Necrotizing Hepatopancreatitis Bacterium Research Articles

Stock Health of Penaeid Shrimps in Sri Lanka: a Comprehensive Molecular-Based Study on Seven Diseases Found in the Asian Region

Purpose: Although shrimp aquaculture in Sri Lanka has mainly been plagued by recurrent outbreaks of White Spot Syndrome Virus (WSSV), recent symptomatic and analytical field data of shrimps were incoherent with typical WSSV infections. As transboundary infection of new diseases is not uncommon in world shrimp aquaculture, the possibility of new diseases established in the country surfaced. Hence, the present study was aimed at evaluating the stock health of shrimps in Sri Lanka for additional six diseases commonly available in the Asian region, namely Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV), Infectious Myonecrosis Virus (IMNV), Taura Syndrome Virus (TSV), Yellow Head Virus (YHV), Acute Hepatopancreatic Necrosis (AHPND) and Necrotizing Hepatopancreatitis Bacterium (NHPB). Research method: Sampling was performed using broodstocks, postlarvae, cultured Penaeus monodon, wild P. semisulcatus, P. indicus and P. monodon from different locations and broodstock collecting areas in the country. Other crustacean species were also collected as possible carrier species. The presence of pathogenic organisms was identified by Polymerase Chain Reaction using respective IQ 2000TM test kits and a total of 2060 PCR tests were conducted. Findings: Of all the samples tested, no samples were positive for IMNV, TSV, YHV, AHPND and NHPB. However, confirmatory evidence was found in Sri Lanka for the presence of IHHNV with the prevalence of 31.3% in broodstock samples, 24.07% in hatchery-produced postlarvae and 18.03% in cultured shrimps. WSSV was also recorded with a prevalence of 10.34% in broodstock samples and 55.56% in cultured shrimp samples collected. None of the wild collected samples was positive for any of the tested diseases. Value: Reinstating the presence of WSSV in the Sri Lankan shrimp industry, provides confirmatory evidence of the presence of IHHNV in the country, highlighting the possibility of the emergence of new diseases in shrimp farming in Sri Lanka. Also, commonly suggested disease carriers seem to be not the major cause of disease spread during the sampling time. This study, therefore, provides much-needed information about the stock health of shrimps in Sri Lanka.

Survey to evaluate the proficiency in the use of antibacterial drugs in shrimp farming in the state of Sonora, Mexico

AbstractAntibiotic drugs (ABD) are often used in shrimp farms to treat the disease when a bacterial diagnosis is presumed. This scenario was the impetus to carry out a field survey to define how ABD are employed in the State of Sonora, the second largest producer of shrimp in Mexico. Twenty‐nine farms were surveyed through their general managers. According to interviews conducted, the most frequent bacterial diseases that were treated with antibiotics in 2020 were: Acute Hepatopancreatic Necrosis Disease (65%), vibriosis (51%), and Necrotizing hepatopancreatitis bacterium (41%). The most common ABDs were enrofloxacin, oxytetracycline and florfenicol, and drug preparations employed were often intended for non‐aquaculture species, mainly poultry, and pigs. The main criteria for selecting a given antibiotic was a direct recommendation, followed by the market price. Neither pharmacokinetics nor pharmacodynamics nor harvesting withdrawal time were taken into account. The absence of protocols to safeguard the health and integrity of workers was detected, training programs for the responsible use of ABD were not encountered, and no data on bacterial resistance to ABD is available in this region. It is concluded that at least formal studies to define the pharmacokinetics of antibiotics are needed to rationalize their use in shrimp production.

Validation of qPCR from a crude extract for the rapid detection of white spot syndrome virus.

White spot disease (WSD) has posed a serious threat to the China and the global shrimp aquaculture. In order to diagnose white spot syndrome virus (WSSV) early and prevent the spread and outbreak of WSD, it is necessary to establish a highly sensitive WSSV diagnosis method suitable for shrimp farming sites. In this study, a pre-amplification qPCR assay from the crude extract of samples heated lysis was established, which was further compared with the universal qPCR assay to verify the shrimp samples. The limit of detection (LOD) of pre-amplification qPCR assay and universal qPCR assay was 2.80 copies and 20.57 copies per reaction at 95% CI, respectively. It had good WSSV specificity and did not show cross-detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV), hepatopancreatic parvovirus (HPV), Enterocytozoon hepatopenaei (EHP), acute hepatopancreas necrosis disease (AHPND), necrotizing hepatopancreatitis bacteria (NHPB), and decapod iridescent virus 1 (DIV1). A total of 36 shrimp samples were detected as WSSV DNA positive by pre-amplification qPCR with crude extract from samples heated lysis and universal qPCR with DNA extraction. The diagnostic sensitivity and specificity were 97.22% (85.5 ~ 99.9%, 95% CI) and 100% (81.5 ~ 100%, 95% CI), respectively. The agreement Kappa value was 0.959 (0.879 ~ 1, 95% CI), and the analysis results were basically consistent. Eliminating the tedious steps of extracting DNA and using pre-amplified qPCR to detect WSSV in shrimp, it is a good choice for aquaculture farms.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s10499-022-00920-9.

Antibacterial effect of biosynthesized silver nanoparticles in Pacific white shrimp Litopenaeus vannamei (Boone) infected with necrotizing hepatopancreatitis bacterium (NHP B)

shrimp necrotizing hepatopancreatitis is caused by a pleomorphic Gram-negative Rickettsialike bacteria known as NHP-B. It is well known that silver is an effective bactericidal, but experiments with aquatic organisms are scarce, even more at nanoparticle level. The objective of this study was to determine the antibacterial effect of silver nanoparticles (AgNP) against NHP-B in the Pacific white shrimp Litopenaeus vannamei. For this, AgNO3 was used as source of silver, and extracts of dried leaves of green tea Camellia sinensis and neem tree Azadirachta indica as reducing agents. Different lots of infected shrimp received 0,5 and 35 μg of AgNP by forced feeding. The differences between the number of bacterial nodules in shrimp hepatopancreas and shrimp mortality relative to the amount of AgNP demonstrated to be effective against this pathogen.

Physiological and immune response of Litopenaeus vannamei undergoing the acute phase of the necrotizing hepatopancreatitis disease and after being treated with oxytetracycline and FF

The physiological and immune responses of adult shrimp (Litopenaeus vannamei) undergoing the acute phase of the necrotizing hepatopancreatitis (NHP) disease and the efficiency of oxytetracycline (OXI) and florfenicol (FF) to eliminate the pathogen were evaluated. Four shrimp groups were considered: three groups infected with necrotizing hepatopancreatitis bacteria (NHP-B) (two treated with antibiotics and a positive control) and one group non-infected (negative control). Hemolymph concentration of glucose, lactate, acylglycerides, cholesterol, total protein, aminotransferases, superoxide dismutase, and the transcriptional expression of several immune related genes were monitored at the acute phase of the disease, and at 15 and 20 days after administration of antibiotics (daa). Shrimp from the positive control registered a mortality of 100%. NHP-B infection affected the immu-nophysiological response of shrimp; herein, most of the parameters were significantly up regulated in infected shrimp before the use of antibiotics, compared to the negative control. Increased transcriptional levels of clotting protein, lipopolysaccharide and β-1-3-glucan binding protein (LGBP), serine protease, peroxinectin, lysozyme, heat shock proteins (HSP) 60 and 70 were detected in shrimp treated with OXI. At 20 daa NHP-B was still detected in FF-treated shrimp, but not in OXI-treated shrimp. It is hypothesized that despite none of the antibiotics per se eliminated the bacterium, both had a negative effect on its virulence. OXI seems to have a greater effect, allowing shrimp to integrate a better immune response at 15 daa.

Gene expression responses of white shrimp (Litopenaeus vannamei) infected with necrotizing hepatopancreatitis bacterium

Abstract Necrotizing hepatopancreatitis bacterium (NHPB) affects the production of shrimp farms along Latin America. NHPB is one of the main threats to shrimp culture; therefore the defense mechanisms and molecular mediators that act against this bacterium ought to be well understood. The aim of this study was to study the changes in the immune response and the expression of genes coding for proteins of the immune system in the white shrimp, Litopenaeus vannamei, infected with NHPB. Shrimp infected with NHPB and healthy shrimp were maintained under laboratory conditions during 24 d. Haemolymph and hepatopancreas were collected from shrimp every six days. Total hemocyte count (THC), activities of α-2-Macroglobulin (A-2-M), phenoloxidase and total phenoloxidase from haemolymph were monitored. The expression of clottable protein (CP) and lipopolysaccharide & β-glucan binding protein (LGBP) were determined from hepatopancreas, whereas the expression of A-2-M, prophenoloxidase (proPO) and transglutaminase (TGase) was determined from hemocytes. THC showed no significant differences among infected and healthy shrimp at any of the four times. The specific activity of A-2-M significantly decreased on days 18th and 24th post-infection. In the case of the specific activity of total phenoloxidase and phenoloxidase a significant decrease was detected only in day 18 post-infection. CP gene expression decreased at 6th day post-infection, while at 18 days showed an increase of about 2.5-fold. LGBP expression increased in the first six days and at the 24th day post-infection had a drastic decline. The expression of A-2-M showed no significant changes in any of the four times. proPO expression increased more than 4-fold at day 12th post-infection but decreased at 18th day post-infection. TGase expression increased 3.5-fold at day 12th post-infection. The results found in this study suggest an active participation of the proPO system and the clotting reaction against NHP infection, mostly on days 12th and 18th post-infection.

Rickettsia‐like organisms from cultured aquatic organisms, with emphasis on necrotizing hepatopancreatitis bacterium affecting penaeid shrimp: an overview on an emergent concern

AbstractAquaculture has expanded significantly in the last two decades. However, mass mortalities of cultured organisms have occurred, sometimes caused by intracellular prokaryotic organisms such as rickettsial‐like organisms (RLO). Different species of bivalves, fish and crustacean considered as commercially important have been affected by RLO. In particular, there are scarce reports of RLO affecting crustaceans; however, the necrotizing hepatopancreatitis (NHPB) bacterium has emerged as a pathogen of concern during the last decade. NHPB thrives in shrimp hepatopancreas, causing physiological alterations and inclusively massive mortalities. In spite of the above, there is still scarce information regarding NHPB and its molecular characterization. The review is focused on the recent advances in the knowledge of RLO of mollusc and fish, with special emphasis on the intracellular necrotizing hepatopancreatitis bacterium (NHPB) of penaeid shrimp.

First record of necrotizing hepatopancreatitis bacterium (NHP-B) associated with the zooplankton samples from the Gulf of California, Mexico

The presence of a shrimp necrotizing hepatopancreatitis bacterium (NHP) in zooplankton samples from the Gulf of California was confirmed by qPCR and DNA sequences analysis. Samples of zooplankton were collected from stations located on the eastern shore of the Gulf of California, an area adjacent geographically to the coast of Sonora, Mexico. Three zooplankton samples (NHP S3, S23 and S24) were detected. These samples were collected in Bahia de Agiabampo and Bahia de Guasimas, an area distributed along the length of the coast in the vicinity of a shrimp farm area. These results clearly indicate that NHP-B may be associated or colonizing zooplankton, which may serve as a potential vector of potential importance in the spread of this disease. The biological meaning of this finding is discussed.

TEXTURAL CHANGES OF RAW AND COOKED MUSCLE OF SHRIMP, LITOPENAEUS VANNAMEI, INFECTED WITH NECROTIZING HEPATOPANCREATITIS BACTERIUM (NHPB)

ABSTRACTAlive white shrimp (Litopenaeus vannamei) were experimentally infected with necrotizing hepatopancreatitis bacteria (NHPB) to evaluate the effect of the infection on the postmortem texture of shrimp muscle. The infection percentage was monitored for 18 days. NHPB was propagated in 16.7, 66.7 and 86.4% of shrimp after 7th, 15th and 18th days from experimental infection. Texture profile analysis (hardness, fracturability, elasticity and cohesiveness), shear force and water‐holding capacity (WHC) were monitored in both raw and cooked shrimp muscle sampled from the live stock. No significant differences were detected among infected and healthy shrimp (raw and cooked) during the first 15 days; however, lower hardness, fracturability, elasticity, cohesiveness and WHC were detected in muscle from infected shrimp (cooked and raw) compared with that from healthy shrimp after 18 days. Results suggest that NHPB have a negative impact on the postmortem textural properties of the white shrimp, but the effect is observed only after certain period.PRACTICAL APPLICATIONSSeveral shrimp farms in Latin American countries are having necrotizing hepatopancreatitis disease problems. As postmortem texture in shrimp muscle is one of the main characteristics for their acceptance in the market and decides its quality, this article attempts to show how the disease affects the postmortem textural properties of the white shrimp muscle.

Experimental Infection and Detection of Necrotizing Hepatopancreatitis Bacterium in the American LobsterHomarus americanus

Necrotizing hepatopancreatitis bacterium (NHPB) is an obligated intracellular bacteria causing severe hepatopancreatic damages and mass mortalities in penaeid shrimp. The worldwide distribution of penaeid shrimp as alien species threatens the life cycle of other crustacean species. The aim of the experiment was to evaluate the possibility of experimentally infecting the American lobster (Homarus americanus) with NHPB extracted from shrimp hepatopancreas. Homogenates from infected shrimp were fed by force to lobsters. Other group of lobsters was fed with homogenates of NHPB-free hepatopancreas. After the 15th day from initial inoculation, the presence of NHPB was detected by polymerase chain reaction in feces and hepatopancreas from lobsters inoculated with infected homogenates. Necrotized spots were observed in the surface of lobster hepatopancreas. In contrast, lobsters fed on NHPB-free homogenates resulted negative for NHPB. Evidence suggests the plasticity of NHPB which can infect crustacean from different species and inhabiting diverse latitudes. Considering the results, the American lobster could be a good candidate to maintain available NHPB in vivo.

Development of a simple method to inoculate necrotizing hepatopancreatitis bacterium in Artemia sp.

Different protocols were performed to evaluate their effectiveness to inoculate necrotizing hepatopancreatitis bacterium (NHPB) into Artemia sp. Protocol-A consisted of adding to water 0.2 mL?L-1 of bacterial inoculum composed of infected hepatopancreas (HPNHPB) and glycerol (1:1 w/v). Protocol-B consisted of adding the same inoculum but at a ratio of 2 mL?L-1, whereas protocol C consisted of adding 0.2 mL?L-1, of HPNHPB+glycerol+formulated feed (0.5:0.5:1 w/v/w). Controls for each treatment consisted of similar inoculums but using healthy hepatopancreas. Artemia from A and B treatments were negative for NHPB after four and eight days post-inoculation, whereas Artemia from treatment C resulted as positive to NHPB after four (75% of individuals) and eight (100%) days post-inoculation. The results suggest that the incorporation of formulated feed into the inoculums induced the bacteria?s consumption of Artemia.

Physiological and immune responses of white shrimp ( Litopenaeus vannamei) infected with necrotizing hepatopancreatitis bacterium

An eighteen day trial was performed to evaluate the effect of necrotizing hepatopancreatitis (NHP) disease on the physiological, nutritional and immunological status of adult white shrimp ( Litopenaeus vannamei). Shrimp were divided into infected and non-infected groups; infected shrimp were fed with hepatopancreas homegenates positive to NHP bacterium, while non-infected shrimp were fed with healthy hepatopancreas. No significant differences were observed for protein, glucose, acylglicerides, cholesterol, ammonia and urea concentrations in haemolymph. However, lactate levels increased by 95% in infected shrimp compared to non-infected shrimp at 18th day post inoculation; similarly, nitrite levels in infected shrimp significantly raised by 141% and 66% at 15th and 18th days respectively. The total phenoloxidase and phenoloxidase activities of infected shrimp increased significantly at 7th day post inoculation, whereas no significant differences were detected for prophenoloxidase and total hemocyte count. The antibacterial activity decreased in infected shrimp at 15th day post inoculation. Total proteinase, trypsin and quimotrypsin activities increased after 15 days from inoculation in infected shrimp; however trypsin and quimotrypsin activities returned to their original levels after 18 days. Results suggest that shrimp maintained a homeostasis status in most of the monitored parameters, while others were modified and returned to their original levels, indicating that shrimp can withstand the disease during the first phase by performing different physiological and biochemical modifications in order to maintain homeostasis (general adaptation syndrome).

Evaluation of different microalgae species and Artemia ( Artemia franciscana) as possible vectors of necrotizing hepatopancreatitis bacteria

Microalgae species commonly used in shrimp aquaculture and observed in shrimp habitat ( Chaetoceros muelleri, Dunaliella sp. and Navicula sp.), and Artemia ( Artemia franciscana) were evaluated as possible vectors of necrotizing hepatopancreatitis bacteria (NHPB). Microalgae species and Artemia were exposed to NHPB. Eight days after inoculation, NHPB were detected (by PCR) only in Navicula sp. Thereafter, the three inoculated microalgae were used to feed white shrimp ( Litopenaeus vannamei): shrimp fed on C. muelleri and Dunaliella sp. resulted negative to NHPB after 12 days, whereas 20% of shrimp fed on Navicula sp. were to NHPB positive. Regarding to Artemia, NHPB were detected in the organisms (dead or alive), four and eight days post inoculation. Microalgae and Artemia controls remained negative for NHPB during the experimental period. The results suggest that planktonic microalgae ( C. muelleri and Dunaliella sp.) cannot be vectors of NHPB, while there is a possibility that benthic microalgae ( Navicula sp.) and Artemia sp. can act as vectors of the bacteria.

A Method for In vivo Propagation of the Necrotizing Hepatopancreatitis Bacterium (NHPB) in Litopenaeus vannamei

The necrotizing hepatopancreatitis bacterium (NHPB) is difficult to preserve and has not been propagated in vitro. NHPB can be maintained continuously by simply adding specific pathogen free (SPF) shrimp to tanks containing NHPB-infected shrimp. However, obtaining large amounts of highly concentrated infectious material free of contaminating bacterial flora for repeating challenge experiments was found to be exceedingly difficult using the current published methods. Therefore, a system was implemented using visible implant elastomer (VIE) tags to identify animals within a propagation tank by introduction group. Utilizing this method allowed for continuous reproduction of NHP within propagation tanks and provided researchers with access to infectious material allowing for consistent replication of challenge experiments and concentrated material for preservation.

Assessing the viability of necrotizing hepatopancreatitis bacterium (NHPB) stored at − 20 °C for use in forced-feeding infection of Penaeus (Litopenaeus) vannamei

Hepatopancreatitis is a severe disease of cultivated penaeid shrimp caused by a bacterium called necrotizing hepatopancreatitis bacterium (NHPB). There are currently no techniques available to recover and isolate NHPB from cell lines or artificial culture medium. The aim of this study was to develop a method for collection and long-term storage of NHP-infected material without ultra freezing processes. Our method involved collection and freezing of NHPB-infected hepatopancreas tissue (HP) at −20°C, using glycerol as a cryoprotectant. This infective material was later used to orally infect shrimp. The viability of the NHPB extracts stored for 14months in cryoprotectant at −20°C was tested by infecting healthy shrimp. Infection progress was monitored by PCR analysis of feces and by scanning electron microscopy. Additionally, we developed a forced-feeding infection procedure to transmit the disease. This type of infection method should allow for greater homogeneity and reproducibility in experimental procedures involving transmission of NHPB to Penaeus (Litopenaeus) vannamei shrimp. Forced-feeding procedure provided more efficient transmission than other previously reported methods. Our findings indicate that in vivo continuous propagation is not required to have a source of viable NHPB. The approach provides an easier, more economically advantageous and more feasible method for experimental infection for NHPB studies.

Quantification of the bacterial agent of necrotizing hepatopancreatitis (NHP-B) by real-time PCR and comparison of survival and NHP load of two shrimp populations

A real-time quantitative PCR (qPCR) assay was developed using a TaqMan probe to detect and quantify the necrotizing hepatopancreatitis bacterium (NHP-B) in Penaeus vannamei. A pair of primers which amplify a 67 bp DNA fragment and a TaqMan probe were selected from the 16 S rRNA gene of NHP-B genome. A positive control plasmid DNA was used to demonstrate that the NHP qPCR assay has a detection limit of 100 copies and a log-linear range up to 10 8 copies. An NHP-B challenge test using two different populations, Colombia (COL) and Specific Pathogen Free KONA line (KONA), was conducted. Higher final survival and a better survival curve were found in the COL population. No differences in NHP-B bacterial load were found in hepatopancreas (HP) analyzed from the survivors from each population. Sensitivity of the qPCR test was much higher than the conventional PCR (100 copies vs 1 × 10 5 copies).

Distribution of necrotizing hepatopancreatitis bacterium (NHPB) in cultured white shrimp, Litopenaeus vannamei, from Mexico

This paper describes the first systematic monitoring conducted in 2002 at shrimp farms in Sinaloa and Sonora (Mexico) for the presumptive and confirmative diagnosis of necrotizing hepatopancreatitis bacterium (NHPB) in Litopenaeus vannamei. Light microscopy (wet mounts and hematoxilin-eosin-stained sections of hepatopancreas) showed atrophy of the hepatopancreas, strangulation and necrosis of the hepatopancreatic tubules, with masses of NHPB within the tubular epithelium, hemocytic infiltrates and tubular melanization. The gene encoding the 16S rRNA (16S rDNA) of NHPB was amplified by polymerase chain reaction (PCR) from clinical specimens and the bacteria isolated using Percoll density gradient centrifugation. Negative-stain transmission electron microscopy revealed pleomorphic bacteria, most of them ovoid or rod-shaped (0.2 µm wide and 0.6–0.9 µm long), and a helical form demonstrating seven to twelve spiral turns (0.23 µm wide and 2.43–5.27 µm long). Several severe epizootics of commercially grown L. vannamei occurred during the study period and the mortality was attributed to NHPB infection. Using PCR and microbiological analyses, other etiologic agents, such as white spot syndrome virus, infectious hypodermal and hematopoietic necrosis virus, hepatopancreatic parvovirus and vibriosis, were ruled out as contributory factors.

Preservation of necrotizing hepatopancreatitis bacterium (NHPB) by freezing tissue collected from experimentally infected Litopenaeus vannamei

Necrotizing hepatopancreatitis (NHP), a severe disease of penaeid shrimp, is caused by bacteria (NHPB) that have previously been demonstrated to reside in tubular epithelial hepatopancreatic (HP) cells of infected shrimp. There has yet to be a successful in vitro culture method to grow the intracellular organism; therefore, it must be propagated in vivo via transmission from NHPB-infected shrimp to healthy individuals. In our studies, NHPB propagation tanks containing infected shrimp were used to maintain a constant supply of organisms for experiments. In order to develop a method for storing infectious NHPB material for future challenge studies, we collected HP tissue containing NHPB by flash freezing whole, fresh HPs at -80 degrees C for up to 80 d and used it to successfully infect specific pathogen-free Litopenaeus vannamei per os in controlled experiments. HP tissue samples were collected from dead shrimp, and PCR was performed to confirm the presence of NHPB. Our results demonstrate that the infectivity of NHPB in tissue is not altered after being frozen at -80 degrees C when compared to NHPB in fresh tissue. Thus, the continual propagation of NHPB in vivo is not required to assure a source of the infectious agent.

Experimental infection of Pacific white shrimp Litopenaeus vannamei with Necrotizing Hepto-pancreatitis (NHP) bacterium by per os exposure

Necrotizing Hepatopancreatitis Bacterium (NHPB), which causes Necrotizing Hepatopancreatitis, was successfully transmitted in individually isolated Kona stock Litopenaeus vannamei through per os exposure. Animals (140) were individually exposed orally to a 0.05 g piece of an NHPB-infected hepatopancreas and 120 control animals were each exposed to a 0.05 g piece of NHPB-negative hepatopancreas. Shrimp were maintained in Sterilite containers with approximately 41 of artificial seawater at 30 per thousand salinity and 30 degrees C for 60 d. Mortality of infected shrimp was observed from Day 16 to Day 51 post-exposure. Infected animals sustained reduced feeding activity and displayed empty guts. Some infected animals developed a pale hepatopancreas noticeable through the carapace. Survival probabilities fit a Weibull distribution and parametric survival analysis revealed lowered survival due to NHPB infection. Median survival time of NHPB-infected animals was 34.5 d. After correcting for background daily mortality in the controls, mean acute daily mortality of NHPB was estimated at 0.09, a value much lower than that estimated for other diseases in Kona stock L. vannamei such as White Spot Syndrome Virus (0.40) and Taura Syndrome Virus (0.30). A chronic, or carrier, state was not demonstrated in NHPB epizootics because all NHPB-positive animals experienced mortality and no animals surviving to 60 d post-exposure were diagnosed NHPB-positive through PCR or histology.

Development of monoclonal antibodies for detection of necrotizing hepatopancreatitis in penaeid shrimp

Monoclonal antibodies (MAbs) were produced against necrotizing hepatopancreatitis bacteria (NHP-B) of penaeid shrimp. The MAbs tested in dot-immunoblot (D-IB) assays were capable of detecting the NHP-B in hepatopancreas samples collected from moribund juvenile Litopenaeus vannamei during an experimentally induced NHP-B infection. The MAbs were also screened by immunohistochemistry (IHC) using case submissions that were determined to be infected not only by histology, but also polymerase chain reaction (PCR) and in situ hybridization (ISH) assays using specific digoxigenin (DIG)-labeled probes on histological sections prepared from naturally infected shrimp. Two of the MAbs were chosen for development of detection methods for NHP. The MAbs were tested using IHC methods on Davidson's alcohol-formalin-acetic acid (AFA) fixed tissue sections and identified NHP-B infected cells and tissues in a pattern similar to that seen with DIG-labeled NHP-specific gene probes. None of the MAbs reacted with tissue from specific pathogen-free (SPF) shrimp or with shrimp tissues infected with a rickettsia-like bacteria, Vibrio sp., Campylobacter sp., and Spiroplasma sp. The MAbs were found to be negative against these other organisms, demonstrating that they are species specific and useful for rapid diagnostic detection of NHP-B.