The greatest risk to the viability and expansion of the global shrimp farming industry is disease, estimated to cause annual losses to shrimp production exceeding US$3 billion. Therefore, proper disease management is crucial to mitigate these losses. Currently, the primary method for managing disease risk involves the use of specific pathogen-free (SPF) or specific pathogen-resistant/tolerant (SPR/T) stocks to produce high-health postlarvae combined with biosecurity and hygiene.
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These strategies rely heavily on access to accurate, rapid, and sensitive pathogen detection methods such as quantitative and real-time PCR (qPCR) assays. PCR (Polymerase Chain Reaction) is a technique that aims to produce a large number of copies of target DNA in vitro based on thermal cycles. And many PCR assays have been developed to detect all major shrimp pathogens. In cases requiring non-destructive testing – such as screening broodstock for the presence of viruses and infected samples – shrimp gill, pleopod, or hemolymph samples are virtually the only practical sources for testing.
Gill-associated virus (GAV) – also known as yellow head virus genotype 2 (YHV2) – is widespread in wild and farmed black tiger shrimp (Penaeus monodon) in Australia. In Australia, there is growing interest in establishing GAV-free or GAV-resistant P. monodon breeding populations. Both require accurate detection and quantification of GAV infection in samples, and gill or pleopod tissues are commonly used for RT-PCR analysis. However, the extent to which GAV infection levels can vary between different gill filaments or pleopods sampled from the same shrimp remains largely undetermined.
This article summarizes the results of a study using RT-qPCR analysis to accurately quantify GAV infection levels in different gill filaments and pleopods sampled from individual P. monodon with natural infections, to compare sensitivity and variability in either tissue type. To understand how GAV can vary in natural infection scenarios, rather than artificial infections, such as screening shrimp from grow-out ponds or wild broodstock – the researchers used naturally infected shrimp. The researchers also sampled both lymphoid organ lobes from a subset of adult P. monodon to compare the sensitivity of GAV detection in gill and pleopod tissues, compared to the lymphoid organ, which is the recommended target tissue for GAV identification.
The lead author of the study was supported by an Australian Government Research Training Program Scholarship, and funding for this research was sourced from the Australian Research Council Industrial Transformation Research Program IH130200013.
Study Setup
The researchers sampled tissues from two independent groups of P. monodon cultured at the Bribie Island Research Centre (BIRC), Queensland, Australia. Group 1 consisted of 10 small shrimp (average weight 10.9 ± 1.4 grams), and eight gill filaments and eight pleopods were sampled from each shrimp. Group 2 had 12 adult shrimp (average weight 40.4 ± 2.3 grams), and 10 gill filaments, 10 pleopods, and both lymphoid organ lobes were sampled from each. Tissues were preserved until processing.
The coefficient of variation (CV) was used to determine whether gill or pleopod tissues differed in the degree of variability of GAV infection detected between individual gill filaments and pleopods sampled from each shrimp.
Results and Discussion
The management of various diseases affecting farmed shrimp relies on reliable and accurate methods for detecting and quantifying infection levels. Our study used RT-qPCR to quantify and compare GAV infection levels among individual gill filaments, pleopods, and lymphoid organ lobes sampled from naturally GAV-infected black tiger shrimp.
The accuracy of technical replicates was generally high, except for some samples with very low GAV infection levels and more variable standard deviation values. Reduced accuracy can affect the precision of GAV infection detection and quantification.
GAV infection levels detected in individual gill filaments and/or pleopods sampled from each shrimp often varied by more than 10-fold, and in some shrimp, up to approximately 3,000-fold. This variability was evident in both P. monodon groups regardless of the severity of GAV infection. Similar variability in infection levels, including false negatives, has been recognized among pleopod samples taken from Pacific white shrimp (Penaeus vannamei) with low-level White Spot Syndrome Virus (WSSV) infections, and can occur with other shrimp pathogens.
The researchers' results highlight the potential for infectious pathogens to be overlooked when using single tissue samples, especially in shrimp with low-level natural infections. It is difficult to predict the minimum required number of gill or pleopod samples, but based on the small shrimp in this study, for example, the minimum number of gill or pleopod samples needed to achieve 100% detection was five and seven samples, respectively. In adult shrimp, a single tissue sample would be sufficient if all samples tested positive, emphasizing the specificity of a minimum sample number for the tested groups.
Misdiagnosis of disease can have serious consequences in black tiger shrimp breeding programs where wild broodstock are often selected for breeding based on negative PCR tests for specific pathogens (SPF). The researchers' data suggest that testing a single gill filament or pleopod can increase the risk of false negatives for disease and overlooking infections, leading to vertical transmission of pathogens to postlarvae and subsequently to grow-out ponds or other SPF broodstock.
For broodstock, performing multiple consecutive PCR tests may be more appropriate, ideally using more than one tissue sample, during the quarantine of wild broodstock before they are selected for use in SPF breeding programs. Alternatively, increasing the number of shrimp tested can enhance the accuracy of infection prevalence estimates when testing at the population level.
Large variability in GAV infection can occur between gill filaments or pleopods in some shrimp – in addition to potential false negatives – which can generate inaccurate data on infection levels and may underestimate the severity of infection when sampling a single gill filament or pleopod. This can affect the accuracy of relative comparisons between individual shrimp, because without accurate data on the infection level of each shrimp, the ability to estimate any genetic contribution to differences between individuals or families will be severely lacking.
GAV infection levels quantified in each lymphoid organ lobe from Group 2 adult shrimp were significantly higher (435- to 856-fold) and showed very little variation compared to pleopods or gill filaments from the same shrimp, consistent with previous analyses of GAV-infected P. monodon detecting higher GAV concentrations in the lymphoid organ.
Although the lymphoid organ is the optimal tissue sample for detecting GAV susceptibility, there are cases where sampling this organ is not suitable, as it requires sacrificing the shrimp (often not an option in breeding situations), is difficult to sample in small animals, and can be very challenging to locate and dissect in high-throughput screening. Therefore, alternative tissues such as gills and pleopods are commonly used because they can be sampled without harming the shrimp and are effective in most scenarios.
Therefore, the researchers' data suggest that neither tissue is inherently more advantageous in providing accurate data on GAV infection levels, so the decision of which tissue type (pleopod or gill) to sample depends on the most suitable collection and laboratory procedures. Differences in total RNA isolated per microgram of tissue may exist and affect relative comparisons between tissue types.
Assessment
Our study results for naturally GAV-infected shrimp demonstrated that infection levels can vary significantly between different gill filaments and pleopods sampled from the same shrimp. Therefore, testing a single sample of these tissues may underestimate the severity of the disease or even lead to misdiagnosis of an infected shrimp.
When lymphoid organ sampling is not feasible, testing pooled tissue samples from two or more gill filaments/pleopods is recommended to overcome this variability and generate more accurate data on the presence and severity of GAV infection.
HNN (Source: GAA)
