Linda M. Nunan

Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp.

A new emerging disease in shrimp, first reported in 2009, was initially named early mortality syndrome (EMS). In 2011, a more descriptive name for the acute phase of the disease was proposed as acute hepatopancreatic necrosis syndrome (AHPNS). Affecting both Pacific white shrimp Penaeus vannamei and black tiger shrimp P. monodon, the disease has caused significant losses in Southeast Asian shrimp farms. AHPNS was first classified as idiopathic because no specific causative agent had been identified. However, in early 2013, the Aquaculture Pathology Laboratory at the University of Arizona was able to isolate the causative agent of AHPNS in pure culture. Immersion challenge tests were employed for infectivity studies, which induced 100% mortality with typical AHPNS pathology to experimental shrimp exposed to the pathogenic agent. Subsequent histological analyses showed that AHPNS lesions were experimentally induced in the laboratory and were identical to those found in AHPNS-infected shrimp samples collected from the endemic areas. Bacterial isolation from the experimentally infected shrimp enabled recovery of the same bacterial colony type found in field samples. In 3 separate immersion tests, using the recovered isolate from the AHPNS-positive shrimp, the same AHPNS pathology was reproduced in experimental shrimp with consistent results. Hence, AHPNS has a bacterial etiology and Kochs Postulates have been satisfied in laboratory challenge studies with the isolate, which has been identified as a member of the Vibrio harveyi clade, most closely related to V. parahemolyticus.

The detection of White Spot Syndrome Virus (WSSV) and Yellow Head Virus (YHV) in imported commodity shrimp

Transmission of exotic pathogens occurs through a variety of means, including migration with humans and animals, rapid transit by land, sea or air or through the shipment of infected frozen food products. White Spot Syndrome Virus (WSSV) and Yellow Head Virus (YHV) have caused mass mortalities of cultured shrimp in Asia beginning in 1992. In 1995, these viruses appeared for the first time in the Western Hemisphere causing high mortalities in farm reared shrimp in Texas, USA. The purpose of this study was to determine if WSSV and YHV are present in frozen shrimp products imported into the United States from Asia. Infectivity assays, transmission electron microscopy (TEM), and polymerase chain reaction (PCR) showed these viruses were detectable and infectious in frozen shrimp imports. Frozen shrimp were used to infect indicator shrimp (Penaeus stylirostris) which resulted in mortalities. The cause of these mortalities was determined by histology and TEM to be by YHV. PCR confirmed the presence of WSSV in the frozen, purchased products. The results from this study indicate that exotic shrimp pathogens can be transmitted via imported frozen products.

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.

Detection of acute hepatopancreatic necrosis disease (AHPND) in Mexico.

Acute hepatopancreatic necrosis disease (AHPND), which has also been referred to as early mortality syndrome (EMS), initially emerged as a destructive disease of cultured shrimp species in Asia in 2009. The pathogen associated with the disease, Vibrio parahaemolyticus, subsequently spread to the Western Hemisphere and emerged in Mexico in early 2013. The spread to the Western Hemisphere is a major concern to shrimp producers in the region. To date, the only peer-reviewed published method for determining whether mortalities are due to AHPND is through histological examination. A novel PCR detection method was employed to assess samples from Mexico in order to confirm the presence of the pathogen in this country. This manuscript details the detection methods used to confirm the presence of AHPND in Mexico. Both immersion and per os challenge studies were used to expose the Penaeus vannamei to the bacteria in order to induce the disease. Histological analysis confirmed AHPND status following the challenge studies. Also provided are the details of the molecular test by PCR that was used for screening candidate V. parahaemolyticus isolates. A rapid PCR assay for detection of AHPND may help with early detection and help prevent the spread of AHPND to other countries.

Use of Polymerase Chain Reaction for the Detection of Infectious Hypodermal and Hematopoietic Necrosis Virus in Penaeid Shrimp.

Abstract: A rapid and reliable polymerase chain reaction (PCR) method was developed for the detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in penaeid shrimp. The oligonucleotide primers amplify a 1681-bp fragment of IHHNV, which encompasses the coding sequence for one of the viral coat proteins. The PCR method detects IHHNV in hemolymph and homogenized tissue obtained from the cephalothorax or pleopods of infected shrimp. The technique was also successfully applied to tissue samples preserved in 70% ethanol. The correct size fragment was amplified using IHHNV obtained from six different geographic regions in three different species of penaeid shrimp. No DNA extraction method was necessary for this technique. The use of hemolymph or pleopods provides a nondestructive screening method by which to test juveniles and adult broodstock for the presence of IHHNV.

Central δ‐opioid receptor interactions and the inhibition of reflex urinary bladder contractions in the rat

1 The in vivo effects of a number of opioid agonists and antagonists were studied on the spontaneous reflex contractions of the urinary bladder recorded isometrically in the rat anesthetized with urethane. All substances were administered into the central nervous system by the intracereboventricular (i.c.v.) or spinal intrathecal (i.t.) route. 2 The conformationally restricted enkephalin analogues [2‐D‐penicillamine, 5‐L‐cysteine] enkephalin (DPLCE), [2‐D‐penicillamine, 5‐L‐penicillamine] enkephalin (DPLPE) and [2‐D‐penicillamine, 5‐D‐penicillamine] enkephalin (DPDPE) produced dose‐related inhibition of reflex bladder contractions when administered by the i.c.v. or i.t. route. 3 Both the novel δ‐opioid receptor antagonist ICI 154,129 (200–600 μg) [N,N‐bisallyl‐Tyr‐Gly‐Gly‐ψ‐(CH2S)‐Phe‐Leu‐OH) and ICI 174,864 (1–3 μg) [N,N‐diallyl‐Tyr‐Aib‐Aib‐Phe‐Leu‐OH: Aib = α‐aminoisobutyric acid] attenuated or abolished the effects of DPLCE, DPLPE and DPDPE when administered by the i.c.v. or i.t. route. The antagonism observed was selective since the equipotent inhibition produced by the μ‐opioid receptor agonist [D‐Ala2, Me‐Phe4, Gly(ol)5] enkephalin (DAGO) was unaffected. Overall, ICI 154,129 was considerably weaker than ICI 174,864 and both antagonists inhibited bladder activity at doses higher than those required to demonstrate δ‐receptor antagonism. 4 Further studies of the agonistic effect of ICI 174,864 showed that it was insensitive to low doses of naloxone (2 μg, i.c.v. or i.t.) but could be abolished by higher (10–15 μg) doses of naloxone. These observations suggested that the agonistic effect of ICI 174,864 was not mediated by μ‐opioid receptor. 5 β‐Endorphin (0.2‐1.0 μg, i.c.v.) inhibited bladder contractions but following recovery from this effect, appeared to prevent the expression of δ‐receptor antagonism by ICI 174,864. In addition a previously subthreshold dose of ICI 174,864 now exhibited marked agonistic activity. The inhibitory effect of a submaximal dose of DPDPE was also potentiated by β‐endorphin under these circumstances. 6 These observations suggest that supra‐spinal and spinal δ‐opioid receptors are involved in the opioid‐mediated inhibition of reflex bladder contractions in the rat. Moreover β‐endorphin may be important in regulating central δ‐opioid receptors.

Optimized PCR assay for detection of white spot syndrome virus (WSSV)

A rapid PCR assay for detection of white spot syndrome virus (WSSV) was developed based on the nested PCR procedure described by Lo et al. (1996) and outlined as the recommended PCR diagnostic assay in the Manual of Diagnostic Tests for Aquatic Animals published by the Office of International Epizootics (OIE, 2009). The optimized procedure incorporated the second step primers used in the nested WSSV PCR. By adjusting the annealing temperature and shortening the cycling times, this modified assay is substantially faster and as sensitive as the recommended OIE protocol. The modified PCR test was compared directly to the two-step nested PCR protocol and a modified nested procedure. The sensitivity of the published assay was determined by template dilutions of semi-purified WSSV virions that had been quantitated using real-time PCR for detection of WSSV. Various isolates were tested using the modified procedure, to ensure that the assay was able to detect WSSV from different geographical locations.

Selective δ-opioid receptor antagonism by ICI 174,864 in the central nervous system

The effects of the novel gamma-opioid receptor antagonist ICI 174,864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH: Aib = alpha-aminoisobutyric acid) have been examined in the CNS in vivo using spontaneous reflex contractions of the rat urinary bladder as an index of activity. Bladder contractions were inhibited by equipotent intracerebroventricular (ICV) doses of the selective mu-agonist DAGO [D-Ala2, MePhe4,Gly-(ol)5]enkephalin and the delta-agonist DPDPE[D-Pen2, D-Pen5]enkephalin. ICI 174,864 (1-3 micrograms) administered by the same route produce a selective and reversible antagonism of DPDPE effects. At higher doses (6-15 micrograms, ICV) ICI 174,864 exhibited marked agonistic activity, producing inhibition of bladder contractions that were resistant to ICV naloxone (1-2 micrograms). Thus ICI 174,864 was considered a selective central delta-opioid receptor antagonist but its usefulness was limited by additional agonistic properties.

The use of an infectious hypodermal and hematopoietic necrosis virus gene probe serodiagnostic field kit for the screening of candidate specific pathogen-free Penaeus vannamei broodstock

Abstract An experimental non-lethal serodiagnostic kit was evaluated for screening candidate specific pathogen-free (SPF) Penaeus vannamei broodstock under typical field conditions. In this trial, a total of 26 adult P. vannamei was screened for the presence of infectious hypodermal and hematopoietic necrosis virus (IHHNV) using an IHHNV dot blot field kit. The non-radioactive IHHNV in situ hybridization method was used as a standard for comparison. The results showed a significant relationship between the outcome of the dot blot kit and the outcome of the in situ method. In particular, the test results using the experimental kit were consistent with the in situ hybridization method. Based on these findings, the experimental serodiagnostic kit may provide a more rapid, cost-effective diagnostic method than traditional techniques for IHHNV detection.

Improvement of a PCR method for the detection of necrotizing hepatopancreatitis in shrimp

Necrotizing hepatopancreatitis (NHP) is considered to be one of the most important bacterial diseases affecting penaeid shrimp culture and is caused by an unclassified Gram-negative, pleomorphic, intracellular Alphaproteobacterium. Due to the enteric nature of the bacteria, PCR is the one non-lethal method available for detection of the pathogen. Over a decade ago, a PCR protocol was developed for detection of NHP, which over the subsequent years was shown to occasionally generate false positive reactions. The University of Arizona Aquaculture Pathology Laboratory has developed a set of primers and PCR cycling parameters that have been tested on a variety of DNA templates, using 2 types of PCR reagent systems, which eliminated the generation of false positive amplicons.