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Inter-calibration of white spot syndrome virus in India

INDIA - The elimination of infected seed prior to stocking is arguably the most important single factor in reducing the risks of diseases in shrimp farming within India and throughout the region.

Packing samples for ring testing.

White spot syndrome virus in India
The elimination of infected seed prior to stocking is arguably the most important single factor in reducing the risks of diseases in shrimp farming within India and throughout the region. This can be achieved by the proficient PCR testing of broodstock and/or seed. In fact in many countries around the region PCR is now used to screen shrimp seed for white spot syndrome virus or WSSV prior to stocking. However, white spot disease (WSD) continues to seriously impact shrimp production and it is suspected that variations in the reliability of screening results, compounded by on-farm factors, may result in the outbreak of disease even when seed has been properly screened. One of the main reasons for this has been a lack of harmonization or an inter-calibration of the PCR testing capabilities of different service providing laboratories. This variance in the quality of testing and accuracy of results has contributed towards an erosion of the confidence shrimp farmers have in laboratory PCR testing.

First PCR training workshop
Under the ACIAR funded Regional Project - Application of PCR for improved shrimp health management in the Asian region – being implemented since January 2005, a 5 day PCR training workshop was jointly organized by MPEDA, CIBA-ICAR, CSIRO and NACA from 17-21 October 2005, at CIBA, Chennai, India. A total of 28 participants representing mainly the private and government PCR service providing laboratories were trained. The training workshop had lecture and practical components. The course curriculum followed a format successfully implemented by ACIAR and CSIRO in several Asian countries and further developed at Mahidol University, Thailand. The participants of the I PCR training workshop also agreed on the modalities for the conduct of I PCR inter-calibration exercise.

First PCR inter-calibration
Under the framework of the ACIAR funded regional project a voluntary PCR inter-calibration programme was developed and executed in June 2006. The technique, which is also known as ring testing aimed to provide an overview of the current quality of WSSV PCR testing in participating laboratories in India. The approach would identify which laboratories might require more assistance in improving their testing procedures and offer individual laboratories the opportunity to compare their results with other laboratories undertaking PCR testing for WSSV. In effect, inter-calibration not only provided a step towards accreditation but also gave participants an opportunity to assess their own performance.

Briefly, 100 sets of ten samples (1,000 samples) were prepared and coded by scientists of CSIRO and CIBA. These included 100 sets of five DNA samples and 100 sets of five tissue samples. This preparation and coding of samples took considerable planning and required two weeks of meticulous work by scientists from CSIRO, Australia and CIBA, Chennai. The DNA samples were derived from WSSV experimental infection in adult Fenneropenaeus indicus, using infected material from a hatchery near Chennai. Batches were sent to forty-nine laboratories throughout India with five batches sent to CSIRO in Australia and six batches in CIBA, India.

Participation in the inter-calibration exercise was voluntary and the results of all testing remained strictly confidential, a fact that was key to the success of the exercise. Participating laboratories were identified by a code number. Throughout the trials the code numbers remained private with participating laboratories only informed of their own identification number. Once completed the laboratories returned their results which were collated into a summary table. Individual laboratories could then view their own results and compare these with results from other facilities; however the confidential nature of the test results meant that the names of the laboratories would never be identified.

The overall performance of the participating laboratories was rated as good with over half of the participating laboratories returning either excellent or acceptable results. This is very reassuring and should help to boost the confidence of the farmers and hatcheries in the seed testing process implemented in India. However, some of the labs reported positive reactions in negative samples (indicative of problems with test contamination) while some have failed to detect positive samples (indicative of a problem with test sensitivity). This is a cause for concern and needs to be addressed on a case by case basis through assistance with resourcing, technical advice and training. The participating labs have been requested to review their individual results, compare the performance with other labs and contact relevant institutions for technical assistance.

2nd PCR Training Workshop
The second PCR training workshop, was held recently in Chennai from 23-26 October 2006. This training workshop was attended by 26 participants (23 from India and one each from Bangladesh, Myanmar and Sri Lanka). The same set of participants had attended the first PCR training workshop completed last year. The training programme provided an opportunity to participants through hands-on training, to further improve their skills in performing PCR. To ensure effective learning and uptake, practical were conducted in 5 small batches of 5 participants and each batch was provided with one expert demonstrator. All the participants were provided with individual operator IDs. A total of 1,056 PCR reactions were run by the participants over a period of four days. Each participant had an opportunity to run over 40 PCR reactions with different types of samples and primers.

2nd PCR inter-calibration
A half day workshop on PCR laboratory accreditation attended by training workshop participants was held on 27th October 2006 at CIBA, Chennai. The workshop provided an opportunity to discuss the results of the first PCR inter-calibration exercise, and also to agree on the modalities for running the second PCR inter-calibration exercise. The second PCR calibration is presently scheduled for January 2007. The PCR laboratory workshop participants agreed that participation in the second PCR calibration exercise would be on a voluntary basis and the identify of the labs should be kept confidential.

Way forward
MPEDA is keen to develop and implement a PCR laboratory accreditation programme in India and is likely to become operational before the end of 2007. The purpose of the two PCR training workshops and two PCR inter-calibration exercises, under the umbrella of the ACIAR regional project is to prepare the PCR laboratories for participation in a future PCR laboratory accreditation programme.

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