Serodiagnosis of Bovine Sarcocystosis by Dot-ELISA

Sarcocystosis is a parasitic zoonosis caused by species of Sarcocystis; an intracellular protozoan parasite in the phylum Apicomplexa and family Sarcocystidae. Sarcocystis has a requisite of two-host life cycle based on a preypredator (intermediate-definitive) host relationship Fayer (2004). Sarcocystosis occur in all of countries Taylor et al., (2007) and Radostits et al., (2008). Sarcocystis spp. are considered one of the most prevalent parasites of livestock and can cause clinical disease in these animals. Three Sarcocystis spp. are known to infect cattle as intermediate hosts, namely Sarcocystis cruzi, S. hirsuta and S. hominis Dubey (1976) and Dubey and Lindsay (2006), whereas buffaloes act as intermediate host for S. fusiformis, S. levinei, S. buffalonis and S.dubeyi Yang et al., (2001).
        
Studies have reported 100% prevalence in adult cattle in the United States Dubey et al., (1989). Although Sarcocystis spp. are generally considered non-virulent for cattle, S. hominis infection was occasionally associated to eosinophilic myositis Wouda et al., (2006). The symptoms of infection in cattle, when apparent, involve reduced weight gain, anorexia, fever, muscle weakness, eosinophilic myositis, reduced milk yield, abortion and death Dubey et al., (1989) and Vangeel et al., (2012). Meats and meat products are the main source of infection in human beings, who become infected when ingesting well-developed tissue cysts containing bradyzoites Juyal and Bhatia (1989). Gross lesions on the meat lead to condemnation of meat resulting in economic losses to the meat industry Bottner et al., (1987). Livestock should be screened routinely and the infected animals must undergo quarantine to prevent the spread of this parasite Hettiarachchi and Rajapakse (2008). As human and animals act as intermediate host and do not shed any diagnostic stages in their faeces and as molecular techniques though sensitive are very costly and not applicable at the field level, hence necessitates the use of serological methods which detect the presence of antibodies in the sera of infected animals. A number of serological tests have been developed for diagnosis of Sarcocystis infection which includes Agargel diffusion tests, Haemagglutination inhibition test (HI), Indirect Fluorescent Antibody Technique (IFAT) and Enzyme Linked Immunosorbent Assay (ELISA) Dubey et al., (1989). Due to lack of standardization and repeatability of the above said tests, the present study aims at optimizing a Dot-ELISA based serodiagnostic assay for epidemiologic evaluation of sarcocystosis at herd level and to take appropriate control measures.

Preparation of the antigen
 
Bovine esophagi containing macrosarcocysts were collected from Chengicherla Slaughter House, Hyderabad, Telangana. Bradyzoites were recovered by gently teasing the cysts and purified by glass wool filtration. The particulate antigen for Dot-ELISA was prepared by fixing the bradyzoites in 1.5% formalin-PBS (pH-7.2) for 1 hour followed by washing thrice in Triethanol buffered saline (TBS, pH-7.5, Triethanolamine- 2.8 ml; NaCl- 7.5g; CaCl₂, 2H₂O- 0.02g; MgCl₂, 6H₂O- 0.1g, make the volume upto 1000ml) and enumerated by using Neaubauer’s haemocytometer. The pure bradyzoite solution was sonicated at 20kHz, 100mA for 3 cycles each lasting for 30 seconds with an interval of 1 min in between, centrifuged at 10,000 rpm for 30 min at 4°C and the protein concentration was measured by using Biospec Nanodrop. The antigen discs for Dot-ELISA were prepared by dotting 1 µl of formalin fixed bradyzoites (1x10³/µl, 2 x10³/µl, 3 x10³/ µl, 4 x10³/ µl, 5 x10³/ µl, 6 x10³/ µl,7 x10³ / µl, 8 x10³ / µl, 9 x10³/ µl , 10 x10³/ µl ) on the nitrocellulose membrane (NCM) of 5mm size. The antigen dotted NCM discs were dried at 56°C for 10 min and stored at -20°C until further use.
 
Collection of reference sera
 
Forty five bovine sera were collected from slaughter house from the animals which are found positive for sarcocystosis on post mortem examination. Similarly 42 known negative sera were collected from infection free animals. For prevalence study, 232 sera (151 cattle and 81 buffalo) were collected from the areas in and around Hyderabad.
 
Standardisation of Dot-ELISA
 
The optimal conditions for Dot-ELISA were standardized as described by Tenter  (1988) with some minor modifications. The cut off values for antigen, serum, Rabbit anti bovine IgG HRPO conjugate were determined by checker board titration. Each antigen concentration was titrated against different dilutions of the serum (1:16,1:32,1:64, 1:128,1:256,1:512) and different dilutions of conjugate (1:100,1:200,1:300). The discs with desired antigen concentration were placed in ELISA plate and the uncoated sides of the antigen dotted NCM discs were blocked by dipping them in 3% BSA-TBS (pH-7.5) and with constant shaking for 1 min. The blocked discs were washed thrice with 0.05% NP-40-TBS each for 5 min. Double fold serial dilution of the serum samples as mentioned above (positive and negative) were done in 1% BSA-TBS and 50µl of each dilution were titrated against each antigen concentration, incubated at 37°C for 40 min. The unbound antibodies were removed by washing as above and then 50µl of Rabbit anti bovine IgG HRPO conjugate diluted to 1:100 in 1% BSA-TBS (pH 7.5) was added to each well, incubated at 37°C for 30 min. The conjugate was removed as per the washing methods mentioned above. Peroxidase mediated colour development was done by dipping the discs in 50µl of chromogen substrate 4-Chloro-1-napthol (Stock solution- 30 mg per ml of methanol, Working solution- 1 ml of stock solution to 5ml of TBS and 4 µl of 30% Hydrogen peroxide) and incubated for 30 min at room temperature. The reaction was stopped by adding distilled water and then the discs were washed as above and visualized for reading the results.
 
Immunofluorescent Antibody Technique (IFAT)
 
The method was adopted from Renterghem and Nimmen (1976) with minor modifications and was earlier standardised by Banothu et al., (2017) in our laboratory.
 
Comparison between Dot-ELISA and IFAT
 
The diagnostic accuracy of Dot-ELISA was tested by comparing it with IFAT. The specificity, sensitivity values were calculated and compared with IFAT.

The protein concentration of the sonicated bradyzoite sample containing around 1.68x10⁸ bradyzoites / ml was 203µg per ml amounting to 0.5 pg per bradyzoite. Formalin (1.5%) fixed antigen containing 2000/µl, 3000/µl, 4000/µl, 5000/µl, 6000/µl, 7000/µl, 8000/µl, 9000/µl and 10,000 bradyzoites/µl amounting to 1 ng, 1.5 ng, 2 ng, 2.5 ng, 3 ng, 3.5 ng, 4 ng, 4.5 ng and 5 ng of protein when titrated against different dilutions of known positive serum and conjugate showed a bluish purple coloured dot. The reaction conditions of 1ng antigen (2000 bradyzoites/µl), 1:32 dilution of serum and 1:100 dilution of conjugate were found optimum for differentiation of positive and negative reaction (Fig 1). In case of IFAT, the Rabbit anti bovine IgG FITC conjugate of 1:40 dilution was considered optimum which gave bright yellowish green over the entire bradyzoite or distinct yellowish green fluorescence around the entire periphery of the bradyzoites (Fig 2) with 1:16 dilution of positive sera and either no fluorescence (faint reddish) (Fig 3a)  or polar fluorescence (Fig 3b) with 1:8 dilution of negative sera. The sensitivity and specificity of Dot-ELISA and IFAT and the prevalence of sarcocystosis in cattle and buffalo are mentioned in Table 1 and 2. The statistical analysis indicated significant association of Sarcocystis infection with cattle than in buffaloes. 
 

 

 

 

 

        
A presumptive diagnosis of sarcocystosis in intermediate hosts can be made by clinical signs, clinical pathology, biopsies etc. The serological diagnostic tests though very helpful for early diagnosis and in prevalence studies, they vary greatly in sensitivity and specificity which may be because various workers used different antigens like particulate and soluble antigens (chemically lysed  bradyzoites, sonicated bradyzoites), used various techniques for bradyzoite extraction (pepsin digestion), purification (percoll gradient method) and protein extraction (freeze thawing, sonication, dialysis, protein elution and concentration). At present, there is lack of standardization of different serological tests like ELISA, IFAT because antigens used vary greatly from one batch to another.
        
Savini et al., (1994) used soluble cystozoite and merozoite antigen of S.cruzi in ELISA and mentioned the superiority of the merozoite antigen in detecting the acute infections. They also informed that the cystozoite antigen was cross reactive and sensitivity and specificity of ELISA was improved using merozoite antigen which may be further used in the diagnosis of acute infection of Sarcocystis in economically important hosts such as cattle and sheep.
        
Singh et al., (2004) studied seroprevalence of sarcocystosis using soluble cystozoite antigen in cattle and reported high sensitivity of dot-ELISA (46%) over DID (26%) and CIE (30%). Mamatha et al., (2006) used cystozoite soluble extract from both macro and microsarcocysts of S bovifelis and S. bovicanis as antigens in ELISA taking EITB as reference test, examined 300 serum samples and found anti-Sacocystis antibodies in 146 (48.66%) and 46 (15.33%) sera for S bovifelis and S. bovicanis respectively. The sensitivity and specificity by ELISA was found to be 76.4 and 66.6 per cent and 81.8 and 71.4 per cent for S.bovicanis and S. bovifelis respectively. The sensitivity and specificity in EITB was found to be 87.5 and 81.8 per cent and 93.5 and 87.5 per cent for S. bovicanis and S. bovifelis respectively.
        
Sabry et al., (2008) crushed the macrosarcocysts in PBS and the released bradyzoites were freeze thawed, sonicated, centrifuged. The supernatant was dialysed and fractionated using SDS-PAGE. Out of all the eluted fractions of Sarcocystis bradyzoite antigen used in micro ELISA technique, the fraction of 53KD was found to be more sensitive (95.83%) and specific as it didn’t show any cross reactivity.
        
Tenter (1988) used different types of antigens such as purified cystozoites without any further treatment and cystozoites fixed in different concentrations of formalin (0.5, 1.0, 1.5, 2.0 or 2.5%).  In a similar study, Hamidinejat et al., (2013) used 2500 whole tachyzoites of Neospora caninum in Dot-ELISA and reported that relative sensitivity and specificity of Dot- ELISA were 92.63 and 89.16% in comparison with ELISA and 93.4% and 90.8% when compared with the DAT (Direct Agglutination Test).
        
However, most of the workers used partially purified cystozoite soluble antigen in the diagnostic procedures because of its ease in preparation. The protein concentration of the antigens used by various authors varied widely. Liang-Zhang and Hui-Yuan (1987) obtained a protein concentration of 897 µg per ml in the soluble antigen of macrosarcocyst of Sarcocystis. However Pandit et al., (1993) achieved a low protein concentration of 232 and 260 µg per ml in one batch and slightly higher concentration of 660 µg and 920 µg per ml in second batch of S. hirsute and S.cruzi soluble antigen respectively by pepsin digestion followed by percoll purification. Singh et al., (2004) obtained a protein concentration of 800 µg per ml by freeze thawing the zoites. However, the above workers either used whole macrosarcocyst antigen or did not mention the number of bradyzoites used in preparing the antigen.The low protein (203 µg/ ml) concentration obtained in our study compared to other workers could be due to less number of bradyzoites (1.6 x10⁸ / ml) used in the extraction of protein. In this current study glass wool purified and 1.5% formalin fixed bradyzoites (2000 bradyzoites/ µl) amounting to 1ng protein gave positive reaction in Dot-ELISA. The antigen used is economical as it excludes the pepsin digestion method Collins et al., (1980) for recovery of bradyzoites and subsequent use of percoll for purification Tenter (1988) and Mamatha (2006). The sensitivity and specificity of Dot-ELISA and IFAT were found to be 86.66% and 83.33% and 88.88% and 100%, respectively. Our observations were almost similar with those of Tenter (1988) who reported 100% and 99% specificity with Dot-ELISA and IFAT, respectively. 
        
In present study, positive reaction was obtained at a serum dilution of 1:32 which could be due to the usage of known positive reference sera collected from naturally infected animal. Hamidinejat et al., (2013) standardized Dot-ELISA at 1:20 serum dilution and 1:1000 conjugate dilution to detect anti-neosporosis antibodies in cattle. In this study, the sera dilution used was comparatively lower when compared to Tenter (1988) who used hyper immune serum. Similar differences in the hyper immune serum and known positive serum in giving a positive reaction in Dot-ELISA were reported earlier by Mamatha (2006) who recorded a positive reaction with hyper immune serum at a dilution of 1:3000 and 1:8000 and with known positive naturally collected serum at a dilution of 1:100 and 1:200 for the diagnosis of S. bovicanis and S. bovifelis, respectively. Purple coloured dot was developed with 4 chloro 1 napthol at conjugate dilution of 1:100. Similar observations were reported earlier by Tenter (1988).
        
The screening of serum samples by Dot-ELISA revealed around 192 positives out of 232 serum samples collected indicating an overall incidence of 82.75%. The rate of prevalence was significantly higher in cattle where 143 out of 151 were found positive indicating 94.70% of infection compared to buffaloes where 49 out of 81 were positive showing 60.49% of prevalence. Our results on the prevalence of bovine sarcocystosis were on line with the observations of Liang-Zhang and Hui-Yuan  (1987) who reported 79.25% seroprevalence of sarcocystosis in cattle by ELISA whereas Fatma et al., (2008) recorded 94% and 98% infection rate by microscopic examination and ELISA respectively in cattle at Assiut abattoir. According to Ellah et al., (2011), the prevalence of macroscopic Sarcocystis was 23% whereas ELISA technique revealed that 94.44% of examined buffaloes were infected with Sarcocystis. Our results on bovine sarcocystosis by Dot-ELISA could be comparable with the observations of Sabry and Shalaby (2004) who examined buffaloes of 2 and over 7 years age group and found 19.33% and 88.5% infection respectively by Dot-ELISA.    

Dot-ELISA is a rapid, less expensive, sensitive and easy to perform test with an advantage that both soluble and particulate antigen can be applied on to the NCM Hawkes et al., (1982) and also the antigen binding capacity of NCM is better than microtitre plates. When compared to ELISA and IFAT, this test does not require any special equipments like ELISA reader and fluorescent microscope and the result can be read with naked eye without the assistance of any experienced personnel. Minute quantity (1µl) of antigen is required whereas 10 µl and 50 µl amounts of antigen are required for IFAT and ELISA respectively. In conclusion, it can be suggested that Dot-ELISA test for the diagnosis of bovine sarcocystosis may be superior to IFAT and ELISA when we have to screen the cattle on herd populations and especially at slaughter houses. This test can be a promising tool in diagnosis of bovine sarcocystosis and can facilitate slaughtering of infected animals, condemnation of infected tissues and minimization of availability of tissues with macro or microsarcocysts to stray dogs and cats under appropriate control measures.