Serodiagnosis of Oesophagostomum Columbianum infection in Goats using indirect ELISA

To determine the seroprevalence of Oesophagostomum columbianum antibodies in goats in Kashmnir, a study was conducted in Srinagar and Ganderbal Districts, located in central parts of Kashmir valley. ELISA was standardized and evaluated to detect goat Oesophagostomosis in experimental and clinical cases using somatic whole adult antigen of Oesophagostomum columbianum. Plate ELISA was standardized using 5μg/well antigen concentration with 1:100 and 1:1000 of sera and conjugate dilution. Indirect plate ELISA was able to demonstrate the antibody titer at different weeks post infection in experimental goats. A comparison of plate ELISA on suspected field sera and faecal sample examination by floatation method revealed that 68 (34.34%) samples were found to be positive using ELISA and twelve by faecal examination. The highest prevalence was found in lower age groups in both the seasons, with the increase in age, the infection level decreased. The prevalence was higher in wet season as compared to dry season. The results were stastically significant (P < 0.05). No sex predisposition was seen. Sensitivity and specificity of ELISA was 80.0%, and 21.42%, respectively. This test is therefore quite sensitive for clinical cases; an early diagnosis, however lacks specificity of faecal examination were 10.81% and 90.0%, respectively.


Introduction
infection is much advanced and the major damage is already done.In order to circumvent these limitations Gastrointestinal nematode infection is arguably there is an acute need for developing a reliable the most serious constraint affecting small ruminant serological assay like Enzyme Linked Immunosorbent production worldwide.Economic losses are caused by Assay (ELISA) for early detection of the infection.decreased production, the costs of prophylaxis and Detection of serum antibodies against parasite by treatment, and the death of the infected animals ELISA is a rapid and simple test with which a (Barger, 1982; Donald and Waller, 1982).
considerable number of samples could be processed at Oesophagostomum columbianum is a common the same time.Furthermore, seroepidemiological and widely prevalent parasitosis with significant studies involving examination of large group of economic importance in small ruminant livestock animals might also benefit from a reliable ELISA.(Olivares et al., 2001) throughout the Asian sub-Usually such a test, in contrast to faecal examination, continent (Khan, 1989;Mohanta et al., 2007).
is less time consuming.Caprine oesophagostomosis, like most of the Therefore an indirect ELISA based on crude helminth infections, is insidious and chronic in nature somatic antigen of O. columbianum was standardized and the migratory prepatent stages of the parasite are and evaluated under field conditions as a diagnostic involved in its major pathogenic effect.Reliable tool for detection of anti-O.columbianum antibodies detection of the active infection of O. columbianum is in sera of infected goats.usually based upon evaluation of clinical signs and faecal examination, which have their inherent Materials and Methods

limitations. Clinical signs usually become apparent
Experimental animals: Four health local goats, only when the infection is heavy and the eggs are 30-45days old, were maintained under intensive passed in the faeces after the prepatent period of rearing conditions precluding accidental parasitic approximately 41 days (Soulsby, 1982) when the infections.At the age of five months two goats were flat-bottom 96-well micro-ELISA plate.The absorbance (optical density; OD) of the wells was used for experimental infection with L3 of O. measured at 492 nm by an ELISA reader (Biorad II).columbianum.

Collection of O. columbianum L3 and
The mean OD plus three times the standard deviation experimental infection : The infective L3 were of the negative control sera was taken as the cut-off obtained by culturing (Soulsby, 1982) the eggs value for considering a sample as test positive (Lejon separated from the adult female worms recovered et al., 2005).from the caecum and colon of slaughtered goats.The Performance of the assay: The sensitivity, specificity L3 at the dose rate of 600 per kg body wt. were orally and accuracy of the ELISA were determined administered in one of the 8 helminthes free goats, (Thrusfield, 2003) using 198 serum samples of field after overnight withdrawal of feed, to serve as donors goats.The parasitological status of animals with O. regard to nematodes trematode and cestodes was for sufficient number of monospecific columbianum carefully examined by floatation technique and eggs for artificial infection.After the recorded.The sensitivity, specificity, positive patency of the infection the faeces of the donor goats predictive value and negative predictive value were was cultured (Soulsby, 1982) and the L3 were calculated from the two way analysis of ELISA test harvested (Anon, 1971).These L3 were used for artificial infection of two goats as stated earlier and faecal examination using following formulas: keeping the remaining two goats as uninfected control.From a total of 198 goats, 68 (34.34%) were 7.2.The worms were washed 3 times in the same positive for Oesophagostomum columbianum.After buffer and finally 200 worms were homogenized in 10 pooling the data, age wise epidemiological observations ml of cooled 0.15 M PBS (pH-7.2) containing 25 mM were made which revealed highest prevalence rate in phenylmethyl sulfonyl fluoride (PMSF) and 24 mM lower age groups in both the seasons, with the increase ethylene-diamine tetraaceticacid (EDTA) in a glass in age, the infection level decreased (Table 1).The tissue homogenizer followed by sonication (Soniprepprevalence was higher in wet season as compared to 150).The disintegrated parasite extract was dry season.The results were statistically significant (P o centrifuged at 4 C at 10000 g for 15 minutes and the < 0.05).No sex predisposition was seen.supernatant was collected as the CSAg with a protein The highest OD value (1.23) in the sera of concentration of 3.48 mg/ml specified according to experimentally infected goats was observed after 33 Lowry et al. (1951).The crude somatic antigens were DPI and the cut-off OD value for the standardized o stored at -20 C for use in the assay.
ELISA as determined was 0.168.O. columbianum Standardization of the assay: Plate ELISA was antibodies in all experimentally infected goats were performed in 96 wells polystyrene microtitre plates detected as early as 18 to 27 DPI.The seroconversion with all incubation times previously determined by was relatively long before the patency of the infection, checkerboard titration following Hudson and Hay which in this study was on 42 DPI.Specific antibodies (1989).The antibody detection was performed were consistently present during the subsequent according to the method of Voller et al. (1976) with observation period i.e. till 33 DPI.some modifications.The optimal concentration of A total of 198 goat samples suspected for ELISA reagents including the concentration of the Oesophagostomosis were examined by indirect plate coating antigen (5µg/well), dilution of the positive and ELISA.Sera of control group (negative goats) and negative reference sera (1:100) as well as rabbit antiexperimentally infected (positive goats) were raised goat IgG-horseradish peroxidase (HRP) conjugate and were taken as reference sera for ELISA.A cut off (1:1000) and the optimal test conditions, respectively value of means of negative controls ±3 SD was taken were determined by checkerboard dilution assay using into account for the detection of each positive case.Sensitivity of plate ELISA was found to be considerable promise for its exploitation in 80.0% whereas specificity was 21.42%, indicating seroepidemiological studies of this economically that this test is quite sensitive for clinical cases: an significant helminthosis.Sequestration of antibodies early diagnosis however lacks specificity.The plate and the formation of circulating immune complexes ELISA positive and negative predictive values were (Gasser et al., 1993) and immune evasion mechanisms found to be 10.81% and 90.0%, respectively. of the parasite (Spinelli et al., 1996) might be A marked difference was observed between the responsible for wide variations in the antibody level in proportion of ELISA positive and faecal examination the necropsy positive samples as shown by indirect (floatation) negative samples.Sixty eight samples were ELISA.Two false negative results in the present assay found to be positive by ELISA but only twelve samples might be due to low worm burden or poor immune were found positive by faecal examination indicating response of the host (Gasser et al., 1994) ).The and it was obviously much earlier than the time standardized assay offers the potential for its required for the infection to reach the patent period (42 development as one of good diagnostic tools for days in the present study).ELISA is known for its Oesophagostomosis.The use of purified and recombinant potential to detect the antibodies at a quite early stage antigens may minimize the cross reactivity with other of the infection.Nevertheless, it could detect the helminthes, thus these antigens might be a good artificially induced Oesophagostomosis only after 17 candidate for immunization and diagnosis of days of the infection.The indirect ELISA was Oesophagostomosis in ruminants.evaluated on field sera and the results were compared with the post-mortem findings with regard to the Sensitivity = Sample positive by both tests / Samples Serum samples of the infected as well as the control positive by reference test (ELISA) X 100 goats were collected on every third day post-infection (DPI) till 33 DPI, following the standard methods and o they were preserved at -20 C for use in the assay.Antigen Preparation: For preparation of crude Predictive value = Sample negative by both tests / Samples somatic antigen (CSAg) O. columbianum from the negative by target (ELISA) X 100 caecum and colon of freshly slaughtered goats was collected (Johnson et al., 2004) in a Petri dish Results containing 0.15 M phosphate buffer saline (PBS), pH- Specificity = Sample negative by both tests / Samples negative by reference test (ELISA) X 100 Positive Predictive Value = Sample Positive by both tests / Samples Positive by Target test (ELISA) X 100 . Besides, host that ELISA is significantly more sensitive method.nutritionalstatus (Jenkins et al., 1991; Gasser et al., 1992) and physiological and environmental factors 2005) might also have an impact on Diagnosis of gastrointestinal nematode infectionsthe antibody levels.False positive result of the assay has conventionally relied upon detection of the with postmortem negative samples might be due to the clinical signs, aided by qualitative detection of the persistence of antibodies to the past infection, which eggs in the faeces of suspected animals.
Discussionlike re-infection or co-infection with other parasites(Carmena et al.,