Optimizing Shrimp Fixation Methods for the Detection of TSV

Donald V. Lightner, PhD., Professor
Ken Hasson, Graduate Research Associate

Proper fixation of penaeid shrimp samples, when utilizing Davidson's AFA fixative, is crucial to obtaining optimum histological sections for disease analysis and diagnosis. The protocol for preparing Davidson's fixative and proper fixation technique for shrimp can be found in Bell and Lightner (1988, A Handbook of Normal Penaeid Shrimp Histology, World Aquaculture Society) and Lightner (1996), A Handbook of Pathology and Diagnostic Proceedures for Diseases of Penaeid Shrimp, World Aquaculture Society). If you do not have access to either of these publications click HERE or, we can fax or mail copies of the sections with the above mentioned protocols at your request. The purpose of the present letter is to briefly review sample fixation times and shipping of preserved samples for disease diagnosis. These protocols should be applied to all shrimp that are preserved with Davidson's fixative. Following the suggested guidelines outlined below is of critical importance for samples that will be analyzed for the presence of RNA viruses such as Taura Syndrome virus (TSV) or Yellowhead virus (YHV).

In-situ hybridization analyses of histological sections of TSV infected Penaeus vannamei have shown that the viral RNA genome is completely degraded in samples stored for one week or longer in Davidson's fixative or Davidson's fixed samples that have been stored for more than a week in ethanol. As a result, TSV genomic probes cannot bind to the viral RNA and no signal is detected in samples that display obvious pathognomonic TSV lesions by routine histology. Degradation of TSV RNA is caused by the high acidity of Davidson's fixative (pH 3.5 to 4), resulting in a chemical process known as acid hydrolysis. Acid hydrolysis of RNA occurs at a pH of less than 5.5 and causes the cleavage of single stranded RNA into individual nucleotides. As a result, genomic probes cannot hybridize to the degraded RNA molecules and false negative in-situ hybridization reactions are produced. As YHV is also an RNA virus, degradation the the viral genome will also occur in YHV infected shrimp during long term storage in Davidson's fixative or in ethyl alcohol after fixation in Davidson's fixative.

If your laboratory is working with Davidson's preserved penaeid shrimp or you plan on submitting samples to the University of Arizona for histological or gene probe analysis, we recommend the following:

1. Following injection (or submersion in the case of postlarval and larval stages) of the sample with Davidson's fixative following standard methods, shrimp less than or equal to 6 grams should be stored in Davidson's for 24 hours and then transferred to a 50 to 70% ethanol solution (especially postlarvae samples). Frequent changes of the ethanol storage solution should be made to maintain the pH between 5.5 to 6.5 (measure with pH paper) and the samples embedded in paraffin as soon as possible. Samples exceeding 6 grams should be maintained in Davidson's solution for 48 hours post fixation and then treated as described above.
   
2. For laboratories with histological capabilities, samples should be mid-sagitally bisected after the appropriate fixation period and submerged in 50 to 70% ethanol (pH 6.5 to 7.0) to permit leaching of the Davidson's from the tissues. Frequent changes of the ethanol storage solution should be made to maintain the pH between 5.5 and 6.5 and the samples embedded as soon as possible. It is recommended that the dissected tissues be submerged for a minimum of 5 hours in ethanol prior to dehydration and embedding.
   
3. For clients submitting samples to the University of Arizona for disease analysis, we recommend that the samples be shipped to us in 50 to 70% ethanol as rapidly as possible after the prescribed time in Davidson's. Samples should not be shipped in Davidson's fixative. Following the protocol described in paragraph 1 above will reduce acid hydrolysis problems. However, all samples that were preserved with Davidson's fixative will still retain the fixative within the tissues and degradation of RNA due to hydrolysis will continue during storage in ethanol. As a result, we want to emphasize the importance of shipping samples (in ethanol) suspected of being infected with TSV or YHV to us as quickly as possible so that they can be processed. Dehydration and embedding of samples in paraffin for sectioning appears to halt RNA hydrolysis.

If you have any questions or concerns, please feel free to contact by the following methods, before preserving your samples:

FAX: (520) 621-4899
Voice: (520) 621-8414
E-mail: aquapath@ag.arizona.edu
Aquaculture Pathology, University of Arizona, Department of Veterinary Science and Microbiology, Veterinary Science/Microbiology Building 90, Room 202, 1117 E. Lowell St., Tucson, AZ 85721 USA

Aquaculture Vol. 193 (1-2) pp. 1-9
Taura syndrome in México: follow-up study in shrimp farms of Sinaloa 
a Martha Zarain-Herzberg
b Felipe Ascencio-Valle

a Centro de Ciencias de Sinaloa, , Culiacán, Sinaloa 012121, Mexico
b Centro de Investigación Biológica del Noroeste, , La Paz, Baja California 021121, Mexico

The Taura syndrome (TS) is one of the viral shrimp diseases that has most affected cultivation of the shrimp Litopenaeus vannamei in America. We analyzed the presence of TS, from its first detection in May 1995 in 75% of the shrimp farms of the Guasave district in Sinaloa State, México, and its progressive spreading from the north toward the central and southern zones of the state, to the districts of Navolato and Elota. The main histopathological findings in shrimp tissues analyzed were necrotic areas in the cuticular epithelium, near the sites where there is melanization, and in various parts of the surface of the body, appendages, gills, hindgut, esophagus, and stomach. As revealed with hematoxylin and eosin--phloxine staining, the cuticular lesions were generally spherical and with cytoplasmic inclusion bodies. Some of the affected cellular nuclei of the tissues were pycnotic or karyorrhectic, giving a "buckshot" appearance to the lesions. During 1996, shrimp samples were collected from 33 shrimp farms in different areas of Sinaloa. The histopathological analysis revealed a 92% prevalence of TS in the central zone, 78% TS in the southern zone, and 73% TS in the northern zone. In 1997, histopathological analysis of shrimp samples collected in the same shrimp farms revealed a decrease in TS. In 1998, a significant reduction in the prevalence of TS was observed, with only 30% incidence of TS in the farms sampled. We concluded that the epidemic of TS in shrimp farms of Sinaloa reached a peak in 1996, followed by a steady decline that closely paralleled the switch by the industry from culturing TS virus (TSV)-susceptible L. vannamei to TSV-resistant L. stylirostris.