonotic importance in transmission of infection to human.
MATERIALS AND METHODS
Fecal and blood samples: This research project was conducted from 1-2011 to 4-2011. A total of 210 quail fecal samples (120 from bobwhite and 90 from brown quails) and 188 quail blood samples (98 from bobwhite and 90 from brown quails) were collected from different quail farms in Giza, Egypt. Sera were separated, labeled and kept at-20°C until use.
Fecal examination: Fine feces smears fixated with methanol spirit and stained with Modified Ziehl-Neelsen Stain (MZN) for detection of Cryptosporidian oocysts according to the procedure described by Henriksen and Pohlenz (1981). The preparations were observed and the oocysts were measured with help of stage micrometer conjugated with the light microscope at the eyepiece 10x and the objective 100x. The all measurements are in micrometers (μm) for about 20-50 oocysts, with the range in parenthesis following the mean (Yatswako et al., 2007; Fayer and Xiao, 2008).
Serological assay
Modified Agglutination Test (MAT): Formalized-fixed whole tachyzoites antigen
was prepared using RH strain of T. gondii secured in Department of Zoonotic
Diseases, National Research Center, Egypt as described by Desmonts
and Remington (1980) and the test procedures was carried out according to
the method of Dubey and Desmonts (1987), considering
1:25 serum dilution as the cut-off point.
Latex Agglutination Test (LAT): The collected quail sera were testing against Toxoplasma infection using latex agglutination test kit (Toxocheck-MT; Eiken Chemical, Tokyo, Japan) following the manufacturer's instructions.
| Table 1: | Prevalence of Cryptosporidium infection among quail |
| Table 2: | The dimensions of Cryptosporidium oocysts detected in quails |
| Table 3: | Prevalence of T. gondii antibodies among quail sera using different serological tests |
| MAT: Modified agglutination test, LAT: Latex agglutination test | |
Sample was considered positive when agglutination observed at dilutions of 1:64 and greater.
RESULTS
Detection of Cryptosporidium oocysts in quails by fecal examination: Quail Fecal samples have been examined concerning the presence of Cryptosporidium spp. oocysts. The extent of invasion was 31.9% (67 out of 210) of quail fecal samples were found to be infected with Cryptosporidium spp. oocysts. The bobwhite quails reveled lower prevalence 30.8% (37 out of 120) while the brown quails showed higher Cryptosporidium infection 33.3% (30 out of 90) (Table 1).
The dimensions of Cryptosporidium oocysts detected in quail: The measurements of detected Cryptosporidium oocysts in the examined quail feces were revealed three Cryptosporidium species of round or slight ovoid shaped oocysts. They were varied from small sized (Cryptosporidium meleagridis) oocysts of 5.2x4.4 μm dimensions and 1.1 μm in shape index; medium sized (Cryptosporidium baileyi) oocysts of 6.4x4.8 μm dimensions and shape index 1.3 μm and large sized (Cryptosporidium galli) oocysts 8.2x6.3 μm in dimensions and 1.3 μm in shape index (Table 2, Fig. 1a-c).
Serological examination for the detection of T. gondii antibodies: Examination of the 188 serum samples from slaughtered quails through MAT and LAT revealed that 56 (29.8%) and 48 (25.5%), respectively had antibodies against T. gondii. The bobwhite quails reveled lower prevalence 25.5 and 22.4% (25 and 22 out of 98) while the brown quails showed higher T. gondii infection 34.4 and 28.8% (31 and 26 out of 90), respectively by MAT and LAT (Table 3).
| Fig. 1 (a-c): | Cryptosporidium oocysts in quail fecal smears stained with Modified Ziehl-Neelsen (MZN) (x100). (a) Cryptosporidium meleagridis, (b) Cryptosporidium baileyi and (c) Cryptosporidium galli |
DISCUSSION
The extent of Cryptosporidium oocysts invasion in quails in this study was found to be 31.9% (lower extent 30.8% in bobwhite and higher 33.3% in brown quails). These finding was higher than (14.9 and 21.7%) detected by Duszynski and Gutierrez (1981) in different quail species from USA and Musaev et al. (1998) in quails from different localities in Azerbaijan.
The variation in the invasion extensities of Cryptosporidium oocysts in quails examined during the present study and those previously surveyed are probably attributed to the number of birds examined, time of feces collection and examination and the hygienic conditions of the quail farms and its surrounding environment. On the same approach, O’Donoghue (1995), Fayer et al. (2000) and Ramirez et al. (2004), also proved that the variation in Coccidian oocysts incidence in birds was associated with the age and sex of the bird examined, the methods used to make the diagnosis, climatic conditions, stresses exposed by birds and differences in management methods.
The three Cryptosporidium species (Cryptosporidium meleagridis, Cryptosporidium baileyi and Cryptosporidium galli) detected in quail fecal smears in this study are morphologically similar with the same species detected in quails and birds in many previous studies (Zha and Jiang, 1994; Fujino, 1996; Ryan et al., 2003). The Cryptosporidium spp. identification was depending upon the conventional criteria, such as oocyst morphology and measurements, this opinion was agreed with Fayer et al. (2000) and Morgan-Ryan et al. (2002), who cited that morphometric measurement of oocysts represents the cornerstone of Cryptosporidium taxonomy and is one of the requirements for establishing a new species.
A coccidian parasite of humans and animals was first believed to be an opportunistic organism but now is recognized as a primary pathogen, several studies have implicated animals as a source of human infection (Levine et al., 1988; Caver et al., 1996). Because strains of Cryptosporidium detected in this study are cross-transmissible especially Cryptosporidium meleagridis, awareness of cryptosporidiosis as a potential zoonotic infection has emerged as a significant public health concern (Hoerr et al., 1986).
Quail in the present study responded serologically to T. gondii through MAT and LAT; high levels of Toxoplasma gondii antibody titers were higher with the MAT (29.8%) than with the LAT (25.5%). These results are nearly agreed with that proved by Dubey et al. (1993) and Dubey et al. (1994) who they found that antibody titers to T. gondii in sera of experimentally infected quails by the MAT using whole tachyzoites were higher than in LAT and Indirect Hemagglutination Tests (IHT) that used soluble antigens and antibodies were not detected in quail sera examined by the Sabin-Feldman dye test.
However, T. gondii prevalence in quails with the MAT (29.8%) and the LAT (25.5%) in this study are slightly lower than that results obtained with Asgari et al. (2008) among free-ranging chickens (27.1%) in Southern Iran while it is largely lower than that obtained with Dubey et al. (2003) in free range chickens (40.4%) from a rural area surrounding Giza, Egypt using MAT and with Butty (2009) among turkeys (76.63%) from ten regions in Ninevah governorate, Iraq using LAT and this finding may due to chickens and turkeys become infected mostly by feeding from the ground contaminated with oocysts and they considered as a good indicator of prevalence of T. gondii in their environment (Ruiz and Frenkel, 1980).
The Modified Agglutination Test (MAT) is considered the more sensitive and recommended test for diagnose the T. gondii infection in several animals and man (Dubey, 1997; Garcia et al., 1999). Moreover the highest sensitivity and specificity among all serological tests of MAT was confirmed by the results obtained by Shaapan et al. (2008) and Shaapan et al. (2010) who demonstrated the benefits of using more sensitive and specific MAT for the detection of T. gondii antibodies in sheep, goat and buffaloes sera which is cheaper, easier than other tests and does not need special sophisticated equipment.
CONCLUSION
It is of interest to point out that, this study is the first report in Egypt for the detection of Cryptosporidium oocysts and Toxoplasma gondii antibodies in native quails. The high prevalence rates obtained by this study indicate the potential risk of its transmission to humans through contact with feces or meat consumption of quails. Also biological and molecular characteristics of the detected Cryptosporidium spp. will necessary to be investigated to confirm their diagnosis.