Serodiagnosis of toxoplasmosis in sheep and goats in Erbil city , Iraq

Sera from (259) sheep and (88) goats from Erbil city were examined for antibodies to Toxoplasma gondii by using latex, MAT, and ELISA. By using latex test, 75 (25.4%) sheep and 25 (28.4%) goats were seropositive. By testing the latex seropositive sheep serum by both MAT and ELISA the distribution of both positive and negative results were as such respectively: 63 (84%), 12 (16%), 11 (14.7%), 64 (85.3%). Using both MAT and ELISA for testing the latex seropositive goats serum, the distribution of both positive and negative results were as such respectively: 21 (84%), 4 (16%), 3 (12%), 22 (88%). There was no significant difference between the results of ELISA vs. MAT.


Introduction
Toxoplasma gondii is an intracellular protozoan organism with a large numbers of intermediate hosts, including all warm-blooded animals, and humans.Felids, particularly the domestic cats, are definitive hosts and the only animal species in which oocyst develop (1,2).Serological surveys done in various parts of the world show that in some countries more than a third of the human population have antibodies against T. gondii (3).This high prevalence of infection in human proves the importance of toxoplasmosis as a zoonotic disease, particularly in pregnant women and immune-compromised patient (4).Toxoplasmosis also causes heavy economic losses to sheep industry worldwide (5).Toxoplasmosis is recognized as one of major cause of infectious reproductive failure in Great Britain, New Zealand, Australia, USA, Uruguay, Norway and other countries (1,2,4,5).Toxoplasmosis was also reported in other parts of Iraq, and in different animals, in sheep and goats (6), in cat (7), in rodent (8) and in cattle (9).About the neighboring countries toxoplasmosis was also a prevalent infection in animals like in Iran (10), in Saudi Arabia (11), and in Jordan (8).Toxoplasmosis causes fetal resorption, abortion at any stage of pregnancy, fetal mummification, stillbirth or birth of weak live offspring in sheeps and goats (12).
The objectives of this study was to determine the prevalence of IgG and IgM in the latex positive sheep and goats sera by using ELISA and MAT (modified agglutination test).

Study protocol and design
This study was carried out during the period November 2009 to April 2010 at the central veterinary laboratory in Erbil city (Iraq).This study was planned by using two stages of simple random sampling procedure (10,11).In the first step, randomly selection of owner or butchers who had given their acceptance, followed by random selection of animals in second step.Five ml blood was taken from jugular vein from each animal (sheep, goat) at the slaughter house.Three hundred eighty three (383) blood samples were collected which were included (295 sheep and 88 goats serum).Each serum was divided into three parts primarily.The first part was examined serologically for detection of antibody to Toxoplasma gondii by (LAT).In the case of the positive latex result, the second part of the serum was tested by MAT while the third part was tested by ELISA method.

Serological tests Latex agglutination test
Determination of Toxoplasma gondii antibody by latex agglutination test, using the kit (Toxocell latex, BIOKIT, S. A. BARCELONA -SPAIN).The principle of the test as follows: If specific anti-Toxoplasma antibodies are present in sera tested they will react with soluble Toxoplasma antigen in the latex reagent and will be visualized by latex particles as whitish granules after five minutes of rotation of the slide.

Modified Agglutination test (MAT)
Treatment of latex positive serum with 2-Mercapto ethanol, (2ME SIGMA-ALDRIC -US A,Bach.125 K 0165) for diagnosis of acute (IgM) or chronic phases of toxoplasmosis (IgG).A stock solution of (0.2) molarities of 2ME in phosphate buffer saline is prepared initially and stored at 4°C in an opaque bottle.It is further diluted to 0.05M with normal saline for use as the diluted in the test.Equal amount of 50 µl of the positive serum with LAT was treated with 50 µl of 2ME then incubated at 37°c for 15 minutes (13).

ELISA
Indirect ELISA (indirect Multi-species, IDVET innovative diagnostics -FRANCE).This kit is used to detect anti-Toxoplasma gondii antibodies (IgM) in ruminant, cat or pig sera, plasma or meat juice.The test is validated if: -the mean value of the positive control O.D is greater than 0.350.-the ratio of the mean O.D values of the positive and negative controls (ODPC and ODNC) is greater than 3.5.All the serological tests above were done in the central veterinary laboratory in Erbil city.
The data analysis was performed by Chi-Square test using Statistical Analysis System (SAS) software program.The differences were considered statistically significant when P ≤ 0.05.

Discussion
This study is an attempt for serodiagnosis of Toxoplasma gondii infection in Erbil, using serological methods including latex agglutination test, modified agglutination test and ELISA techniques in slaughtered animals used for human consumption.The procedures used were LAT, MAT and ELISA.The choice of ELISA technique for this study is justified since it is considered to be one of the most sensitive immunological technique (14).A study followed the kinetics of IgG and IgM antibodies in 20 sheep fed oocysts, T. gondii IgM antibodies peaked at 3 weeks and precedes IgG response (15).In sheep inoculated intravenously with tachyzoites, IgM was detected by one month and persisted for three months (16).A study had reported that two sheep fed 5000, 50000 oocysts develop IgM antibodies detected by ELISA by 14 days (17).
The MAT and ELISA tests detected similar proportion of Toxoplasma positive serum samples.Therefore, both are reliable for screening tests.However, both tests have their own advantages and limitations.The need for species specific conjugates, and automatic processor to increase the efficacy and spectrophotometer for quantifying the activity of antibodies by ELISA test may limit its use.On the other hand, the MAT is safe and does not require species specific conjugate and can be used on any species (18).It was noted that that standardization of ELISA is a major problem because only a few kits are commercially available for use in animals (19).The diagnostic accuracy of these two tests (MAT, ELISA) were superior when performed on sheep (20).Unlike ELISA, which usually is developed to be used on particular species, MAT is commonly used to test sera from any species because it does not require species specific conjugates.The agreement between both tests (MAT and ELISA) was almost perfect and the accuracy would not justify by themselves the selection of either MAT or ELISA for its use in toxoplasma control schemes.Other aspects such as possibility of standardization, rapidity, economy should be considered as well (19).MAT is simpler to perform but involves more subjectivity when interpreting the results than the ELISA.ELISA can be easily automated, enabling the rapid screening of a large number of animals.However, they should be valuable tools for prevalence studies at flock or population levels (21).