Detection of Infectious Pancreatic Necrosis Virus from the Leeches Hemiclepsis marginata and Hirudo medicinalis

Leeches have been reported to harbor several important fish pathogens, including spring viremia of carp virus, infectious hematopoietic necrosis virus (IHNV), and viral hemorrhagic septicemia virus (VHSV), and also may contain blood protozoa. In the present study, leeches were collected from water bodies located in Kurdistan province, Iran. The specimens were tested for IHNV, VHSV, and infectious pancreatic necrosis virus (IPNV) using the PCR method. The results showed that two different species of leeches, Hemiclepsis marginata and Hirudo medicinalis, were infected by IPNV among the seven species studied. The infected leeches were found in areas that were polluted with untreated sewage coming from upstream fish farms culturing Rainbow Trout Oncorhynchus mykiss. In addition, the fish at fish farms in the vicinity had been infected with IPNV 9 months previously. Our results showed that the virus causing infectious pancreatic necrosis is present in the leeches H. marginata and H. medicinalis, suggesting that leeches are a potential source of IPNV in fish farms.

Received October 14, 2015; accepted June 1, 2016 Published online September 29, 2016     

Simulating the effect of eliminating routine bacterial screening on the negative predictive value of the Ontario fish hatchery disease monitoring program.

We determined the impact of eliminating routine screening for Aeromonas salmonicida and Yersinia ruckeri on the efficacy of the Ontario Ministry of Natural Resources (OMNR) fish disease monitoring program, using Monte Carlo simulation. Because the main purpose of the program is to prevent transferring infected fish among OMNR hatcheries, or to wild fish populations through stocking waterways, the hatchery-level negative predictive value (HNPV) was used as an indicator of monitoring efficacy. The present program (which includes both routine screening of asymptomatic hatchery fish, and diagnostic testing of hatchery mortalities and clinically diseased fish) was confirmed to have a high median HNPV (0.999) for both study pathogens. Simulations suggested that the median probabilities that a hatchery would be pathogen-free if only diagnostic testing were continued (i.e. if no asymptomatic lots were screened), and all diseased lots tested negative for A. salmonicida and Y. ruckeri would be 0.994 for both pathogens (with <5% probability that HNPV would be less than 0.953 and 0.957, respectively) - indicating acceptable monitoring efficacy. However, limitations of the theoretical monitoring model must be considered.     

Rearing unit-level factors associated with bacterial gill disease treatment in two Ontario, Canada government salmonid hatcheries

Early-rearing salmonids in Ontario Ministry of Natural Resources (OMNR) fish hatcheries have been consistently affected by bacterial gill disease (BGD) (causative agent: Flavobacterium branchiophilum) for many years. Separate retrospective epidemiological investigations of BGD treatments at two OMNR fish hatcheries (Hatcheries A and B) for the 1999 production year were conducted using on-site hatchery records. Both investigations were carried out at the rearing unit-level, with early-rearing (<9 months of age) “tank-lot” as the unit of analysis to identify unique fish populations over time. Multivariable repeated measures logistic regression models were created for both hatchery datasets, controlling for lot-level and species effects. For Hatchery A, the species brook trout (Salvelinus fontinalis) and brown trout (Salmo trutta) were significantly associated with BGD treatment, as well as lower water exchange rate, and higher feeding and mortality percentages during the 2 weeks previous to BGD treatment. At Hatchery B, the species brook trout (S. fontinalis) and splake (Salvelinus namaycush × S. fontinalis) were significantly associated with BGD treatment, as well as lower individual fish weights and treatment for BGD during the previous week. These results emphasize the importance of water quality, feeding rate, fish size and prior mortality on the development of BGD. Significant hatchery and species effects were evident, and future observational research on BGD must account for these factors in their design and analysis.     

Prevalence of Infectious Pancreatic Necrosis Virus on Salmonid Fish Farms in Spain

Abstract

In Spain, salmonid fish farming was commercially developed in the 1960s, and now there are 140 private farms that depend heavily on imported embryonate eggs. Infectious pancreatic necrosis was first clinically diagnosed in Spain in 1970, but the virus (IPNV) was not isolated and identified until 1980. Since that time, researchers have isolated IPNV from other samples in Spain. A diagnostic survey was conducted to determine how prevalent IPNV is on fish farms in Spain and whether the virus has been responsible for some of the major financial losses occurring every year on these farms. In total, 236 samplings of rainbow trout Oncorhynchus mykiss from 31 farms in eight hydrographic areas were done over a 3-year period. Infectious pancreatic necrosis virus was isolated in 94 cases, and serotyping of the viral strains revealed that 81% of these isolates were strain Sp and 19% were strain Ab. Neither IPNV strain VR-299 nor rhabdovirus (as infectious hematopoietic necrosis virus or viral hemorrhagic septicemia virus) was detected in any of the samples.     

Infectious Pancreatic Necrosis Virus: Transmission from Infectious to Susceptible Rainbow Trout Fry

Abstract

Fry of rainbow trout Oncorhynchus mykiss were exposed to serotype VR-299 of infectious pancreatic necrosis virus (IPNV) by using a standardized immersion challenge. In concurrent experiments, fish were monitored for 11 d for excretion of IPNV or monitored for 9 d for excretion and transmission of IPNV to susceptible rainbow trout fry. Immersion-challenged fish began excreting virus within 2 d after challenge. The rate of IPNV excretion per fish increased steadily from about day 4 to day 8 and then decreased. Virus concentrations in tissues of immersion-challenged fish increased exponentially. Susceptible fish became infected with IPNV within 4 d after being introduced to immersion-challenged fish (e.g., 2 d after the challenged fish began excreting virus). By 9 d, 84% of the susceptible fish were infected with IPNV.     

Effects of Elevated Water Temperature and Immunosuppressant Treatment on Prevalence and Titer of Infectious Pancreatic Necrosis Virus in Naturally Infected Brook Trout

Abstract

Using fingerlings of brook trout Salvelinus fontinalis naturally infected with infectious pancreatic necrosis virus (IPNV), we demonstrated that elevated water temperature and treatment with the immunosuppressant triamcinolone acetonide (generic Kenalog®) significantly increases the titer of IPNV and probably also the prevalence of the infection. Stress-promoting treatments could potentially enhance the capability to detect various fish viral agents.     

Factors associated with the incidence of bacterial gill disease in salmonid lots reared in Ontario, Canada government hatcheries

Bacterial gill disease (BGD) (causative agent: Flavobacterium branchiophilum) has been a persistent problem in early-rearing salmonids in the Ontario Ministry of Natural Resources (OMNR) fish hatchery system. Retrospective epidemiological investigations of BGD diagnoses and treatments in OMNR fish hatcheries during the period 1991–2001 were conducted using University of Guelph Fish Health Laboratory and OMNR central office data. All investigations were conducted at the lot-level, which is the major within-hatchery-level of population aggregation. Survivorship of BGD diagnosis in early-rearing lots within seven individual hatcheries ranged from 84.2 to 100%; within individual species groups, survivorship was lowest (84.6%) in brook trout (Salvelinus fontinalis) lots. Annual risk percentages (cumulative incidence) for BGD diagnosis within hatchery and species groups varied considerably among years. Multivariable proportional hazards survival analysis indicated that the species brook trout, and the Spring (March–May) season, were significantly associated with treatment for BGD. Combined, these results emphasize the importance of hatchery, species, and time on the development of BGD. Future observational research on this disease must consider these factors in their design and analysis.     

Distribution of Infectious Pancreatic Necrosis Virus (IPNV) Based on Surveillance Programs in Freshwater Trout Farms of Mexico

Diagnostic testing was performed between 2000 and 2012 to determine the distribution of infectious pancreatic necrosis virus (IPNV) in the main states of the Mexican Republic with freshwater Rainbow Trout Oncorhynchus mykiss (Walbaum) farms. This virus was positively identified from Rainbow Trout farms in seven of the eight states assessed. Due to nonnormal data distribution, a logistic regression model was applied for statistical analysis, the results of which indicated that virus prevalence was variable between states, with moderate but significant differences. Regarding the time periods evaluated, IPNV prevalence was higher during the first years of the study. The susceptible, infected, removed model was used to examine this phenomenon, which indicated that the decreased prevalence during the latter years of the study could be associated with a real elimination of the infection. The information of the cases analyzed also suggests a relationship with the irregularity in the submission of samples to the laboratory and emphasizes other factors that have contributed to the transmission of IPNV throughout the country.

Received November 10, 2014; accepted December 5, 2015.     

Assessment of the Risk of White Sturgeon to become Infected and Potential Carriers of Infectious Pancreatic Necrosis Virus

Abstract

Little scientific information is available to assess whether White Sturgeon Acipenser transmontanus can become infected and potential carriers of infectious pancreatic necrosis virus (IPNV). To assess this risk, monitoring results of adult and progeny White Sturgeon were examined from waters historically stocked with salmonid fish known to be IPNV carriers. From 1999 through 2004 White Sturgeon from a total of 30 separate families whose parentage came from waters historically stocked with IPNV carrier fish were tested. Duplicate groups of 25 juvenile Snake River White Sturgeon were waterborne exposed to 1.0×10⁴ 50% tissue culture infective dose (TCID50)/mL of water for 1 h and an additional group was injected intraperitoneally with 1.0×10⁵ TCID50/fish. A negative control group was handled similarly but was not exposed to the virus. No morbidity was detected in any of the treatment groups or the negative control. At 34, 40, 47, and 54 d postexposure to IPNV, virus reisolation was attempted on five fish from each group, and an additional five fish from each group were examined for histological changes consistent with an IPNV infection. At 34 and 40 d postinjection with IPNV, 20% (one of five) of the fish tested positive for the virus per sample interval; however, fish from groups that were waterborne-exposed to IPNV were all negative. At 47 and 54 d after exposure or injection with IPNV an additional five fish from each group were tested at each sample interval and all results were negative. Histological analysis of target tissue obtained from five fish per group at 34 and 54 d postinfection also failed to detect any consistent change associated with an IPNV infection. These results suggest that the risk of White Sturgeon to become infected and develop into potential carriers of IPNV is negligible.

Received May 21, 2013; accepted July 8, 2013     

Prevalence of Chlamydia psittaci in different cat populations in Britain

The prevalence of Chlamydia psittaci infection in household, feral and farm cats in Britain was investigated. Chlamydia were isolated from 30 per cent of conjunctival swabs collected from 753 household cats with conjunctivitis. The prevalence of active chlamydial infection was highest in cats in the age group five weeks to nine months. Males were more frequently infected than females. Cats with chlamydial conjunctivitis usually had antibody titres greater than 1024 as assessed by indirect immunofluorescence. Chlamydia appeared to be endemic in two out of three feral cat colonies on the basis of serological evidence and occasional isolations. Cats on 10 of 22 sheep farms (45 per cent) had serological evidence of chlamydial infection, and this was confirmed on two farms by isolation of the organism from conjunctival and, or, rectal swabs. This is the first survey of infection with Chlamydia psittaci in cat populations in Britain.     

Fish Processing Facilities: New Challenge to Marine Biosecurity in Canada

Abstract

The transmission of pathogens is a common consequence of animal food production. Marine salmon farms and their processing facilities can serve as sources of virulent fish pathogens; our study is the first to confirm the broadcast of a live fish pathogen from a farmed salmon processing facility into the marine waters of Canada's Pacific coast. We found live salmon lice Lepeophtheirus salmonis, mucus, and fish tissue in effluent from the processing facility. Sea lice transmitted from this source may pose a threat to wild salmon populations, and the release of untreated offal, including blood water, is of considerable concern. Further research is needed to quantify the extent to which processing facilities release sea lice and to determine whether more virulent fish pathogens are present in effluent. These data underscore the need for fish farming nations to develop mandatory biosecurity programs to ensure that farmed salmon processing facilities will prevent the broadcast of infectious fish pathogens into wild fish habitat.

Received April 26, 2012; accepted September 7, 2013     

Persistence of infectious pancreatic necrosis virus (IPNV) in Scottish salmon (Salmo salar L.) farms

Infectious pancreatic necrosis is a disease that is causing increasing loses to Scottish Atlantic-salmon farms. I asked the question: is infection of individual salmon farms persistent or transient? Using Fisheries Research Services' Fish Health Inspectors' data, conditional probabilities that a farm would (time 0) be infected were estimated for farms that had been infected [P(I(0)|I(-T))] or free [P(I(0)|F(-T))] from infectious pancreatic necrosis virus (IPNV) when a sample was taken at some earlier time (-T). A logistic-regression model was used to estimate these conditional probabilities with T; this model was multilevel to account for regional and inter-annual level differences in prevalence of IPNV. In freshwater, conditional probabilities remained substantially different for periods of at least 4 years, so, although many farms did change infection status, IPNV either persisted or recurred at specific freshwater farms. Marine farms showed similar conditional probabilities after about 2 years following a positive or a negative sample, indicating that infection was transient. Management of larger areas and exchanges between farms might be more effective than farm-level management at controlling marine IPNV.     

Journal of Aquatic Animal Health: Guide for Authors 2012

Abstract

A virus adsorption-elution (viradel) method for use with positively charged microporous filters was previously developed in our laboratory to concentrate waterborne infectious pancreatic necrosis virus (IPNV). In the present study, this method was evaluated for the detection of IPNV in water of laboratory aquaria containing IPNV-infected rainbow trout Onchorhynchus mykiss. Waterborne IPNV samples concentrated by the viradel method were compared with samples of sediment, fish tissue homogenate, and untreated water to determine how much the viradel method could improve rapidity and efficiency of virus detection. Results of three separate experiments indicated a significant, positive correlation between the virus titers in fish tissue and in the viradel method samples and between the sediment and untreated water samples.     

Detection of infectious pancreatic necrosis virus (IPNV) RNA by hybridization with an oligonucleotide DNA probe

Marine farming of Atlantic salmon (Salmo salar) is growing rapidly in Norway, and fish diseases have now become one of the biggest obstacles for this new industry. Infectious pancreatic necrosis virus (IPNV) is commonly found in fish from Norwegian sea farms. Diagnosis of IPNV infection is, at present, mainly based on virus isolation in cell cultures and identification by neutralization tests (NT). A DNA-RNA hybridization assay was developed using a 24 base DNA oligonucleotide probe. This is homologous to a part of the nucleotide sequence of the IPNV genome coding for a protease. RNA extracted from IPNV and harvest from infected cell cultures were fixed to nylon filters and hybridized with the 32P end-labelled probe. The results showed that the probe specifically identified IPNV from these two materials, both for the three different virus strains (Ab, Sp and VR-299) used, and for several different field isolates. It did not hybridize with reoviruses or non-infected cell cultures used as controls. These results indicate that the probe is not serotype specific, and furthermore that RNA extraction is not required before hybridization. This method may be a useful alternative to NT for routine identification of IPNV, particularly when non-radioactive labelling of the probe is introduced.     

Host factors associated with the detection of Aeromonas salmonicida and Yersinia ruckeri in Ontario, Canada government fish hatcheries

This paper presents an epidemiological investigation of Ontario Ministry of Natural Resources Fish Health Laboratory data from 1981 to 1997, to determine whether fish species and age were associated with lot-level detection of Aeromonas salmonicida and Yersinia ruckeri in hatchery fish. In stepwise logistic regression, the species brook trout and back-cross (lake trout crossed with the hybrid “splake”) were more likely to test A. salmonicida-positive compared to all other species reared in the hatcheries. Similarly, the species brook trout was significantly more likely to test Y. ruckeri-positive compared to all other species. For both pathogens, the 1–5-month age group was associated significantly with detection. These findings suggest that purposive sampling of higher-risk fish lots could increase the likelihood of detecting both study pathogens.     

Infecting Pacific Herring with Ichthyophonus sp. in the Laboratory

The protistan parasite Ichthyophonus sp. occurs in coastal populations of Pacific Herring Clupea pallasii throughout the northeast Pacific region, but the route(s) by which these planktivorous fish become infected is unknown. Several methods for establishing Ichthyophonus infections in laboratory challenges were examined. Infections were most effectively established after intraperitoneal (IP) injections with suspended parasite isolates from culture or after repeated feedings with infected fish tissues. Among groups that were offered the infected tissues, infection prevalence was greater after multiple feedings (65%) than after a single feeding (5%). Additionally, among groups that were exposed to parasite suspensions prepared from culture isolates, infection prevalence was greater after exposure by IP injection (74%) than after exposure via gastric intubation (12%); the flushing of parasite suspensions over the gills did not lead to infections in any of the experimental fish. Although the consumption of infected fish tissues is unlikely to be the primary route of Ichthyophonus sp. transmission in wild populations of Pacific Herring, this route may contribute to abnormally high infection prevalence in areas where juveniles have access to infected offal.

Received June 25, 2015; accepted September 12, 2015