Department of Microbiology and Immunology


Current Research Highlights

 

Strontium Chloride Target Animal Safety Study

There is a need to develop alternative, reliable, and cost-effective methods of marking otoliths in hatchery-raised salmonids as well as other cultured fish species. Fish use otoliths for balance, orientation, and sound detection. They function similarly to the inner ear of mammals. Chemical marking involves incorporating biologically rare elements or compounds into body tissues and is an efficient means of marking large numbers of small fish. This work addressed the need for alternative marking methods to replace the potential loss of oxytetracycline as an FDA-approved marking compound for fish. The study is significant because otolith marking has proven to be an effective tool to determine the hatchery origin of individual fish in high seas, coastal waters, and the lakes and rivers around the globe. Strontium chloride hexahydrate has been used as a skeletal marking agent for Pacific salmon fry administered at a dosage of 3,000 ppm by immersion for 24 hours. Strontium chloride appears to be an ideal chemical mark.

Chinook Salmon fry were tested because they are representative of the freshwater-reared, Pacific salmon and life stage most likely to be treated with strontium chloride. A strontium chloride concentration of 3000 ppm was used as the 1x dose as it has been shown to be an efficacious dose for Sockeye Salmon fryOncorhynchus nerka during 10 years of INAD (10-536) permitted use at the Gulkana Hatchery in Alaska. This study tested whether fish immersed in strontium chloride hexahydrate for three times the standard duration (24 hours) and one, three and five times the standard concentration (3,000 ppm) showed adverse signs, mortalities, gross or histological lesions. This project provides needed safety data to support approval for a new marking compound, strontium chloride. 

The mortality for fish immersed in the 5x strontium chloride exposure group was higher than that observed in the other exposure groups. A dose-related effect on general fish behavior and feeding behavior was observed. Fish in all test tanks consumed 100% of feed, except during days 2 and 3 for the 15,000 ppm concentration. Fish in all other test tanks behaved normally.  No dose-related effect was detected on fish growth. Fish in the 5x exposure group had a significantly higher number of gill lesions as compared to the 0x group (see gill photo). The margin of safety for strontium chloride when administered to Chinook Salmon fry by immersion for 3 consecutive days extends to at least 9,000 ppm.

Gill Separation
Gill epithelial separation (H&E 40X)

Recent Strontium Chloride Research Presentation:

R.G. Getchell, E.R. Cornwell, H. Marquis, and P.R. Bowser.  2015. The safety of strontium chloride as a skeletal marking agent for Pacific Salmon.  21th Annual USFWS Drug Approval Coordination Workshop. 30 July 2015. Bozeman, MT.

 

Role of Microbiota in the Control of Bacterial Infections in Aquaculture

The skin of fish and its mucus layer play an important role in maintaining homeostasis.  This tissue is populated with communities of bacteria and bacteriophages, identified as the microbiota.  Some of the bacterial species that form the microbiota are likely to modulate the fish susceptibility to bacterial infections, whereas the bacteriophage population has the power of influencing the dynamics of the bacterial communities within the microbiota.  Our lab is interested in (1) defining the effects of specific environmental changes on the composition of the skin mucus microbiota, and the impact of these changes on the fish susceptibility to bacterial infections; and in (2) identifying bacterial species and bacteriophages that have an impact on the growth/survival of bacterial pathogens causing diseases in fish.  We hope that results from this study will lead to the development of sustainable approaches for preventing and controlling outbreaks of bacterial infections in aquaculture.

 

Development of a Phage-Based Diagnostic Test for the Rapid Detection of Pathogenic Vibrio species in Bivalves

The prevalence of gastrointestinal illnesses associated with the consumption of Atlantic coast shellfish contaminated with Vibrio parahaemolyticus and V. vulnificus has increased over the years, having a negative impact on the industry due to recalls and loss of consumer confidence in the product.  We are developing a rapid and highly sensitive bacteriophage-based diagnostic test for early detection of shellfish contamination by virulent strains of these two bacterial pathogens.  The first objective of this project is to isolate and characterize phages that infect V. parahaemolyticus and V. vulnificus.  These phages are being isolated from water samples in which these bacteria thrive along the Atlantic northeast coast.  Isolated phages will be characterized to differentiate those that infect environmental bacterial strains not associated with infection from those that infect isolates from clinical cases (virulent strains).  In the second phase of this project, selected phages will be modified to express a marker that will be easily detectable in infected samples, bypassing the requirement for an enrichment phase.  The ultimate goal of this research project is to develop a test that will be specific, sensitive, cost-effective, and rapid for the detection of shellfish contaminated with V. parahaemolyticus and V. vulnificus.  This test will be beneficial to the industry and the consumers because it will provide a reliable and rapid tool to monitor contamination at any step of the process from pre-harvest to table.

This project is a collaboration between Dr. Helene Marquis, Dr. Martin Wiedmann, and Dr. Rod Getchell from Cornell University, Mr. Greg Rivara from Cornell University Cooperative Extension Suffolk County, and Dr. Cheryl Whistler and Dr. Stephen Jones from the University of New Hampshire.

 

Insect Larvae As A Substitute For Fishmeal In Aquaculture Feed: Effects On Fish Growth, Health, Taste And Nutritional Value

The long-term goal of this project is to develop a sustainable diet that decreases the need for fish-based ingredients in the aquaculture industry.  For this purpose, we are assessing the value of fly larvae as a substitute for fishmeal used in fish diets.  In preliminary studies, the potential of larva meal in rainbow trout diet was evaluated: it assessed that the nutritional value of larva meal closely matched with the fishmeal one.  Also, the diet was palatable for fish, sustained fish growth, and did not cause intestinal pathology.  Hence, we are expanding the validation of larva meal as a replacement for fish meal in rainbow trout and Atlantic salmon diets.  1: Fish growth rate will be monitored until market size for trout or smolt stage for Atlantic salmon.  2: Gross and histological evaluation of tissues will be performed,  the effect of diet on intestinal and skin mucus microbiota will be defined, and the competence of fish to fight disease and mount a protective immune response to vaccination will be evaluated.  3: The palatability and consumer acceptance of the product will be studied.  4: The nutritional value will be determined.  At completion of this study, we will know whether larva meal can sustain fish growth, health, palatability, and nutritional value. 

This project is a collaboration between Dr. Helene Marquis, Dr. Vimal Selvaraj, Dr. Robin Dando, and Dr. Paula Ospina from Cornell University.

 

Effects of Sub-Inhibitory Levels of Antibiotics in Water on Colonization and Transmission of Waterborne Bacterial Pathogens and Enrichment of Resistant Microbiota

Exposure of bacteria to sub-inhibitory levels of antibiotics induces changes in bacterial gene expression promoting biofilm formation, mutagenesis, and horizontal gene transfer, as well as modulating bacterial metabolism and virulence amongst other effects.  Moreover, sub-inhibitory levels of antibiotics promote the emergence of resistance in bacterial populations.  Considering that sub-inhibitory levels of antibiotics are commonly found in natural water sources, it is reasonable to speculate that the aquatic microbial community is under substantial pressure.  Therefore, we hypothesize that (i) chronic exposure to sub-inhibitory levels of antibiotics perturbs the make-up of the fish gut microbiota, enhancing the probability of colonization with and transmission of zoonotic waterborne bacterial pathogens as well as modulating the level, and duration of infectiousness post colonization; and (2) significantly promote the acquisition of antibiotic resistance in the intestinal microbiota of exposed fish.  To address these hypotheses, we will be using zebrafish as an animal model.  We hope that the information obtained from these studies will be instrumental (i) in modeling the risks of zoonotic waterborne infections, (ii) in shaping policies regarding the monitoring of antibiotics in natural water, (iii) and the design of measures to prevent disease, and (iv) in mitigating the emergence of antibiotic resistance.  These goals are imperative for sustaining animal and human health.

This project is a collaboration between Dr. Helene Marquis and Dr. Renata Ivanek from Cornell University.

 

Viral Hemorrhagic Septicemia in New York State

Viral Hemorrhagic Septicemia Virus (VHSV) was identified in the freshwater environment of North America for the first time in 2005. The virus was found in two separate instances: in muskellunge from Lake St. Clair, Michigan by researchers at Michigan State University and in freshwater drum from the Bay of Quinte, on the Canadian shore of Lake Ontario by researchers from the University of Guelph in Ontario, Canada.

VHSV was first found in New York State in round gobies collected during a mortality event in the St. Lawrence River. The fish were submitted by the NYS DEC to the Aquatic Animal Health Program at Cornell University in May, 2006. Since that time the Aquatic Animal Health Program has had confirmed cases of VHSV in numerous other fish species from the St. Lawrence River, Lake Ontario, Lake Erie, Conesus Lake, Skaneatles Lake, and the Canal between Cayuga Lake and Seneca Lake. During surveillance efforts, VHSV also was confirmed in baitfish, including bluntnose minnows from the St. Lawrence River and emerald shiners from the Niagara River and Lake Erie. VHSV has also been found and confirmed in Lake Huron, Lake Michigan, and Lake Superior. Inland water bodies outside of New York State with confirmed cases include:  Winnebago chain of lakes (Wisconsin); Budd Lake (Michigan), Clear Fork Reservoir (Ohio), and Baseline Lake (Michigan).

Muskellunge
Muskellunge (57", 45 lbs.) mortality due to VHSV from the St. Lawrence River, NY

Background on VHSV

VHSV is a Rhabdovirus. There are other known fish rhabdoviruses (Infectious Hematopoietic Necrosis Virus, Spring Viremia of Carp Virus), but the most well known member of the Rhabdovirus family is the rabies virus. It is important to note here that VHSV does not infect humans. As a Rhabdovirus, VHSV is an RNA virus that has an envelope. Viruses, in general, must reside in a living cell and can survive outside of a living cell for a relatively short time. Historically, VHSV has been known in Europe as the most serious viral disease of rainbow trout reared in freshwater. More recently, information suggests that VHSV may have actually been a disease of a variety of saltwater fish species and gained access to the freshwater environment in Europe when it was common for unpasteurized fish caught in the marine environment to be used to feed rainbow trout. In 1988 and 1989 VHSV was found in apparently healthy returning Chinook and coho salmon in the Puget Sound area of Washington State. Those isolations constituted the first documentation of VHSV in the Western Hemisphere. Since 1988, VHSV has been isolated from a variety of marine fish species on both the Pacific and Atlantic coasts of North America. The identification of VHSV in 2005 were the first documentations of VHSV in the freshwater environment of North America.

Detection Methods for VHSV

Procedures used to detect VHSV require that a laboratory be appropriately equipped to work with fish viruses. Presence of the virus cannot be determined without rather sophisticated laboratory testing. The specific methods used to detect VHSV are provided in two resources: the OIE (World Animal Health Association) Manual and the Fish Health Section/American Fisheries Society document titled "Suggested Procedures for the Detection and Identification of Certain Finfish and Shellfish Pathogens." There is a two-step procedure used to detect the virus: (1) The virus is first isolated in a cell culture. In this procedure the cell culture is examined for destruction of the cells, called cytopathic effects or CPE. Since there are several different viruses that may cause CPE, the specific identity of the virus must then be determined. (2) The specific identity of the virus is determined by either the use of a specific antibody against the virus or by some kind of molecular technique where a specific portion of the genetic structure of the virus is detected. In the former case, a test called a serum neutralization is performed and in the latter case, a Polymerase Chain Reaction (PCR), or in the case of VHSV as an RNA virus, an RT-PCR is performed. Our Aquatic Animal Health Program at Cornell developed a Quantitative RT-PCR (qRT-PCR) for VHSV. Our use of the qRT-PCR showed that it is far more capable of detecting the virus even when it is present in low quantities. The test also lends itself to the processing of large quantities of samples. The qRT-PCR has gone through a process called "validation," where it was compared against the current accepted testing methodologies (OIE Manual and Fish Health Section Blue Book) for sensitivity and specificity.

VHSV Transmission

VHSV can be transmitted by several means including -- an infected fish shedding virus into the water to infect an non-infected fish, a predatory fish eating a prey species of fish that is infected, infected brood fish that shed the virus with the reproductive products to result in infected young fish, movement of virus in water that is used to transport infected fish, contamination of fish handling equipment (nets, buckets, hauling tanks, etc.) with virus, movement of virus by animals other than fish (amphibians, reptiles, birds, small mammals). There may be other means of movement of the virus that will result in infecting fish. In general it is not advisable to move fish, that are known to be infected, to a new location whether that new location is disease free or that new location contains infected fish. In the case of VHSV this should not be done until we know a lot more about this virus as well as such things as the dynamics of how this virus can survive in the environment and what constitutes an infectious dose under different conditions.

Recent VHSV Research References: 

R.G. Getchell, T. Erkinharju, A.O. Johnson, B.W. Davis, E.E. Hatch, E.R. Cornwell, P.R. Bowser.  2015.  Goldfish Carassius auratus susceptibility to viral hemorrhagic septicemia virus (VHSV) genotype IVb depends on route of exposure.  Diseases of Aquatic Organisms 115:25–36.

Cornwell, E.R., G. B, Anderson, D. Coleman, R.G. Getchell, G.H. Groocock, J.V. Warg, A.M. Cruz, J.W. Casey, M.B. Bain, P.R. Bowser.  2015.  Applying multi-scale occupancy models to infer host and site occupancy of an emerging viral fish pathogen in the Great Lakes.  Journal of Great Lakes Research 41:520-529.

J.V. Warg, T. Clement, E.R. Cornwell, A. Cruz, R.G. Getchell, C. Giray, A.E. Goodwin, G.H. Groocock, M. Faisal, R. Kim, G.E. Merry, N. Phelps, M.M. Reising, I. Standish, Y. Zhang, K. Toohey-Kurth. 2014. Detection and surveillance of viral hemorrhagic septicemia virus using real-time RT-PCR. I. Initial comparison of four protocols. Diseases of Aquatic Organisms 111:1-13.

Imanse, S.M., E.R. Cornwell, R.G. Getchell, G. Kurath, and P.R. Bowser.  2014.  In vivo and in vitro phenotypic differences between Great Lakes VHSV genotype IVb isolates with sequence types vcG001 and vcG002.  Journal of Great Lakes Research 40:879-885.

R.G. Getchell, E.R. Cornwell, G.H. Groocock, P.T. Wong, L.L. Coffee,  G.A.Wooster, and P.R.Bowser  2013.  Experimental transmission of VHSV genotype IVb by predation. Journal of Aquatic Animal Health 25:221-229. 

E.R. Cornwell, C.A. Bellmund, G.H. Groocock, P.T. Wong, K.L. Hambury, R.G. Getchell, Paul R Bowser. 2013. Fin and gill biopsies are effective nonlethal samples for detection of Viral hemorrhagic septicemia virus genotype IVb. Journal of Veterinary Diagnostic Investigation.

 

Recent VHSV Research Presentations:

R.G. Getchell, E.R. Cornwell, and P.R. Bowser.  2014.  Recurrence of VHS outbreaks in the Lower Great Lakes. International Symposium on Aquatic Animal Health Aug 31 – Sept 4, 2014. Portland, OR. 

R.G. Getchell, E.E. Hatch, A.O. Johnson, E.R. Cornwell, G.A. Wooster, T. Erkinharju, and P.R. Bowser.  2014. Susceptibility of goldfish to viral hemorrhagic septicemia virus type IVb by intraperitoneal injection. 39th Annual Eastern Fish Health Workshop.  28 April – 2 May 2014.  Shepherdstown, WV.

R.G. Getchell, E.R. Cornwell, G.H. Groocock, P.T. Wong, L.L. Coffee, G.A. Wooster, and P.R. Bowser.  2012.  Consumption of infected fathead minnows transmits viral hemorrhagic septicemia virus genotype IVb to tiger muskellunge. American Fisheries Society Fish Health Section Annual Meeting.  31 July – 3 August 2012.  La Crosse, WI.

VHSV Articles in the Cornell Chronicle:
14 June 2006
19 July 2006
14 February 2007
17 May 2007
1 January 2010
5 May 2010
20 October 2010
18 March 2013

References Describing Initial VHSV Outbreaks: 
USDA APHIS Emerging Disease Notice (July 2006)
USDA APHIS Industry Alert (August 2006)
USDA APHIS Questions and Answers about the VHSV Federal Order (November 2006)
Fish Species Affected by the VHSV Federal Order (effective 8 November 2007)
VHSV Slide Presentation 20 March 2007