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.

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.

Experimental infection of Pacific white shrimp Litopenaeus vannamei with Necrotizing Heptopancreatitis (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.

Seasonal and yearly population dynamics of two exotic helminths, Camallanus coti (Nematoda) and Bothriocephalus acheilognathi (Cestoda), parasitizing exotic fishes in Waianu Stream, O'ahu, Hawaii.

Exotic poeciliid fishes introduced into Hawaiian freshwaters are responsible for the introduction of several exotic parasites, of which the most important are Camallanus cotti and Bothriocephalus acheilognathi in terms of potential disease threat to native stream fishes. This roundworm and tapeworm are the most prevalent and abundant freshwater fish helminths in Hawaiian streams. This study examined the seasonal and yearly population structure of C. cotti and B. acheilognathi to determine if the tropical Hawaiian environment characterized by low climatic variability permits continuous opportunities for parasite transmission regardless of time of year. Camallanus cotti displayed seasonal differences in prevalence and mean abundance, whereas B. acheilognathi did not. Camallanus cotti prevalence and mean abundance were higher in the Hawaiian summer (47.7%, 0.79) than in winter (25.8%, 0.36). A seasonal relationship of C. cotti levels is likely explained by extensive rains associated with the Hawaiian winter season, which may act to decrease parasite transmission by flushing infected poeciliid hosts, intermediate copepod hosts, and possibly free-living infective worm stages downstream. Bothriocephalus acheilognathi displayed low prevalence and mean abundance in both summer (4.0%, 0.06) and winter (6.5%, 0.07), and it may be difficult to detect seasonal changes due to these low levels. Camallanus cotti prevalence and mean abundance remained relatively constant from the summer of 1995 to the summer of 1999, indicating that levels of this roundworm are stable in Waianu Stream. Whereas B. acheilognathi prevalence and mean abundance were low during the summer of 1995 and the summer of 1997, a dramatic peak in prevalence and mean abundance was observed in the summer of 1998 (41.2%, 1.06), with levels decreasing sharply in the summer of 1999 (4.4%, 0.07). It appears that B. acheilognathi also is present in stable populations at low levels, even though levels rose sharply during a single year.

Host specificity and population structure of two exotic helminths, Camallanus cotti (Nematoda) and Bothriocephalus acheilognathi (Cestoda), parasitizing exotic fishes in Waianu Stream, O'ahu, Hawai'i.

Introduction of exotic fishes into Hawai'ian streams has resulted in the simultaneous introduction of exotic parasites. Camallanus corti (Nematoda) and Bothricephalus acheilognathi (Cestoda) are the most prevalent and abundant fish helminths in Hawai'ian streams. The population structure and host specificity of C. cotti and B. acheilognathi in exotic poeciliids were examined during May and June 1995 in Waianu Stream, O'ahu, Hawai'i. Prevalence and mean abundance of C. cotti were significantly different among Poecilia reticulata (65.2%, 1.05), Poecilia mexicana (49.0%, 0.87), and Xiphophorus helleri (32.3%, 0.44). Prevalence of B. acheilognathi was significantly higher in P. mexicana (6.1%) than in P. reticulata (2.1%) and X. helleri (1.6%). However, tapeworm differences in mean abundance were not significant among P. mexicana (0.08), P. reticulata (0.04), or X. helleri (0.03). Nematode and tapeworm prevalence and mean abundance were not significantly different with regard to fish sex. Camallanus cotti prevalence and mean abundance increased as P. mexicana body size increased (r2 = 0.84 and r2 = 0.72, respectively), whereas B. acheilognathi displayed no significant trend with respect to poeciliid body size.