Application of PCR for improved shrimp health management in the Asian region

Objective 1 Reduce the risk of WSD in shrimp farms through the application of PCR-based detection tests and epidemiological probes.
A major experimental component of this project is a large longitudinal study of shrimp ponds in India. The aim is to obtain information on the quality of PCR screening results available to farmers and to use molecular epidemiological analysis to trace the sources of disease outbreaks in ponds. The longitudinal study was conducted at a NACA/MPEDA/ACIAR study area in the West Godavari District of Andhra Pradesh. The site comprises 27 farm clusters around 14 villages between Bhimavarum and the mouth of the Godavari River. Farmers participating in the study are members of "aquaclubs" formed as part of an ongoing extension program of best management practices. The farms were stocked in February - April 2005 with PCR-screened post-larvae (PLs) obtained from local hatcheries. Some farmers employed nursery ponds to improve survival rates during grow-out. Each nursery pond served to seed a number of grow-out ponds at the same location. A total of 27 nursery ponds were sampled on stocking; 19 of these were also sampled at harvest. A total of 457 grow-out ponds were sampled at the time of stocking. Samples were also collected during disease outbreaks from 14 ponds, emergency harvests from 52 ponds, and planned harvests from 277 ponds. Wild shrimp were collected from 23 ponds and crabs from 65 ponds during the period between stocking and harvest.

The shrimp samples were preserved in alcoholic fixative for subsequent analysis by PCR. Other samples collected from disease outbreaks and emergency harvest ponds were preserved in Davidson's fixative for histological analysis. All samples were transported to CIBA in Chennai and catalogued. Samples for PCR analysis were divided into two equivalent sets. Testing of samples in the first set will be conducted at CIBA and the College of Fisheries in Mangalore. The second set of samples will be transported to CSIRO in Australia. All histology samples were retained for preliminary analysis at CIBA. Laboratory analyses on all of these samples will be reported during the next period.

Objective 2 Reduce the risk of yellow head and other diseases in shrimp farms through the application of PCR-based detection tests and epidemiological probes.

Monodon slow growth syndrome (MSGS) is a newly emerging disease that has impacted severely on P. monodon production in Thailand with losses in 2004 estimated at ~40 million baht. Work at Centex Shrimp in Thailand has suggested that the disease is infectious. Examination of shrimp displaying signs of the disease has identified three infectious agents - a yellow head virus genotype, a new shrimp virus (Laem Singh virus - LSNV) and a microsporidium. It is not yet clear which, if any, of these agents is the primary cause of MSGS. LSNV has been partially sequenced and a PCR detection test developed. Using this test, the virus can be detected in healthy shrimp as well as those showing signs of slow growth. In June-July 2005, Dr Sitidilokratna of Centex Shrimp visited CSIRO to apply the PCR detection test to P. monodon samples from throughout the Indo-Pacific region. LSNV was detected in shrimp from Thailand, Malaysia and Indonesia but not in samples from Australia, India, Vietnam, Mozambique or Fiji.

Objective 3 Improve the effectiveness of PCR-based viral screening in hatcheries and service laboratories in India. Indonesia and other countries in the Asian region.
The project also aims to enhance technical capabilities in India and Indonesia through PCR training workshops and the inter-calibration of PCR testing performance between laboratories. The first PCR training workshop was held in Bogor, Indonesia in August 2005. The workshop was attended by 24 participants from laboratories throughout the Indonesian archipelago. A second PCR training workshop was held at CIBA in India in October 2005 with financial support from MPEDA. The workshop was attended by 25 participants from hatcheries and government, private and research laboratories in seven States, and 3 international participants (Sri Lanka, Bangaldesh and Myanmar) supported by ACIAR. Each workshop included practical sessions and an open Seminars which were attended by representatives from the research, government and industry sectors.

Mr Agus Sunarto and Mrs Isti Koesharyani from the Fish Health Research Laboratory, Jakarta, and Mrs Christina Handayani from the Center for Brackishwater Aquaculture Development, Japara, Indonesia visited AAHL in July 2005. These scientists assisted with preparation for the training workshop in Bogor and participated as trainers in the workshop. They were also instructed in the use of real-time PCR during the visit to AAHL. Dr Nursa Sittidilokratna of Centex Shrimp, Thailand visited AAHL in June-July 2005 to work with CSIRO staff on studies of LSV. Dr Sitidilokratna also advised on the implementation of training workshops and participated as a trainer.

This project aims to reduce risks of disease outbreaks in small-holder shrimp farms by a combination of strategies involving training of PCR laboratory technicians, the inter-calibration of PCR laboratories test performance and improved health management practices derived from a better understanding of sources of white spot disease on farms. A major aspect of the project is a large field study at a site in Andhra Pradesh, India. The study is the first in a series of investigations that aim to assess the quality of PCR screening available to small holder shrimp farmers and to identify the sources of disease outbreaks in shrimp ponds. The study involved a total of 457 ponds from 28 clusters in 15 villages. The farms were stocked in early 2005 with PCR-screened PLs obtained from local hatcheries. Samples of shrimp post-larvae (PLs) were collected from farmers at the time of stocking and from juvenile shrimp and wild crustaceans from the ponds during grow-out and at the time of disease outbreaks and planned or emergency harvest. Samples collected from the site were tested by laboratory staff in India and Australia by PCR to identify infected stock and assess the source of infection. Samples from disease outbreaks were also examined by histology to determine the cause of disease. The results indicate that very few of the PL batches used by the farmers in the study were positive by PCR tests conducted in India and Australia by the project team, suggesting that PCR screening conducted by local laboratories was relatively effective. However, PCR testing of samples collected from shrimp at planned or emergency harvest indicated there was a very high prevalence of WSSV infection in the study ponds with a high proportion of moderate-severe infections. There was also evidence that a small number of nursery ponds were the source of seed for a high proportion of disease outbreaks during grow-out. There was also evidence of clustering according to the village under study with 2 villages in particular being major foci of infection and disease. The data suggests that nursery ponds may be a weak link in the current disease management practice.

Work has also continued in the search for infectious agent associated with monodon slow growth syndrome (MSGS), a newly emerging disease that has impacted severely on P. monodon production in Thailand. At Centex Shrimp in Bangkok, a new shrimp virus (Laem Singh virus - LSNV) has been investigated. Although LSNV occurs commonly in P. monodon in Thailand, it is thought that progression of the infection in the optic nerve may be the cause of slow growth. To determine if LSNV also occurs in India, 205 samples collected in the field study in Andhra Pradesh were screened for the presence of the virus using PCR test developed at Centex Shrimp. Although no evidence of infection was detected, we have concerns about the stability of the LSNV RNA after prolonged storage in ethanol and new set of samples will be collected in early 2007.

To improve the reliability of PCR-based screening, the first of two inter-laboratory calibrations of WSSV PCR testing was conducted in India in June 2006. Forty-nine service laboratories from the government and private sectors and shrimp hatcheries received equivalent sets of randomly coded positive and negative samples comprising fixed shrimp tissues WSSV DNA for testing. Seventeen laboratories correctly identified all positive and negative samples and 3 laboratories failed to detect only one low positive sample. Six laboratories reported negative results for positive samples indicating problems with test sensitivity. Nine laboratories reported positive results for negative samples indicating problems with test contamination. Two laboratories reported incorrect results for both positive and negative samples. The results were collated returned to all participating laboratories is a form that allowed them to see the results of all laboratories but identify only themselves by code. A second inter-calibration is planned following the second PCR training workshop in early 2007 and it is expected that a national PCR laboratory accreditation program will commence in 2008. A similar inter-calibration commenced in Indonesia in December 2006.

The second in a series of 2 PCR training workshops has been completed in India. The second workshop was conducted in October 2006 for essentially the same group that attended the first workshop in 2005 including 23 from hatcheries and government, private and research laboratories in India and 3 participants from Sri Lanka, Bangaldesh and Myanmar. The second workshop provided more focussed training and assessment with each participant required to perform every step in the process at least once during the 4-day program and feedback was given on the basis of individual rather than group performance.