Leone L. Mohney

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.

Qualitative and quantitative studies on the relative virus load of tails and heads of shrimp acutely infected with WSSV

There is currently concern and controversy in the shrimp industries of the Americas about the risk posed by the importation and reprocessing of shrimp infected with white spot syndrome virus (WSSV) and yellow head virus (YHV). To further understand the risk, more knowledge concerning the quantitative virus load of infected shrimp is needed. The present study was carried out to better define, using qualitative and quantitative methods, the relative virus load of shrimp heads and tails. For these studies, specific pathogen-free (SPF) Penaeus vannamei were infected with WSSV. Emergency harvest of these shrimp was simulated by collecting the infected shrimp at the onset of postinfection mortalities and determining the relative virus loads of the head and tails by quantitative real-time PCR and histology methods. Routine histology and in situ hybridization assay with a WSSV-specific DNA probe demonstrated qualitatively similar levels of WSSV infection in the heads and tails of experimental infected shrimp. The novel real-time PCR method demonstrated quantitatively that the head had a slightly higher WSSV load than did the tail. However, since the tail represents 58% of the total body weight, the total virus load on a per weight basis turns out to be similar in the head (49%) and tail (51%) of the same shrimp with acute phase WSSV infections. In proportion to the total tail weight, the virus load of the peeled shell represents 55% of the total viral load in tail.

The geographic distribution of Taura Syndrome Virus (TSV) in the Americas: determination by histopathology and in situ hybridization using TSV-specific cDNA probes

Abstract Representative archived Litopenaeus vannamei samples (117 total), originating from 13 different countries and collected between 1992 to 1996, were analyzed by in situ hybridization to verify the presence of Taura Syndrome Virus (TSV) within pathodiagnostic acute phase TS histological lesions. The in situ assay results showed that TSV was present in one or more representative samples analyzed from each country (76 of 117 samples or 65%), thus, confirming the original histological diagnosis of TSV infection. The false negative in situ hybridization results, obtained for 35% of the samples assayed (41 in total), were attributed to over-fixation with Davidsons AFA (acetic acid, formaldehyde, alcohol) solution and consequent acid hydrolysis of TSV genomic RNA within pathodiagnostic TSV lesions. The collective findings of this disease survey assisted in documenting the spread and current distribution of TSV over a 5-year period and definitively established the presence of TSV within TS diseased shrimp originating from Ecuador when and where the disease was first recognized in 1992. These findings further strengthen the existing evidence that TS has a viral, not a toxic, etiology and indicate that either a single TSV strain, or very similar strains of the same virus, are responsible for the TSV panzootic that has been expanding in the Americas since 1992.

Induced resistance to white spot syndrome virus infection in Penaeus stylirostris through pre-infection with infectious hypodermal and hematopoietic necrosis virus—a preliminary study

Abstract White spot syndrome virus (WSSV) is highly virulent to penaeid shrimp and has been responsible for serious economic losses on shrimp farms throughout the world. Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a small DNA virus that also infects penaeid shrimp; and, although once virulent to culture stocks of Penaeus stylirostris , it has not been associated with mass mortalities in recent years. Through three laboratory challenge studies, we discovered an interference interaction between these two viruses in P. stylirostris. In the first bioassay, juvenile P. stylirostris were infected with IHHNV by feeding them with IHHNV-infected tissue. These shrimp, along with a group of non-infected control shrimp, were then fed WSSV-infected tissue. Two days after the WSSV challenge, mortalities began to occur. All of the control shrimp, which were not exposed to IHHNV, died within 5 days. In contrast, replicated challenge groups of the IHHNV pre-infected shrimp had 31% and 44% survival. Quantitation by real-time PCR determined that the surviving shrimp had high levels (10 9 copies per μg DNA) of IHHNV and very low levels (50–400 copies per μg DNA) of WSSV DNA. Moribund shrimp had high levels (10 6 –10 7 copies per μg DNA) of WSSV and low levels (10 4 –10 6 copies per μg DNA) of IHHNV DNA. A second challenge study with P. stylirostris that were heavily infected with IHHNV showed 21 of 23 (91%) shrimp survived 19 days after exposure to WSSV. Ten of these surviving shrimp were re-exposed to WSSV-infected tissue, and six were still alive after 3 weeks. The third challenge study showed that 28% of the IHHNV pre-infected P. stylirostris survived 22 days after exposure to WSSV. In situ hybridization analysis in all the challenge studies confirmed that the surviving shrimp were strongly infected with IHHNV and had, at most, only low levels of WSSV infection. Laboratory WSSV challenges showed that there were no survivals in either IHHNV pre-infected Penaeus vannamei or in IHHNV-resistant P. stylirostris (SuperShrimp™). In conclusion, juvenile P. stylirostris that are highly infected with IHHNV show resistance to WSSV infection.

Shrimp Antimicrobial Testing. I. In Vitro Susceptibility of Thirteen Gram-Negative Bacteria to Twelve Antimicrobials

Abstract Standard minimum inhibitory concentration (MIC) studies were conducted in vitro on eleven prospective antimicrobials and one reference antimicrobial. The compounds were tested against a standardized battery of 13 gram-negative bacterial isolates associated with shrimp disease. Two additional bacterial organisms not associated with shrimp, Escherichia coli and Pseudomonas aeruginosa, were also tested as system controls. The compounds tested were chloramphenicol (reference), enrofloxacin, erythromycin, florfenicol, oxytetracycline, paromomycin, four experimental fluoroquinolines (PD124816, PD127391, PD131628, PD132133), Romet-30®, and Sarafin®. All compounds, with the possible exception of paromomycin, had significantly better overall MIC ranges than did chloramphenicol.

Residues of oxytetracycline in cultured juvenile blue shrimp, Penaeus stylirostris (Crustacea: Decapod), fed medicated feed for 14 days

Abstract Juvenile shrimp fed for 14 days on oxytetracycline (OTC) medicated feed with a potency of 1500 mg OTC kg −1 of feed reached a level of 3.3 μg OTC g −1 of shrimp tail muscle within 4 days, as measured by the microbial agar plate diffusion assay method for oxytetracycline in tissues, approved by the Association of Official Analytical Chemists. The concentration of OTC remained between 3.3–5.2 μg OTC g −1 of shrimp tail muscle from Day 4 through Day 14 of the feeding period. These concentrations were greater than the Minimum Inhibitory Concentration for sensitive organisms. By the fifth day following cessation of feeding medicated feed the drug content in shrimp tail muscle was less than the detectable limit for the method, or 0.4 μg of OTC g −1 shrimp tissue.

Isolation and identification of mycobacterium peregrinum from the pacific white shrimp penaeus vannamei

Abstract Mycobacterium peregrinum was isolated from multifocal, melanized nodular lesions in the carapace of the cultured marine shrimp Penaeus vannamei. This is the first identification of this species of Mycobacterium in shrimp. The lesions appeared to be the result of opportunistic infections in otherwise healthy animals. The presence of M. peregrinum in cultured shrimp has a direct negative impact on the marketability of shrimp because it causes obvious black lesions. The potential for accidental infections of shrimp farm or packing plant workers from handling infected shrimp is of concern since nodular skin lesions induced by the bacterium are difficult to treat.

Evaluation of Difloxacin for Shrimp Aquaculture: In Vitro Minimum Inhibitory Concentrations, Medicated Feed Palatability, and Toxicity to the Shrimp Penaeus vannamei

Abstract Standard in vitro minimum inhibitory concentrations (MICs) were determined for difloxacin and compared with the MICs of several other antimicrobials, against a standardized battery of 13 gram-negative bacterial isolates associated with shrimp disease. The palatability and safety (toxicity) of difloxacin to the shrimp Penaeus vannamei were also evaluated during 15 d of medicated feeding at 1× (100 mg/kg of feed), 2×, and 4× treatment levels to give doses of approximately 5, 10, and 20 mg difloxacin/kg body weight. A significant reduction (P ≤ 0.05) in difloxacin-medicated feed palatability was noted in the 2× and 4× trials. However, differences were still acceptable, because more than 80% of the feeds were consumed in both treatments relative to the control diet. Shrimp mortality rates increased with difloxacin level from 7% for the control treatment to 20% for the 4× treatment. Differences in percent survival were not significant (P > 0.05) by the Williams test; however, analysis of mean survival...