Historic emergence, impact and current status of shrimp pathogens in the Americas.

Shrimp farming in the Americas began to develop in the late 1970s into a significant industry. In its first decade of development, the technology used was simple and postlarvae (PLs) produced from wild adults and wild caught PLs were used for stocking farms. Prior to 1990, there were no World Animal Health Organization (OIE) listed diseases, but that changed rapidly commensurate with the phenomenal growth of the global shrimp farming industry. There was relatively little international trade of live or frozen commodity shrimp between Asia and the Americas in those early years, and with a few exceptions, most of the diseases known before 1980 were due to disease agents that were opportunistic or part of the shrimps' local environment. Tetrahedral baculovirosis, caused by Baculovirus penaei (BP), and necrotizing hepatopancreatitis (NHP) and its bacterial agent Hepatobacterium penaei, were among the "American" diseases that eventually became OIE listed and have not become established outside of the Americas. As the industry grew after 1980, a number of new diseases that soon became OIE listed, emerged in the Americas or were introduced from Asia. Spherical baculovirus, caused by MBV, although discovered in the Americas in imported live Penaeus monodon, was subsequently found to be common in wild and farmed Asian, Australian and African penaeids. Infectious hypodermal and hematopoietic necrosis virus (IHHNV) was introduced from the Philippines in the mid 1970s with live P. monodon and was eventually found throughout the Americas and subsequently in much of the shrimp farming industry in the eastern hemisphere. Taura syndrome emerged in Penaeus vannamei farms in 1991-1992 in Ecuador and was transferred to SE Asia with live shrimp by 1999 where it also caused severe losses. White Spot Disease (WSD) caused by White spot syndrome virus (WSSV) emerged in East Asia in ∼1992, and spread throughout most of the Asian shrimp farming industry by 1994. By 1995, WSSV reached the eastern USA via frozen commodity products and it reached the main shrimp farming countries of the Americas located on the Pacific side of the continents by the same mechanism in 1999. As is the case in Asia, WSD is the dominant disease problem of farmed shrimp in the Americas. The most recent disease to emerge in the Americas was infectious myonecrosis caused by IMN virus. As had happened before, within 3years of its discovery, the disease had been transferred to SE Asia with live P. vannamei, and because of its impact on the industry and potential for further spread in was listed by the OIE in 2005. Despite the huge negative impact of disease on the shrimp farming industry in the Americas, the industry has continued to grow and mature into a more sustainable industry. In marked contrast to 15-20years ago when PLs produced from wild adults and wild PLs were used to stock farms in the Americas, the industry now relies on domesticated lines of broodstock that have undergone selection for desirable characteristics including disease resistance.

Application of molecular diagnostic methods to penaeid shrimp diseases: advances of the past 10 years for control of viral diseases in farmed shrimp.

The most important diseases of farmed penaeid shrimp have infectious aetiologies. Among these are diseases with viral, rickettsial, bacterial, fungal and parasitic aetiologies. Diagnostic methods for these pathogens include the traditional methods of gross pathology, histopathology, classical microbiology, animal bioassay, antibody-based methods, and molecular methods using DNA probes and DNA amplification. While methods using clinical chemistry and tissue culture are standard methods in veterinary and human diagnostic laboratories, the former has not been routinely applied to the diagnosis of penaeid shrimp diseases and the latter has yet to be developed, despite considerable research and development efforts that have spanned the past 40 years. No continuous shrimp cell lines, or lines from other crustaceans, have been developed. Hence, when molecular methods began to be routinely applied to the diagnosis of infectious diseases in humans and domestic animals in the mid- to late 1980s, the technology was applied to the diagnosis of certain important diseases of penaeid shrimp for which only classical diagnostic methods were previously available. A DNA hybridization assay for the parvovirus IHHNV was the first molecular test developed for a shrimp disease. This was followed within a year by the first PCR test for MBV, an important baculovirus disease of shrimp. Today, shrimp disease diagnostic laboratories routinely use molecular tests for diagnostic and surveillance purposes for most of the important penaeid shrimp diseases.

Identification of genomic variations among geographic isolates of white spot syndrome virus using restriction analysis and Southern blot hybridization.

White spot syndrome virus (WSSV) is widely distributed in most of the Asian countries where penaeid shrimp are cultured, as well as in some regions of the USA. Six geographic isolates of WSSV-1 each from penaeid shrimp from China, India, Thailand, and the US states of Texas and South Carolina, and 1 isolated from crayfish at the National Zoological Park in Washington, DC-were compared by combining the methods of restriction analysis and Southern blot hybridization. DNA was extracted from purified viruses and then digested with selected endonucleases: AccI, BglII, ClaI, BamHI, EcoRI, HindII, HaeI, SacI and XhoI. The blots were detected with digoxigenin-11-dUTP-labeled WSSV genomic probes: LN4, C42 and A6. No distinctive differences among the 5 WSSV isolates from penaeid shrimp were detected; however, differences in the WSSV isolate from crayfish were observed. A 2.8 kb DNA fragment originating from the crayfish isolate and encompassing the LN4 region was subcloned into pBluescript and sequenced for comparison with the LN4 fragment from the Thailand WSSV isolate. The results indicate that some genomic components of WSSV from different geographic regions share a high degree of homology. This method can be used to distinguish between the WSSV isolate from crayfish and the WSSV isolates from penaeid shrimp.

Reverse transcription polymerase chain reaction (RT-PCR) used for the detection of Taura syndrome virus (TSV) in experimentally infected shrimp.

Taura Syndrome Virus (TSV) has adversely affected the shrimp culture industries of the Americas. First recognized in 1992, this viral agent has spread throughout the shrimp growing regions of South and Central America to become established in North America in the short span of 5 yr. Diagnostic methods for TSV include histopathology, bioassay using susceptible Penaeus vannamei as the indicator species and in situ hybridization with TSV specific complimentary DNA (cDNA) gene probes. An additional method for detecting TSV is through the use of reverse transcription polymerase chain reaction (RT-PCR). Two oligonucleotide primers were selected using the sequence information from a cloned cDNA segment of the TSV genome. The primers, designated 9195 and 9992, used in the RT-PCR procedure amplify a 231 base pair (bp) fragment of the cDNA. Using the RT-PCR technique, TSV has been detected in the hemolymph of P. stylirostris and P. vannamei with experimentally induced TSV infections.

Risk of spread of penaeid shrimp viruses in the Americas by the international movement of live and frozen shrimp.

Within the past decade, viral diseases have emerged as serious economic impediments to successful shrimp farming in many of the shrimp-farming countries of the world. In the western hemisphere, the viral agents of Taura syndrome (TS) and infectious hypodermal and haematopoietic necrosis have caused serious disease epizootics throughout the shrimp-growing regions of the Americas and Hawaii, while in Asia the viral agents of white spot syndrome (WSS) and yellow head (YH) have caused pandemics with catastrophic losses. The international transfer of live shrimp for aquaculture purposes is an obvious mechanism by which the viruses have spread within and between regions in which they have occurred. Shrimp-eating gulls, other seabirds and aquatic insects may also be factors in the spread of shrimp viruses between and within regions. Another potentially important mechanism for the international spread of these pathogens is the trade in frozen commodity shrimp, which may contain viruses exotic to the importing countries. The viral agents of WSS, YH and TS have been found, and demonstrated to be infectious, in frozen shrimp imported into the United States market. Mechanisms identified for the potential transfer of virus in imported frozen products to domestic populations of cultured or wild penaeid shrimp stocks include: the release of untreated liquid or solid wastes from shrimp importing and processing plants directly into coastal waters, improper disposal of solid waste from shrimp importing and processing plants in landfills so that the waste is accessible to gulls and other seabirds, and the use of imported shrimp as bait by sports fishermen.

Development of a non-radioactive gene probe by PCR for detection of white spot syndrome virus (WSSV).

Combining primers created from the sequence information of two baculo-like viruses of penaeid shrimp, Baculovirus penaei (BP) and Monodon baculovirus (MBV), produced a 750 bp band on a 0.8% agarose gel using White Spot Syndrome Virus (WSSV), from Penaeus monodon, as the DNA template. The PCR fragment was ligated to a plasmid vector, (pGEM-T) and transformed, creating a 3.7 Kbp clone. The DNA insert was sequenced, and the original primer pair was located. Using restriction enzymes, the insert was isolated, excised and non-radioactively labeled. This cloned labeled fragment was tested by in situ hybridization for specificity and reactivity with BP, MBV and WSSV-infected shrimp tissues. The major advantage of this novel method of gene probe development is that no DNA sequence information of the targeted infectious agent needed to be known or available. In addition, tedious viral isolation and purification was circumvented. In this study, knowledge of the possible viral strain was important in limiting the PCR primer pairs investigated. The use of arbitrary primers designed for PCR assays from two other possibly related shrimp viruses, increased the likelihood that a generated PCR product would be specific for WSSV.

A minority of muscarinic receptors mediate rabbit tracheal smooth muscle contraction.

To enhance our understanding of cholinergic mechanisms and muscarinic receptors in bronchoconstriction, we have characterized the muscarinic receptor subtypes in rabbit tracheal smooth muscle using radioligand binding and functional assays. The Kd for [3H]quinuclidinyl benzilate ([3H](-)QNB) binding determined from saturation isotherms was 12.6 x/divided by 1.1 pM (geometric mean x/divided by SEM), and the Bmax was 269 +/- 7 fmol/mg protein (arithmetic mean +/- SEM). Competitive inhibition studies with the muscarinic antagonists pirenzepine (PZ), 11[[2-[(diethylamino)-methyl]1-piperidinyl]acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX116), 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP), and hexahydrosiladifenidol (HHSiD) demonstrated heterogeneity of muscarinic receptor subtypes in rabbit tracheal smooth muscle. PZ bound with low affinity to a single receptor site, indicative of an absence of M1 receptors. AF-DX116 (M2 selective) bound with high affinity to approximately 83% of muscarinic binding sites, and 4-DAMP and HHSiD (M3 antagonists) bound with high affinity to approximately 24 and 28% of muscarinic binding sites, respectively. Additionally, direct binding studies with [3H]4-DAMP demonstrated high-affinity binding with 23% of muscarinic binding sites. Thus, the majority of muscarinic receptors in rabbit tracheal smooth muscle bound with high affinity to an M2-selective antagonist, and the remaining receptor sites bound with high affinity to M3 antagonists. The inhibitory effects of atropine, PZ, AF-DX116, and 4-DAMP on methacholine-induced contraction of rabbit tracheal rings were compared. 4-DAMP was a potent inhibitor of methacholine-induced contraction, but PZ and AF-DX116 demonstrated low potency.(ABSTRACT TRUNCATED AT 250 WORDS)