Necrotizing Hepatopancreatitis Research Articles

2020년 국내 양식 흰다리새우(Litopenaeus vannamei)의 TS, IMN, NHP 및 DIV1 모니터링

Disease monitoring in aquatic animals is important to minimize disease transmission and provide important information for effective quarantine measures. In this study, Taura syndrome (TS), infectious myonecrosis (IMN), necrotizing hepatopancreatitis (NHP), and decapod iridescent virus 1 (DIV1) were monitored in 2,592 whiteleg shrimp (Litopenaeus vannamei) collected from 108 shrimp farms in Korea from September to October 2020. A disease detection and monitoring experiment was conducted according to the WOAH

Molecular identification of a novel intracellular proteobacteria from scallop Chlamys farreri

The scallop Chlamys farreri is an important bivalve species cultured in the north of China. With the large expansion of culturing, mass mortalities have occurred persistently and caused great economic loss since 1996. An intracellular Rickettsiales-like prokaryote (RLP) was proposed as the causative agent. To identify the RLP, a partial 16S rDNA sequence of 1319 base pairs was amplified, cloned and sequenced. Phylogenetic analysis indicates that the prokaryote is a member of the α subclass of the proteobacteria. In the phylogenetic tree, it forms an independent and novel clade, which shares an evolutionary line of descent with a group of uniquely obligate intracellular organism comprised of Caedibacter caryophilus, an endosymbiont of Acanthamoeba sp., and the necrotizing hepatopancreatitis (NHP) bacterium of shrimp. Based on the obtained 16S rDNA sequence, three specific RNA targeted oligonucleotide DNA probes were designed, synthesized and labeled with digoxigenin for in situ hybridization detection. Results showed that the probes specifically bonded to the RLP inclusion in infected C. farreri tissues, indicating the amplified, cloned and sequenced 16S rDNA originated from the intracellular RLP parasitizing the C. farreri. We propose the name Sinorickettsia chlamys sp.nov. to C. farreri RLP.

Diseases of shrimps and natural methods to protect their health

Shrimp farming is an economically important part of aquaculture. Shrimps constitute the largest portion of shellfish intended for human consumption. According to the World Organization for Animal Health (OIE), viral diseases pose the most serious threat to shrimp farming. There are currently six viral and two bacterial diseases on the OIE list. The most serious viral diseases are white spot disease (WSD), Taura syndrome (TS), infection with yellow head virus genotype 1 (YHV), infectious hypodermal and haematopoietic necrosis (IHHN), infectious myonecrosis (IMN) and white tail disease (WTD), whereas the most dangerous bacterial diseases are acute hepatopancreatic necrosis disease (AHPND) and necrotizing hepatopancreatitis (NHP). All these diseases result in high mortality, thus limiting shrimp production and causing large economic losses. Therefore, effective preparations are constantly sought to control infectious diseases in shrimps. Probiotics and immunostimulants, thanks to their demonstrated antibacterial and antiviral effects, as well their positive impact on the immune system, may play a crucial role in protecting the health of shrimps.

Detection and quantification of Hepatobacter penaei bacteria (NHPB) by new PCR and quantitative PCR assays.

Necrotizing hepatopancreatitis (NHP) is a bacterial disease caused by a Gram-negative bacterium classified as Hepatobacter penaei. H. penaei affects cultured penaeid shrimp in several countries from the western hemisphere, including the USA, and most Central and South American countries that farm shrimp. The current PCR and quantitative PCR (qPCR) assays based on the amplification of the 16S rRNA gene developed at the University of Arizona Aquaculture Pathology Laboratory (UAZ-APL) are the only techniques recommended in the World Organisation for Animal Health (OIE) manual for H. penaei detection. Although these techniques are quite sensitive and specific to H. penaei detection in shrimp, in recent years, rare non-specific amplifications have been observed in the end-point PCR when screening for H. penaei in Artemia cyst samples submitted to the UAZ-APL. To avoid these non-specific amplifications, new end-point PCR and qPCR assays were developed based on the H. penaei flagella gene, flgE. Unlike the current OIE methods, the new H. penaei PCR assay did not provide any non-specific amplification, and the qPCR assay had a detection limit of 100 copies and a log-linear range up to 108 copies. Because the previous PCR-based assay using the 16S rRNA was showing non-specific amplification, the new non-specific product of around 400 bp was sequenced to determine its identity. A phylogenetic analysis revealed 2 clusters of H. penaei: Ecuador and Central-North America. This information will enable us to determine the genetic diversity and possible origin of H. penaei and emphasizes the need to evaluate H. penaei PCR detection methods to avoid inaccurate detection of H. penaei.

Identifying the causal agent of necrotizing hepatopancreatitis in shrimp: Multilocus sequence analysis approach

The causal agent of the necrotizing hepatopancreatitis (NHP) disease in penaeid shrimp and other crustaceans was studied by sequencing its genome and locating particular biomarker genes that could contribute to a better taxonomic classification. Hepatopancreas of shrimp with clear signs of NHP was used as infective inoculum. The disease was reproduced and the causal agent was isolated by Percoll gradient. Genomic DNA was extracted, purified, submitted to high throughput sequencing and de novo assembled. Thereafter, strategies aimed to improve the taxonomic classification of the pathogen were used. A phylogenetic classification by multilocus sequence analysis (MLSA) was performed using the complete gene sequences of 16S rRNA, gyrase subunit alpha (gyrA), gyrase subunit beta (gyrB), RNA polymerase subunit beta (rpoB), ATP synthase subunit beta (atpD) and recombinase subunit alpha (recA). Phylogenetic analysis confirmed with a greater degree of confidence that the causal agent of NHP in shrimp belongs to the Holosporaceae family, with similarity to some species considered as pathogens of other arthropods.

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.

White Spot Syndrome Virus (WSSV) and Necrotizing Hepatopancreatitis (NHP) detection in wild shrimp of the San Andrés Lagoon, Mexico

The presence of diseases caused by virus and bacteria pose a threat to the capture and commercialization of shrimp, and may cause significant economic damage. Nocturnal monthly sampling were conducted to detect the presence of IHHNV, WSSV, and NHP in San Andres Lagoon in Tamaulipas, Mexico, an important coastal ecosystem due to its shrimp fishery and the existence of shrimp farms in the area. Polymerase chain reaction (PCR) analysis in the shrimp tissue did not detect the presence of IHHNV, however, WSSV was detected, as well as NHP during July and August, when low salinities and high temperatures were recorded.

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).

Detection of Necrotizing Hepatopancreatitis (NHP) in Wild Shrimp from Laguna Madre, Mexico by a Multiplex Polymerase Chain Reaction

Detection of Necrotizing Hepatopancreatitis (NHP) in Wild Shrimp from Laguna Madre, Mexico by a Multiplex Polymerase Chain Reaction

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.

Effect of salinity on transmission of necrotizing hepatopancreatitis bacterium (NHPB) to Kona stock Litopenaeus vannamei

Elevated salinity and temperature have been observed prior to devastating necrotizing hepatopancreatitis (NHP) outbreaks in several geographically isolated shrimp ponds. These observations have led to the hypothesis that the NHP-bacterium (NHPB) is hindered by reduced salinity, even though the mechanism is not understood. The objective of this research was to examine the effect of salinity on transmission of NHPB. The transmission rate of NHPB was estimated through laboratory experiments whereby individuals of Kona stock Litopenaeus vannamei were orally exposed to a dead NHPB-infected shrimp. For each replicate, 12 susceptible shrimp were placed with a dead NHPB-infected shrimp in a 1 m2 bottom area cylindrical tank maintained at 30 degrees C for a period of 24 h. Four salinities of 10, 20, 30, and 40 per thousand were replicated 2 times in 2 trials, giving a total of 192 shrimp exposed per os to infective material. In each trial, a negative control group was included at each salinity, giving a total of 96 shrimp exposed orally to uninfected material. After the 24 h exposure period, susceptible shrimp were individually isolated at the same physical conditions for up to 60 d to determine NHPB transmission. The NHPB was transmissible regardless of salinity: nearly a quarter of susceptible shrimp exposed to NHPB at the lowest (10 per thousand) and highest (40 per thousand) salinity examined acquired NHPB. Transmission rates were highest at the intermediate salinities of 20 and 30 per thousand, suggesting that those salinities are optimal for NHPB transmission. The observed association between high salinity and NHP outbreak in a shrimp pond is not explained by these results because reduced transmission occurred at very low and very high salinities.

Advances in Research of Necrotizing Hepatopancreatitis Bacterium (NHPB) Affecting Penaeid Shrimp Aquaculture

Necrotizing hepatopancreatitis (NHP) is a severe bacterial disease affecting penaeid shrimp aquaculture. NHP is caused by the gram-negative, pleomorphic, obligately intracellular NHP-bacterium (NHPB) that targets the hepatopancreas tissue of shrimp. NHPB is classified as an α -proteobacterium and is related to members of the Rickettsia. First reported in 1985 in a Texas shrimp farm, NHP has since affected shrimp farming in several North and South American countries, causing mortalities up to 95% and devastating economic losses to aquaculture crops. Because NHPB remains unculturable through traditional in vitro methods, the development of an in vivo cultivation system of NHPB in susceptible, specific pathogen-free Litopenaeus vannamei has allowed advancement of experimental research, including NHPB population biology and transmission dynamics. Current research on NHPB is discussed relative to management strategies of NHP disease in a shrimp pond. Compared to other important shrimp pathogens, NHPB is geographically limited, like that of yellowhead virus (YHV), but in contrast to the global distribution of Taura syndrome virus (TSV) and white spot syndrome virus (WSSV). Additionally, NHPB is less virulent than TSV and WSSV, and the transmission dynamics of TSV and YHV are much more complicated than that of NHPB and WSSV in penaeid shrimp.

Necrotizing hepatopancreatitis (NHP) infected Penaeus vannamei female broodstock: Effect on reproductive parameters, nauplii and larvae quality

Necrotizing hepatopancreatitis (NHP) is a disease of cultured Penaeus vannamei caused by a gram-negative intracellular bacterium rickettsia-like organism. NHP was first reported in Texas in 1985, as being responsible for mortalities in shrimp ponds in Central and South America. The growth and proliferation of NHP within the hepatopancreatic epithelial cells is associated with anorexia, lethargy, abdominal muscle atrophy, softened exoskeleton, growth retardation and mortalities ranging from 20% to 95%. Although NHP can cause high mortalities in commercial ponds, it has been considered a problem only in broodstock ponds and maturation laboratories due to an increase in broodstock mortalities in Colombia. In order to evaluate additional negative effects at this level, we investigated the effect of NHP infection upon the reproductive behavior of females and the effect of maternal infection on nauplii and larval quality. Broodstock from NHP affected ponds were transferred to a maturation laboratory. After copula, females were transferred to individual spawning tanks. After spawning, females were sacrificed for histopathology and PCR analysis, while the eggs were placed in individual tanks for hatching. Females were classified into three groups according to their histopathological findings: NHP negative ( n = 23) had no NHP lesions, positive for NHP with lesions grade 1 ( n = 20) and NHP positive females with lesions grade 2 ( n = 12). For each group, we analyzed the number of eggs, hatching percentage and number of nauplii per female. Our results show that females with NHP lesions grade 2 presented a significant reduction in the number of eggs and nauplii ( P < 0.05) as compared with NHP negative females and females grade 1 NHP disease. No significant differences were found in hatching percentage among the three groups. Triglyceride levels in nauplii II were significantly higher in NHP negative females than in females with grade 2 NHP ( P < 0.05), whereas there was no difference between NHP negative and grade 1 females. No significant differences were found for glucose and cholesterol levels in nauplii among the three groups. No differences were found in survival at zoea I, mysis I, PL-1 and PL-10 for the three groups. However, after a salinity stress test, the PL-10 survival decreased significantly in larvae from NHP positive females. Also a significant decrease in PL-10 length was also observed in the progeny of these females (grades 1 and 2). This study demonstrated that NHP disease in female shrimp spawners affects both maturation and larviculture causing a decrease in the number of eggs and nauplii per female, a reduction in the levels of triglyceride in nauplii II and decreasing larvae growth and resistance to osmotic stress test at PL-10 stage.

Prevalence of Necrotizing Hepatopancreatitis in Female Broodstock of White Shrimp Penaeus vannamei with Unilateral Eyestalk Ablation and Hormone Injection

AbstractThe present study analyzed the relationship between unilateral eyestalk ablation and steroid hormone injection in female white shrimp Penaeus vannamei, the prevalence of necrotizing hepatopancreatitis (NHP) caused by the intracellular rickettsia‐like bacterium NHP‐B. Two simultaneous bioassays determined the onset and development of NHP in broodstock. Wet‐mount analysis, conventional histopathology, and in situ hybridization with an NHP‐B‐specific DNA probe were used to assess the prevalence and degree of severity of NHP and to confirm NHP‐B infection. Wet‐mount and histopathological results showed that females with unilateral eyestalk ablation had a 64% prevalence of NHP at grades 2 and 4, while females receiving the hormone injection showed a 23% prevalence of NHP at grades 3 and 4; control shrimp injected with phosphate‐buffered saline (PBS) displayed a 9% prevalence of NHP at grade 3, whereas nonablated controls showed no signs of disease. The rickettsia‐like specific DNA probe demonstrated a positive hybridization signal in NHP‐B, and the hepatopancreas, the target tissue for the bacterium, showed a strong positive signal. Significant differences (P &lt; 0.05) were found in females with unilateral eyestalk ablation compared with nonablated controls, in hormone‐injected females compared with PBS‐injected controls, and in females with unilateral eyestalk ablation compared with the hormone injection group. We conclude that unilateral eyestalk ablation enhances NHP disease prevalence in broodstock reared under maturation conditions.

A necrotizing hepatopancreatitis (NHP) outbreak in a shrimp farm in Campeche, Mexico: A first case report

Shrimp culture is the most profitable type of aquaculture in Mexico and it is highly developed in the northern states of the Pacific coast. A reduced number of commercial shrimp farms have been established along the coast of the Yucatan Peninsula and have initiated operations with Litopenaeus vannamei. The Federal Government is concerned about the use of exotic species for aquaculture and has prompted research on domestication of Gulf of Mexico native penaeids. This paper reports the first outbreak of necrotizing hepatopancreatitis (NHP) in a farm culturing L. vannamei in Campeche State, Southern Mexico. The implications of this finding with respect to native shrimp species and aquaculture development for the region are discussed.

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.

Time course of necrotizing hepatopancreatitis (NHP) in experimentally infected Litopenaeus vannamei and quantification of NHP-bacterium using real-time PCR

Necrotizing hepatopancreatitis (NHP), a severe bacterial disease affecting penaeid shrimp aquaculture, is caused by a gram-negative, pleomorphic, intracellular alpha-proteobacterium referred to as the NHP-bacterium (NHPB). The time course of NHP was investigated in experimentally infected juveniles of Kona stock Litopenaeus vannamei. Susceptible animals were individually isolated in 41 of aerated artificial seawater at salinity 30 +/- 1 ppt and maintained in a water bath at 30 +/- 1 degree C for 60 d. A total of 120 individuals were exposed per os to a 0.05 g piece of NHPB-infected hepatopancreas and 100 controls were exposed to uninfected tissue. At intervals of 3, 6, 9, 16, 23, 30, 37, 44, and 53 d post-exposure, 6 shrimp exposed to NHPB-infected tissue and 4 controls were randomly removed from the experiment; hepatopancreas samples were processed for histological and molecular analysis, and feces were processed for molecular diagnosis of NHPB infection. NHPB was first detected in the hepatopancreas through histology at 6 d post-exposure. All control shrimp were diagnosed as NHPB negative. NHPB infections classified as stage I (scattering of hepatopancreatic tubules with adjacent epithelial cells containing NHPB) and stage II (numerous infected tubules with occasional hemocyte infiltration) were observed from 6 to 37 d post-exposure. All animals that experienced NHPB-induced mortality from 16 to 51 d post-exposure were at stage III (numerous necrotic tubules, dense hemocyte infiltration, and presence of granulomas). NHPB is capable of infecting all hepatopancreatic cell types including embryonic, resorptive, fibrillar and blister-like cells. The percent of hepatopancreatic tubules containing NHPB in epithelial cells increased over time, representing bacteria multiplication and spread. Real-time PCR allowed for quantification of NHPB in hepatopancreas and feces. Over the course of infection, NHPB was present at 10(3) to 10(7) copies mg(-1) of hepatopancreas and 10(1) to 10(5) copies mg(-1) of feces. Lethal infections contained 10(6) to 10(7) copies mg(-1) of hepatopancreas and 10(3) to 10(6) copies mg(-1) of feces.

Fecal samples as DNA source for the diagnosis of Necrotizing Hepatopancreatitis (NHP) in Penaeus vannamei broodstock.

Necrotizing Hepatopancreatitis (NHP) is a severe disease of cultivated penaeid shrimp caused by a pleomorphic, gram-negative, intracellular rickettsia-like bacterium. Current diagnostic methods for this disease are invasive, requiring dissection of the animal to perform histopathological analysis. In Colombia, NHP affects mainly broodstock, being a major cause of mortalities in maturation laboratories. In order to identify the presence of NHP without having to dissect the animal, we developed a PCR-based method using fecal samples as the DNA source. The DNA was extracted using a quick isolation protocol followed by amplification with primers specific for 16S ribosomal RNA gene sequences. To verify the sensitivity and specificity we analyzed samples from the same animal by PCR and in situ hybridization, and found 100% agreement. In addition, we amplified DNA extracted form paraffin blocks to confirm NHP diagnosis. PCR amplification from fecal samples and paraffin blocks yielded the expected 440 bp fragment. We conclude that PCR amplification from fecal samples is a valuable tool for the diagnosis of NHP in broodstock organisms, and that paraffin-fixed tissues can be used as a source of DNA for PCR amplification of NHP.

Molecular phylogeny and in situ detection of the etiologic agent of necrotizing hepatopancreatitis in shrimp.

Necrotizing hepatopancreatitis (NHP) is a severe disease of farm-raised Penaeus vannamei that has been associated with mortality losses ranging from 20 to 95%. NHP was first recognized in Texas in 1985 (S. K. Johnson, p. 16, in Handbook of Shrimp Diseases, 1989) and is an economically important disease that has limited the ability to culture shrimp in Texas. The putative cause of NHP is a gram-negative, pleomorphic, intracellular, rickettsia-like bacterium that remains uncultured in part because of the absence of established shrimp cell lines. The inability to culture the NHP bacterium necessitated the use of molecular methods for phylogenetic placement of the NHP bacterium. The gene encoding the 16S rRNA (16S rDNA) of this shrimp pathogen was amplified by PCR, cloned, and sequenced. Sequence analysis of the cloned 16S rDNA indicates that the NHP bacterium is a member of the alpha subclass of the Proteobacteria. Within the alpha subclass, the NHP bacterium is shown to be most closely related to bacterial endosymbionts of protozoa, Caedibacter caryophila and Holospora obtusa. Also, the NHP bacterium is distinct from but related to members of the typhus group (Rickettsia typhi and R. prowazekii) and spotted fever group (R. rickettsii) of the family Rickettsiaceae. Fluorescently labeled oligonucleotide DNA probes that bind to variable regions (V2, V6, and V8) of 16S rRNA of the NHP bacterium were used to detect the bacterium in infected shrimp by in situ hybridization. This technique provided direct visual evidence that the 16S rDNA that was amplified, cloned, and sequenced was derived from the intracellular bacterium that infects the hepatopancreas of farm-raised P. vannamei shrimp.

Specific, nonradioactive detection of the NHP bacterium in Penaeus vannamei by in situ hybridization.

Necrotizing hepatopancreatitis (NHP) is a disease of farm-raised Pacific white shrimp (Penaeus vannamei) caused by a pleomorphic intracellular bacterium. A DNA probe that is specific for the etiologic agent of necrotizing hepatopancreatitis was devised and tested in an in situ hybridization assay. A procedure was developed for labeling a single-stranded DNA probe with digoxigenin by the polymerase chain reaction. The DNA probe encompasses the V1 and V2 variable regions of the 16S ribosomal RNA (rRNA) gene and is designed to hybridize to complementary sequences of the 16S rRNA of the NHP bacterium. The probe was tested on fixed, paraffin-embedded specimens, and an intense, specific hybridization signal was localized to the cytoplasm of hepatopancreatic epithelial cells that were infected with the NHP bacterium, as demonstrated by serial sections stained with hematoxylin and eosin or the Steiner and Steiner method. Negative results were obtained from normal shrimp and from shrimp infected with Vibrio spp. The specificity of the probe was confirmed using either mammalian or avian tissues infected with other intracellular bacteria, including Ehrlichia canis, Salmonella enteritidis, Brucella abortus, and Chlymidia spp., and using another species of shrimp (P. monodon) infected with a different rickettisa-like intracellular bacterium.