Experimental Transmission and Pathogenicity of a Viral Erythrocytic Infection in Sea Bass Dicentrarchus labrax

Abstract

The experimental transmission of a viral erythrocytic infection (VEI) in sea bass Dicentrarchus (=Morone) labrax of the Mediterranean is reported. Transmission procedures included the intraperitoneal inoculation of healthy fishes with blood and hematopoietic tissue extracts from VEI-affected fishes, direct contact between VEI-affected and unaffected fishes, and rearing of fishes in unfiltered, nonirradiated seawater that had been used for holding VEI-affected fishes. Successful transmission of the infection was achieved in all experimental conditions, as ascertained by the presence of inclusion bodies in Giemsa-stained blood cells and subsequently confirmed by indirect immunofluorescent staining. Transmission of VEI by the inoculation of filtered cell-free extracts from affected tissues confirmed not only the infectious character of the disease but also its viral etiology. Two different patterns of effects on the hematological variables were observed: an erythroblastic polycythemia occurred 3 months after infection, whereas a moderate anemia appeared after 5–6 months. Fish growth was also negatively affected by VEI. Sea bass infected with VET were found to be more susceptible to bacterial challenge infection with Vibrio anguillarum isolate AO 18 than were control fish; the V. anguillarum dose that was lethal to 50% of the test fish was lower for VEI-affected fish, and their cumulative mortality in the bacterial challenge was higher. The injection of cyclophosphamide produced immunosuppression in unaffected sea bass and an increase in the level of infection in VEI-affected fishes; these results suggest the role of the immunological status of the host sea bass in the development of VEI.     

Communications: Protective Immunity Induced by a Commercial Vibrio Vaccine in Hybrid Striped Bass

Abstract

Commercial Vibrio anguiliarum-V. ordalii bacterin was used to vaccinate hybrid striped bass (Morone saxatilis ♀ × M. chrysops ♂) to test the vaccine efficacy against vibriosis. Vaccination by direct immersion of fish in diluted Vibrio vaccine for 20 s resulted in increased protective immunity. The relative percent survival of hybrid striped bass challenged 35 d after vaccination was 66.7% for those challenged by 1-h immersion exposure to 7.03 × 10⁷ V. anguillarum cells/mL, 75.0% for those challenged by injection with 3.51 × 10⁵ cells/fish, and 86.7% for those challenged by injection with 3.51 × 10⁴ cells/fish.     

Experiments on Virulence Dose and Portals of Entry for Aeromonas hydrophila in Walking Catfish

Abstract

Aeromonas hydrophila, isolated from chevron snakehead Ophicephalus (=Channa) striatus affected with epizootic ulcerative syndrome (EUS), was injected intramuscularly into healthy walking catfish Clarias batrachus at varying 10-fold serial dilutions from 10⁸ to 0 colony-forming units (cfu) per fish. Only 10⁶ or more cfu/mL induced dermomuscular lesions. Initial healing of lesions was observed by day 7 but complete healing was not apparent until day 16. Experiments were also conducted on possible portals of entry of A. hydrophila into walking catfish: Intramuscular (IM) injection, gastric gavage, fish food, and immersion of injured fish in rearing water inoculated with the test bacteria. Injuries were caused by skin or muscle cut, dermal scraping or incision, fish bite, and cohabitation of fish with golden snails Ampullarius sp. Only IM injection treatment induced dermomuscular pathology in the test catfish. This suggests that a localization of A. hydrophila to a level of 10⁶ cfu/mL in the musculature must be established for dermal lesions to develop.     

Effect of Nanophyetus salmincola and Bacterial Co-Infection on Mortality of Juvenile Chinook Salmon

The freshwater trematode Nanophyetus salmincola has been demonstrated to impair salmonid immune function and resistance to the marine pathogen Vibrio anguillarum, potentially resulting in ocean mortality. We examined whether infection by the parasite N. salmincola similarly increases mortality of juvenile Chinook Salmon Oncorhynchus tshawytscha when they are exposed to the freshwater pathogens Flavobacterium columnare or Aeromonas salmonicida, two bacteria that juvenile salmonids might encounter during their migration to the marine environment. We used a two-part experimental design where juvenile Chinook Salmon were first infected with N. salmincola through cohabitation with infected freshwater snails, Juga spp., and then challenged with either F. columnare or A. salmonicida. Cumulative percent mortality from F. columnare infection was higher in N. salmincola-parasitized fish than in nonparasitized fish. In contrast, cumulative percent mortality from A. salmonicida infection did not differ between N. salmincola-parasitized and nonparasitized groups. No mortalities were observed in the N. salmincola-parasitized-only and control groups from either challenge. Our study demonstrates that a relatively high mean intensity (>200 metacercariae per posterior kidney) of encysted N. salmincola metacercariae can alter the outcomes of bacterial infection in juvenile Chinook Salmon, which might have implications for disease in wild fish populations.

Received February 24, 2015; accepted September 7, 2015     

Effects of Practices Related to Catch-and-Release Angling on Mortality and Viral Transmission in Juvenile Largemouth Bass Infected with Largemouth Bass Virus

Abstract

Largemouth bass virus (LMBV; family Iridoviridae) has recently emerged as a causative agent in fish kills of largemouth bass Micropterus salmoides. Little is known about how the virus is transmitted or what factors predispose fish to mortality subsequent to infection. Concern has nevertheless arisen that activities related to recreational angling may affect transmission dynamics and may alter the susceptibility of infected fish to clinical disease. This study examined the separate effects of two angling-related factors on the susceptibility of juvenile largemouth bass to mortality from LMBV infection and on the transmission of LMBV from infected to uninfected fish. The first factor was hook-and-line angling. Infected fish that underwent a simulated angling treatment did not experience higher mortality or have higher viral loads in their tissues than those that were not angled. The second factor was direct contact between infected and uninfected fish, as would occur in live wells and holding tanks. The LMBV was transmitted from infected to uninfected fish through water, even when direct contact was prevented. Transmission of LMBV between infected and uninfected fish separated by a fenestrated barrier was nearly as efficient as LMBV transmission between infected and uninfected fish that were allowed direct contact. These results imply that angling itself may have only minimal effects on the survival of largemouth bass infected with LMBV but that angling-related practices that place infected and uninfected fish together in a limited water volume may facilitate viral transmission. Partitioning or cooling of live wells and holding tanks, as well as limiting their use, could reduce LMBV-associated mortality and viral transmission.     

Cutaneous Listeriosis in a Veterinarian with the Evidence of Zoonotic Transmission – A Case Report

A case of Listeria monocytogenes skin infection in a man is presented. A 54‐year‐old male veterinary practitioner developed pustular changes on the skin of arms and hands after assisting with the delivery of a stillborn calf. Listeria monocytogenes was isolated from the skin lesions on the arms and from the bovine placenta. Listeria monocytogenes isolates were serotyped and genotyped with pulsed‐field gel electrophoresis (PFGE) to confirm the suspected transmission of the pathogen from animal to human. All isolates were of serotype 4b with identical pulsotype. To the best of our knowledge, this is the first case of cutaneous listeriosis in which the evidence for zoonotic transmission of L. monocytogenes is supported by genotyping methods.     

Disease Susceptibility of Hatchery Snake River Spring–Summer Chinook Salmon with Different Juvenile Migration Histories in the Columbia River

Abstract

Various methods have been developed to mitigate the effects of dams on juvenile Pacific salmon Oncorhynchus spp. migrating to the Pacific Ocean through the Columbia River basin. In this study, we examined the health of hatchery Snake River spring and summer Chinook salmon relative to two mitigating strategies: dam bypass and transportation (e.g., barging). The health of out-migrants was assessed in terms of the difference in the incidence of mortality among fish, categorically grouped into no-bypass, bypass, and transportation life histories, in response to challenge with the marine pathogen Listonella anguillarum during seawater holding. These three life histories were defined as follows: (1) fish that were not detected at any of the juvenile bypass systems above Bonneville Dam were classified as having a no-bypass life history; (2) fish that were detected at one or more juvenile bypass systems above Bonneville Dam were classified as having a bypass life history; and (3) fish that were barged were classified as having the transportation life history. Barged fish were found to be less susceptible to L. anguillarum than in-river fish—whether bypassed or not—which suggests that transportation may help mitigate the adverse health effects of the hydropower system of the Columbia River basin on Snake River spring–summer Chinook salmon. The findings of this study are not necessarily transferable to other out-migrant stocks in the Columbia River basin, given that only one evolutionarily significant unit, that is, Snake River spring–summer Chinook salmon, was used in this study.     

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.     

Iridovirus Infections among Missouri River Sturgeon: Initial Characterization,Transmission, and Evidence for Establishment of a Carrier State

Abstract

Iridovirus infections of the integument were associated with disease and mortality among hatchery-reared populations of juvenile pallid sturgeon Scaphirhynchus albus and shovelnose sturgeon S. platorynchus from the Missouri River. Virus-infected cells in the integument of fins and body were greatly enlarged, possessed pleomorphic and eccentric nuclei, and exhibited an amphophilic to eosinophilic staining of the cytoplasm in hematoxylin-and-eosin-stained sections. Virus particles found in the host cell cytoplasm were composed of an outer hexagonal capsid measuring 254 nm in diameter and surrounding a dense nucleoid. Despite numerous attempts, the virus could not be propagated on routine cell lines used in fish viral diagnostics or from established cell lines from white sturgeon Acipenser transmontanus, pallid sturgeon, or shovelnose sturgeon. Bath exposures of healthy juvenile pallid sturgeon to a crude extract or a 0.45-μm-filtered extract from the fins of infected fish resulted in transmission of the virus and mortality. At water temperatures of 15°C, the first deaths occurred at approximately 1 month; mortality peaked between 50 and 60 d postexposure, after which surviving fish recovered. Presence of the virus was confirmed among dead and moribund pallid sturgeon by both histology and detection of viral DNA by polymerase chain reaction methods. Feeding of infected tissues and cohabitation with virus-infected shovelnose sturgeon also resulted in successful virus transmission to juvenile pallid sturgeon. Virus infections among experimentally exposed pallid sturgeon that recovered from clinical episodes persisted for at least 8.5 months, and these apparently healthy fish transmitted the virus and disease to juvenile pallid sturgeon by cohabitation. The newly described Missouri River sturgeon iridovirus (MRSIV) as found in pallid sturgeon and shovelnose sturgeon shares many properties with a group of iridoviruses associated with serious skin and gill infections in several species of sturgeon.

Received October 5, 2010; accepted October 26, 2010     

Efficacy of Vaccination against Vibriosis in Cultured Kuruma Prawns Penaeus japonicus

Abstract

Cultured kuruma prawns were experimentally vaccinated against vibriosis with formalin-killed Vibrio sp. Application of the vaccine by injection, immersion, and spray techniques all reduced mortality of the prawns when they were challenged by Vibrio injection 30 d later. Hemocytes in contact with blood homogenate from immunized prawns produced a chemokinetic factor that activated other hemocytes to migrate through a membrane in a Boyden chamber.     

Transmission of Edwardsiella ictaluri from Infected,Dead to Noninfected Channel Catfish

Abstract

Enteric septicemia of catfish (ESC) was transmitted horizontally from channel catfish Icialurus punctatus that had died from Edwardsiella ictaluri infection to contact channel catfish during 2 d of habitation in a tank. The contact channel catfish became positive for E. ictaluri antibody, became infected with this bacterium, and had signs of ESC and died within 12 d postexposure. Edwardsiella ictaluri was recovered from 24 of the 30 contact channel catfish that died from ESC, as well as from 9 of the 25 tested contact survivors. The cannibalizing of E. ictaluri-infected fish, or the shedding of E. ictaluri from dead fish, or both, were shown to be mechanisms of horizontal transmission of ESC among channel catfish.     

Mortality of American Bullfrog Tadpoles Lithobates catesbeianus Infected by Gyrodactylus jennyae and Experimentally Exposed to Batrachochytrium dendrobatidis

Abstract

The fungal pathogen Batrachochytrium dendrobatidis (Bd), which causes the disease chytridiomycosis in postmetamorphic amphibians, has been linked to amphibian population declines. Different amphibian species, however, exhibit different susceptibility to Bd pathogenicity. At the same time, agricultural pesticides commonly found contaminating aquatic habitats have been reported to increase the susceptibility of amphibians to pathogens. To investigate whether certain pesticides are able to alter the pathogenicity of Bd to larval amphibians, we exposed larval American bullfrogs Lithobates catesbeianus to end-use formulations of the herbicides atrazine or glyphosate, and then exposed them to Bd. Following the experimental exposures, a preexisting infection of the tadpoles by the monogenean ectoparasite Gyrodactylus jennyae was detected in all experimental and control tadpoles. Gyrodactylus jennyae infection intensity varied, and individuals with heavy G. jennyae infections suffered more skin erosion due to grazing by the parasite. Tadpoles experimentally exposed to Bd, or to Bd and either herbicide, had significantly reduced survival rates compared with untreated tadpoles that were only infected by G. jennyae. Increased mortality was also correlated with degree of skin erosion; survival of tadpoles with severe skin erosion was significantly reduced compared with that of tadpoles with no, or mild, skin erosion. While infected with G. jennyae, the group of tadpoles with the lowest survival rate (exposed only to Bd) included significantly more individuals exhibiting severe skin erosion and significantly fewer individuals without skin erosion, compared with the control group. These results emphasize the potential pathogenicity of gyrodactylid infections in larval amphibian hosts and suggest that concomitant exposures to Bd may enhance infections and effects of G. jennyae in bullfrog tadpoles.

Received February 3, 2012; accepted August 10, 2012     

Serological Analysis of Thermolabile Antigens of Vibrio anguillarum

Abstract

Serological analysis of thermolabile antigens of representative strains belonging to the J-O-1, J-O-2, and J-O-3 serotypes of Vibrio anguillarum, which are the major serotypes of thermostable antigens (O-antigens) in Japan, showed that the strains belonging to each O-serotype possessed a common thermolabile antigen designated as k-1. In addition to the k-1 antigen, strains of the J-O-1 serotype of biovar II V. anguillarum possessed another antigen designated as k-2. These findings were confirmed by examining additional strains belonging to each O-serotype. The k-1 antigen was found in all the strains of J-O-4, J-O-5, J-O-6, J-O-7, and J-O-8 serotypes, which are minor serotypes in Japan. The same antigen was also found in some strains of V. metschnikovii, Aeromonas hydrophila, and A. salmonicida.     

Transmission of Walleye Dermal Sarcoma and Lymphocystis via Waterborne Exposure

Abstract

A study was conducted to determine if walleye dermal sarcoma could be experimentally transmitted by waterborne exposure of tumor-free walleyes Stizostedion vitreum to tumor-positive walleyes by cohabitation. Uninfected walleyes were placed in a common raceway with tumor-positive fish for either 5 or 15 d. Direct contact between the two groups of fish was prevented by two screen barriers. The exposed fish were then maintained in the laboratory for a total of 20 weeks. Walleye dermal sarcoma developed in 89% and 71% of the fish exposed for 5 and 15 d, respectively. There was no significant difference (P > 0.05) in incidence of tumors between the two groups of fish exposed for different lengths of time.     

Physiological and Immunological Effects of Adjuvanted Aeromonas salmonicida Vaccines on Juvenile Rainbow Trout

Abstract

The effects of injectable vaccines against Aeromonas salmonicida on oxygen consumption, growth, kidney lysozyme activity, and anti-A. salmonicida plasma antibody titers of juvenile rainbow trout Oncorhynchus mykiss were examined. The vaccines were A. salmonicida bacterin only, bacterin adjuvanted with levamisole, bacterin in emulsified oil, microencapsulated bacterin, microencapsulated bacterin with muramyl dipeptide, microencapsulated bacterin with β-1,3-glucan, and microencapsulated bacterin with Vibrio anguillarum lipopolysaccharide (LPS). The greatest and broadest ranges of responses were caused by the microencapsulated bacterin with V. anguillarum LPS. Oxygen consumption rates and specific growth rates were significantly higher over the course of 1 month among fish treated with the LPS vaccine. These fish also maintained a higher anti-A. salmonicida plasma antibody titer and kidney lysozyme activity for a substantially longer period than fish receiving the other treatments.     

The Effect of High Total Ammonia Concentration on the Survival of Channel Catfish Experimentally Infected with Flavobacterium columnare

Abstract

Ammonia concentrations in water can affect the severity of Flavobacterium columnare infections in fish. Two trials lasting 7 d each were conducted to determine the effect of a single immersion flush treatment of total ammonia nitrogen (TAN; 15 mg/L) on the survival of channel catfish Ictalurus punctatus infected with F. columnare; the chemical was added while the water flowed continuously through the tanks. Both trials consisted of four treatments: (1) no ammonia exposure and no bacterial challenge (control), (2) ammonia exposure only, (3) bacterial challenge only, and (4) both ammonia exposure and bacterial challenge. Two hours after exposure to ammonia, the highest un-ionized ammonia level was 0.43 mg/L. The percent un-ionized ammonia is based on TAN, temperature, and pH. Caudal fins from three fish in each treatment were sampled at 24 h posttreatment to be analyzed by quantitative real-time polymerase chain reaction (qPCR). No significant difference in survival (mean ± SE) was noted between the channel catfish in treatment 1 (95.2 ± 1.2%) and those in treatment 2 (95.6 ± 1.0%); however, survival in both treatments 1 and 2 differed significantly from that in treatments 3 (8.5 ± 4.5%) and 4 (41.8 ± 12.7%). Treatment 4 catfish had significantly higher survival than treatment 3 catfish. Quantitative PCR data showed that treatment 4 fish had significantly less F. columnare (7.6 × 10⁵) than did treatment 3 fish (1.2 × 10⁷), and treatment 2 fish (8.5 × 10³) had significantly less bacteria than did treatment 1 fish (6.9 × 10⁴), indicating that ammonia limited the F. columnare infection. The highest mean concentration of the bacteria (3.9 × 10⁷) was found on moribund fish. The ammonia concentrations tested did not negatively influence fish survival but interfered with the infection process. An in vitro assay was also conducted to evaluate the direct effects of ammonia on F. columnare.

Received September 15, 2010; accepted May 7, 2011     

Horizontal transmission dynamics of White spot syndrome virus by cohabitation trials in juvenile Penaeus monodon and P. vannamei

White spot syndrome virus (WSSV), a rod-shaped double-stranded DNA virus, is an infectious agent causing fatal disease in shrimp farming around the globe. Within shrimp populations WSSV is transmitted very fast, however, the modes and dynamics of transmission of this virus are not well understood. In the current study the dynamics of disease transmission of WSSV were investigated in small, closed populations of Penaeus monodon and Penaeus vannamei. Pair cohabitation experiments using PCR as a readout for virus infection were used to estimate transmission parameters for WSSV in these two species. The mortality rate of contact-infected shrimp in P. monodon was higher than the rate in P. vannamei. The transmission rate parameters for WSSV were not different between the two species. The relative contribution of direct and indirect transmission rates of WSSV differed between the two species. For P. vannamei the direct contact transmission rate of WSSV was significantly lower than the indirect environmental transmission rate, but for P. monodon, the opposite was found. The reproduction ratio R₀ for WSSV for these two species of shrimp was estimated to be above one: 2.07 (95%CI 1.53, 2.79) for P. monodon and 1.51 (95%CI 1.12, 2.03) for P. vannamei. The difference in R₀ between the two species is due to a lower host mortality and hence a longer infectious period of WSSV in P. monodon.     

H5N1亚型禽流感病毒感染海兰白鸡模型的建立

为建立H5N1亚型禽流感病毒感染海兰白鸡模型,本研究选取1株鹅源H5N1高致病性禽流感病毒A/goose/guangdong/1/96(H5N1)(简称GD1/96),测定其对4周龄海兰白鸡的半数致死量.感染模型试验中,将30只4周龄海兰白鸡随机分成3组,每组10只,5只直接感染,5只同居,试验组设置一个重复,将病毒液稀释至10^4.5EID₅₀,滴鼻、点眼各0.1 mL,对照组接种PBS,感染后24 h放入同居鸡;感染后连续观察14 d,记录死亡时间,每天采集咽喉拭子和泄殖腔拭子;感染组和同居组第3、5 天各剖解3只鸡,采集气管、肺脏、脑、脾脏、肾脏和十二指肠,进行病毒分离;qRT-PCR法分析感染组和同居组第3、5 天鸡肺组织中IFN-α和TNF-α的相对表达量.结果显示,GD1/96株的鸡胚半数感染量(EID₅₀)为10^-8.167/0.1 mL,对4周龄海兰白鸡的半数致死量为10^4.5 EID₅₀.感染模型试验结果显示,以10^4.5EID₅₀的攻毒剂量感染海兰白鸡,感染组鸡在感染后8 d全部死亡;在感染和同居3 d后,各组鸡的咽喉拭子和泄殖腔拭子均可检测到病毒;感染和同居后第3、5 天,各组鸡的6种组织中均可分离到高滴度的病毒;IFN-α和TNF-α在感染组和同居组的鸡肺脏组织中的表达量均显著增加(P <0.05).本试验建立了海兰白鸡的H5N1亚型禽流感病毒感染模型,为H5N1亚型禽流感病毒的致病机理及表达抗流感基因转基因鸡的研究奠定了基础.