Recipient Organization
UNIV OF MASSACHUSETTS
(N/A)
AMHERST,MA 01003
Performing Department
Microbiology
Non Technical Summary
The world fisheries production has levelled off and most of the main fishing areas have reached their maximum potential. In contrast, the global human population is increasing; thus, the demand for aquatic food products also increase. Global aquaculture production attained 90.4 million tons in 2012, generating an incomes US$ 144.4 billion, and the production of food fish was 66.6 million tons. Epitheliocystis is a serious skin and gill disease in fish, believed to be caused by pathogenic intracellular bacteria. The disease has been reported in at least 90 species of marine and freshwater fish in both the southern and northern hemisphere. It affects a number of commercially important aquaculture species, including salmon, kingfish, trout and bream. Infection in these fish species is characterized by the development of cysts, typically in the gill epithelia, promoting the fusion of gill lamellae. This condition is usually benign; however, sometimes it can be associated with a high mortality, particularly in cultured fish. Infections can lead to respiratory distress and death, particularly in cultured and juvenile fish. While fish mortality data attributed to epitheliocystis is sparse, a remarkably broad range of 4%-100% observed mortality rates has been reported. Even with the recent molecular advances in identifying the pathogens, the reservoirs and modes of transmission of the etiologic agent(s) of this disease remain largely unknown. Bacteria belonging to the order Chlamydiales are an extremely important and diverse group of pathogens of vertebrates, which include respiratory diseases of fishes. Chlamydial infections of fish are emerging as an important cause of epitheliocystis in new and established aquaculture industries [6, 8]. However, empirical data and direct evidence relating to the etiology, treatments and epidemiology remain limited, highlighting the need for more work to better characterize this disease across the different hosts and locales affected.We hypothesize that Chlamydia-like organisms are an important etiologic agent of epitheliocystis. We believe that a better understanding of disease transmission, mechanism of disease process and host range will provide a basis for preventing this emerging disease in aquaculture. Upon successful completion of this project, we will (1) have a better understanding of the etiologic agent(s) of epitheliocystis; (2) have an understanding of how the disease is transmitted and possible reservoirs in the aquaculture setting for common fish species; (3) determine the treatment regimen most effective at preventing and eliminating epitheliocystis in commercial fisheries settings and (4) pilot an effective screening/diagnostic test for early detection and confirmation of epitheliocystis
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The major goals of this project are:1. Determine the etiologic agent(s) of epitheliocystis in specific fish species i. We plan to use a combination of electron microscopy and molecular tools, such as qPCR and sequencing to identify pathogens associated with outbreaks of epitheliocystis. ii. We will utilize modern tissue culture techniques to cultivate isolated microbes in the lab for further analysis2. Determine the mode of disease transmission i. We will utilize a fish model of infection to determine if the culture expanded isolates can cause epitheliocystis in an experimental lab setting, thereby establishing required dose, transmission and associated pathology. ii. Determine if environmental conditions that approach or exceed animals' physiological tolerances (e.g. atypical temperature, salinity or pH levels) contribute to disease development and progression.3. Determine the most effective prevention and treatment methods We hope to establish intervention strategies to prevent or reduce the impact of epitheliocystis on wildlife and the aquaculture industry. We will pilot different antimicrobial treatment regimens based on the mode of transmission and progression of disease.4. Development of an effective diagnostic/screening test for epitheliocystis i. We will explore the use of a combination of 16S rRNA, FISH and easy to use LAMP assays to determine early infection in aquaculture settings. ii. Pilot the most effective assay in small and large fisheries settings
Project Methods
1. Determine the etiologic agent(s) of epitheliocystis in specific fish species We will obtain biopsy samples from fish diagnosed with epitheliocystis through histological examination and electron microscopy. Infection characteristically presents as severe cellular hypertrophy, forming cysts within the skin epithelium or on the gill filaments. The presence of the microbes will be confirmed using molecular tools and would entail sequencing to determine the identity of the epitheliocystis agent (s). Chlamydiae thrive in amoebae naturally and in vitro, therefore we will initially try to cultivate epitheliocystis agents in amoebae. We will utilize Acanthamoeba species as the initial method of cultivation. We will simultaneously attempt to cultivate the epitheliocystis agent in fish epithelial cell lines (RTgill-W1/ATCC CRL-2423, EPC-175 (fathead minnow, RTG-2 (rainbow trout) and G1B/ATCC CRL2536). A mouse macrophage cell line (J774A.1) that has been used to cultivate Chlamydia from human clinical samples will also be utilized to maximize our chances of cultivating the etiologic agent(s).Direct infection: In order to determine if the microbe(s) isolated and cultivated above play a role in the development of epitheliocystis, we will introduce the microbes to three susceptible fish species (salmon, kingfish and trout) in regulated fish tanks. Each control and test tank will have 10 fish. We will place four different inclusion forming units (IFU) of the intracellular microbes in these tanks and a killed control, maintaining a steady temperature, pH and salinity. We will later repeat these experiments on two additional fish species, to determine species-specific variations in infection and pathology. W. Webley, Department of Microbiology Infected fish transmission: In order to determine different modes of transmission among fish, we will place one diseased fish with epitheliocystis in a tank with 10 healthy fish and observe for signs of disease transmission as well as microbes in the water through microscopy and molecular genetics tools (qPCR). We will closely monitor time to first notable transmission as well as all the environmental conditions of each tank. Amoeba-mediated infection model: Free-living amoebae are ubiquitous in the environment, especially water [23]. The Chlamydiales order contains many amoeba resistant bacteria, such as Parachlamydia acanthamoebae or Simkania negevensis that have previously been implicated in human respiratory infections [23]. We hypothesize that the agent of epitheliocystis could be transmitted via infected amoeba. We will therefore infect Acanthamoeba with Chlamydia-like bacteria and once we confirm infection through qPCR we will introduce them into tanks of healthy fish (10 fish in each tank and all three species as outlined above). This will allow us to determine if they can infect the fish when taken up, or when the bacteria are introduced into the water through infected the amoeba. Alternative experiments: Since the etiologic agents of epitheliocystis appear to have species-specific infectivity[2], it is likely that the species of fish we have chosen in this set of infection might now be compatible and therefore not show cysts typical of the disease pathology. We will there utilize histology and molecular techniques to confirm infection, even in the absence of obvious pathology. We will also explore the role of vertical transmission from eggs.2. Determine the mode and efficiency of disease transmission It is possible that various environmental conditions affect transmission of the epitheliocystis agent. We will therefore test the effect of pH, temperature variations and salinity on rates of transmission and severity of disease presentation. We will utilize 10 fish per tank as above for each pH range from neutral to basic and slightly acidic and introduce the infecting agent at a concentration that we would have previously determined can readily cause disease. We will then observe them for rate of disease transmission across the range of pH, compared to a normal pH control. Similarly, we will establish a temperature range that allows the fish to still remain comfortable and compare disease transmission rates and severity of symptoms through histology and microscopy findings. Finally, we will examine the effect of salinity in different tanks on transmission of epitheliocystis among fish from different species, while all other environmental parameters are held constant.3. Determine the most effective prevention and treatment methods One of our goal for this study is to find meaningful ways to prevent epitheliocystis in aquaculture settings and also to stop the spread of the disease once it has started. To determine the effectiveness of these treatment interventions, we will add increasing concentrations of azithromycin, doxycycline or a combination of these antibiotics to tanks of 10 uninfected fish. We will then introduce an optimal amount of the epitheliocystis bacteria into the tank to see if a specific concentration of antibiotics would prevent infection. We will repeat these experiments in tanks containing one, three and five infected fish respectively, to determine if after an infection has been established, antimicrobial treatment could halt W. Webley, Department of Microbiology the spread or lead to recovery from the disease. Fish will be examined through gross observations, histopathology evaluations as well as molecular and culture techniques for the presence of epitheliocystis.4. Development of an effective diagnostic/screening test for epitheliocystis Early diagnosis of epitheliocystis will no doubt be an important step in preventing further spread and could save farmers millions of dollars each year. Currently, diagnosis is initially based on the presence of macroscopic lesions or changes in fish behavior. Later, many have used transmission electron microscopy of molecular methods to confirm the presence and type of the etiologic agent. However, there is no standard, consistent method to confirm epitheliocystis, especially in early disease. Since there are most likely several species of microbes causing epitheliocystis and determine which species could significantly impact intervention methods and treatment course, having a sensitive, specific and simple system would be desirable. We will explore the use of 16S rRNA-based primers that contain both variable and conserved domains to provide the flexibility of differentiating species, genus, family and even phylum level differences in isolates. We will determine if a loop isothermal amplification (LAMP Assay) method can be adopted for this purpose since it would not require sophisticated equipment and specialized personnel to complete the screening task. This assay only requires a heating block and results can be obtained in less than two hours, once the assay is optimized.