Abstract: Serodiagnosis of Haemonchus contortus by ELISA using adult crude somatic and excretory-secretory antigens of Haemonchus contortus was tested in the present study. The diagnostic sensitivity of ELISA using excretory secretory antigens was 87.5% which was significantly higher compared to crude somatic antigen 72.22%. Mean ELISA absorbance value of excretory-secretory antigens was significantly higher corresponding to crude somatic antigens. Excretory secretory antigens showed 92.02% specificity compared to 76.81% of crude somatic antigens (p<0.05). The results revealed that excretory secretory antigens are very sensitive and may be useful as a supplementary method for diagnosis of Haemonchosis in ruminants.
INTRODUCTION
Haemonchosis is one of the most important diseases of livestock through out the world. The disease has a cosmopolitan distribution but more prevalent in most sheep-raising countries. The parasite inhabits in the abomasa of sheep, bores its walls and feeds on blood. It causes great economic losses in sheep industry including decreased weight gain (Ploeger et al., 1990), decreased milk yield Gross et al. (1999). Young lambs are very sensitive to Haemonchus contortus while as older sheep acquire immunity after continuous or seasonal exposure to the parasite. The mechanism underlying immunity is still not completely understood. Analysis of host immune response to helminth parasites is hampered by two main factors (i) the complexity of antigen profile of parasites (ii) the presence of cross-reactive determinants on antigens (Maizels et al., 1987; Gualzata et al., 1988). To identify specific antigens, Excretory-Secretory (ES) products of helminth parasites have received increasing attention. This is due to the fact that excretory-secretory products of helminthes usually display a relatively simple antigenic composition compared to the somatic worm antigens. Diagnosis of haemonchosis is based on clinical findings as well as laboratory tests. The most reliable method is the finding of egg in the stool of infected animals. This method cannot be used until the parasite attains sexual maturity. Therefore, it is necessary that these parasites should be controlled before they can cause damage to the stomach. Hence serological diagnosis should be preferred because antihaemonchus antibodies can be detected as early as one week post infection and thus can facilitate early chemotherapeutic intervention. Despite of the recent studies involving the diagnosis of haemonchosis using Haemonchus contortus antigens in ELISA test, the effectiveness of Haemonchus contortus crude somatic antigens and excretory-secretory antigens have not been studied adequately especially in Kashmir as this is the first report in this case.
The present study was carried out from October 2005 to January 2006 and the purpose of this investigation was to compare the specificity of crude somatic and excretory-secretory antigens in ELISA test for serodiagnosis of sheep haemonchosis.
MATERIALS AND METHODS
Parasites collected from the infected abomasa of sheep were washed with 0.01 M phosphate buffer pH 7.2. Freshly collected worms were incubated in 20 mL phosphate buffer saline for 5 h at 37°C. After incubation, worms were removed and the suspension containing excretory secretory products was centrifuged at 15000 rpm for 15 min at -20°C. The supernatant obtained was concentrated in dialysis bags under moving fan. The concentrated samples were stored at -20°C till further use.
Preparation of Crude-Somatic Antigens
Adult worms were collected from infected animals cleaned in normal
saline solution. Later, worms were mixed with Phosphate Buffer Solution
(PBS), pH 7.4 homogenized in Teflon tissue homogenizer at 4°C for
1 h. The homogenate was kept undisturbed for 24 h in refrigerator. Supernatant
collected form homogenate was centrifuged in cooling centrifuge at 12000
rpm for 15 min. The supernatant was stored as crude solubilized antigen
at -20°C till further use. Protein concentration of crude somatic
and excretory-secretory antigens was determined by Lowry et al.
(1951).
Collection of Sera Samples
A total of 432 sera samples collected from sheep confirmed positive
for Haemonchus contortus by coprological examination were tested
for sensitivity in ELISA. To see the specificity of excretory-secretory
antigens 138 sera samples were collected from the abattoirs. Out of 138
sera samples, 50 sera samples were collected from the healthy uninfected
sheep, treated as control, 88 sera samples were collected from sheep infected
with nematode parasites like Ostertagia circumcincta (46), Bunostomum
trigonocephalum (26) and Trichuris ovis (18). All the sera
samples were stored at -20°C.
ELISA
ELISA was performed in polystyrene EIA micro titration 96-welled flat-bottomed
plates. Buffers (carbonate-bicarbonate pH 9.6 and phosphate buffer saline
pH 7.2 containing teween 20 were used for coating and washing procedures.
Each well of micro titration plate was coated by 1 mL of antigenic solution,
protein concentration of which was 2 μg mL-1. The plates
were then sealed and incubated at 4°C overnight. After incubation
the plates were washed with phosphate buffer saline containing teween
20 (PBST) and then blocked by PBS solution containing 1% bovine serum
albumin and 0.5% NaN3. After over night incubation diluted
test samples (1:25) were added to the wells and the plates were again
incubated at 37°C for 3 h and then washed with PBST. Thereafter 50
μL of diluted antirabbit IgG conjugated with horseradish peroxide
was added to each well and then again incubated for 3 h at room temperature.
After incubation the plates were washed three times with PBST. The reaction
was developed by adding 100 μL of freshly prepared solution (tetramethlbenzidine
and 1%. H2O2) to each well. Finally reaction was
stopped by the addition of 50 μL of 3 N Na OH solutions. The optical
density was recorded at 450 nm on ELISA reader. Samples with an absorbance
of <0.25 were deemed negative and those ≥0.25 as positive.
Statistical Analysis
The differences observed in the data were statistically analyzed by
student t-test and Pearson’s chi-square test using SPSS 10.0 for windows.
RESULTS AND DISCUSSION
In order to test the potential diagnostic value of excretory secretory
and crude somatic antigens, 432 sera samples of sheep naturally infected
with Haemonchus contortus were collected from abittors. ELISA
analysis showed that excretory-secretory antigens were more sensitive
compared to crude somatic antigens. Out of 432 samples collected 378 (87.5%)
showed positive reaction with excretory-secretory antigens which was significantly
higher corresponding to 312 (72.22%) of crude somatic antigens (Table
1).
| Table 1: | Comparison of sensitivity between crude somatic and excretory-secretory antigens of Haemonchus contortus |
| Pearson’s chi-square test was applied using SPSS 10.0 for windows, *: p<0.05 | |
| Table 2: | Comparison between mean absorbance of crude absorbance and excretory antigens |
|
"(Standard deviation), The differences were statistically analyzed by Students t-test using SPSS 10.0 |
|
| Table 3: | Comparison of specificity between crude somatic antigens and excretory-secretory antigens of Haemonchus contortus |
| Pearson’s chi-square test was applied using SPSS 10.0 for windows | |
The results of the present study revealed that excretory-secretory antigens proved to be better antigens than crude somatic antigen for serodiagnosis of haemonchosis. The diagnostic sensitivity of excretory-secretory antigens observed in ELISA was increased to 87.5 from 72.22% of crude somatic antigen, which was statistically significant. Increased sensitivity of ELISA using excretory-secretory antigens compared to crude somatic antigens may be attributed to strong recognition of excretory-secretory antigens by the sera of infected animals. It has been demonstrated that excretory-secretory antigens elicit strong immune response (Ogilvie and De Savigny, 1982) and may be potential source of diagnostics (Lightowlers and RicKard, 1988; Brandt et al., 1992; Mahannop et al., 1992) or protective antigens (O’ Donnell et al., 1989; Savin et al., 1990). Somatic antigens have been considered poorly immunogenic compared to excretory-secretory antigens (Parkhouse et al., 1987). The excretory-secretory product of Haemonchus contortus was found to include antigenic polypeptides of 10, 15, 16, 20, 30, 40, 46, 52, 66, 68 and 93 kDa. (Schalling et al., 1994). The 15 and 24 kDa antigenic proteins present in the excretory-secretory products could be specifically recognized by Haemonchus contortus primary sera (Schalling and Leeuwen, 1997). Torgerson and Lloyd (1993) demonstrated that Haemonchus contortus antigens with molecular weight less than 30 kDa present in excretory-secretory products can induce strong lymphocyte responses. This might indicate that low molecular weight excretory secretory products have protective value. The diagnostic specificity of ELISA using excretory-secretory antigens was increased to 92.02 from 76.81%. The cross reactivity of excretory secretory antigens with the sera samples of Ostertagiasis, Bunostomiasis and Trichuriasis was reduced compared to that of crude somatic antigens. This reduced cross reactivity may be attributed to high levels of 15 and 24 kDa antigenic proteins present in excretory-secretory products which could be specifically recognized by Haemonchus contortus primary sera (Schalling and Leeuwen, 1997).
CONCLUSION
In conclusion the present study demonstrated that E/S products of Haemonchus contortus are highly antigenic contains antigenic peptides of potential diagnostic and protective value.