Koi Herpes Virus (KHV)Koi Herpes Virus (KHV)Allen C. Riggs DVM, MSAllen C. Riggs DVM, MS

Aquaculture Development Program- Aquaculture Development Program- Disease Prevention (ADP-DP)Disease Prevention (ADP-DP)

Hawaii Department of AgricultureHawaii Department of Agriculture

What is KHV?What is KHV?

• Cyprinid herpesvirus-3 or CyHV-3Cyprinid herpesvirus-3 or CyHV-3• DNA virus – family HerpesviridaeDNA virus – family Herpesviridae• Susceptible species – Common carp Susceptible species – Common carp

(Cyprinus carpio carpio), Koi (Cyprinus (Cyprinus carpio carpio), Koi (Cyprinus carpio koi) and Ghost carp (C. carpio carpio koi) and Ghost carp (C. carpio goi)goi)

• Resistant species – common goldfish Resistant species – common goldfish (Carassius auratus) and grass carp (Carassius auratus) and grass carp (Ctenopharyngodon idella)(Ctenopharyngodon idella)

KHV mortality patterns:KHV mortality patterns:

• 80 – 90% mortality in susceptible 80 – 90% mortality in susceptible populationspopulations

• Clinical disease occurs must Clinical disease occurs must commonly when water temperatures commonly when water temperatures are between 22 – 27C (72 – 81F)are between 22 – 27C (72 – 81F)

• However new cases have been However new cases have been reported in lower water temperatures reported in lower water temperatures down to 18C (64F)down to 18C (64F)

Signs of KHV:Signs of KHV:

• Non-specific Non-specific

• Gill mottling with white necrotic Gill mottling with white necrotic patches dispersed between normal red patches dispersed between normal red gill tissuegill tissue

• Bleeding gillsBleeding gills

• Sunken eyesSunken eyes

• Skin lesionsSkin lesions

• Notched noseNotched nose

Methods of transmission:Methods of transmission:

• Direct contact with infected fishDirect contact with infected fish

• Direct contact with fluids from Direct contact with fluids from infected fishinfected fish

• Direct contact with water or mud Direct contact with water or mud which infected fish had been exposed which infected fish had been exposed toto

• Fomites/vectors from contaminated Fomites/vectors from contaminated systemssystems

How do fish become How do fish become infected?infected?

• Infective virus enters susceptible fish Infective virus enters susceptible fish through gills, skin and possibly gutthrough gills, skin and possibly gut

• Depending upon water temperature, Depending upon water temperature, susceptible fish that get exposed susceptible fish that get exposed may become infected and diemay become infected and die

• OR may survive the initial outbreak OR may survive the initial outbreak and become carriers and become carriers

Latent carriersLatent carriers

• Survivors of initial infection typically Survivors of initial infection typically never show clinical signs againnever show clinical signs again

• However they are still infected and However they are still infected and can spread disease to susceptible can spread disease to susceptible fishfish

• This latent carrier possibility is what This latent carrier possibility is what makes KHV especially problematic to makes KHV especially problematic to commercial dealers *commercial dealers *

Water temperature and Water temperature and KHV:KHV:• Clinical KHV disease has an average Clinical KHV disease has an average

incubation period of 14 days following incubation period of 14 days following naïve fish exposed to infected or carrier naïve fish exposed to infected or carrier fishfish

• Most mortalities occur between 22 and Most mortalities occur between 22 and 25.5C (72 -78F)25.5C (72 -78F)

• Virtually no mortalities above 30C (86F)Virtually no mortalities above 30C (86F)

• New cases have shown clinical outbreaks New cases have shown clinical outbreaks in temperatures below 21C (70F)in temperatures below 21C (70F)

How to confirm KHV How to confirm KHV infection:infection:• No one can tell by just looking at the No one can tell by just looking at the

fish!fish!

• Laboratory tests are requiredLaboratory tests are required

• Direct methods – actually detect the Direct methods – actually detect the virus or “pieces” of its genetic materialvirus or “pieces” of its genetic material

• Indirect methods detect antibodies Indirect methods detect antibodies produced by the fish after exposure to produced by the fish after exposure to the virusthe virus

Direct methodsDirect methods

• Virus isolation (growing the virus) on Virus isolation (growing the virus) on KoiFin (KF) cell linesKoiFin (KF) cell lines

• Polymerase chain reaction (PCR) Polymerase chain reaction (PCR) • Best samples from alive then Best samples from alive then

euthanized fish – posterior kidneyeuthanized fish – posterior kidney• Non-lethal samples of blood, feces or Non-lethal samples of blood, feces or

gill clips can be used but results are gill clips can be used but results are often less reliableoften less reliable

Indirect methods:Indirect methods:

• Enzyme-linked immunosorbent assay Enzyme-linked immunosorbent assay (ELISA)(ELISA)

• Virus neutralization (VN)Virus neutralization (VN)

• Non-lethal sample using bloodNon-lethal sample using blood

• A positive ELISA or VN indicates that A positive ELISA or VN indicates that a fish has antibodies ( protective a fish has antibodies ( protective immune product) against KHV virus*immune product) against KHV virus*

No test answers all KHV No test answers all KHV questions!questions!

• Negative tests by either direct or Negative tests by either direct or indirect methods do NOT mean fish indirect methods do NOT mean fish are not carriersare not carriers

• No test definitely detects all carriers No test definitely detects all carriers or survivors of outbreaksor survivors of outbreaks

• Using diagnostic tests as a part of a Using diagnostic tests as a part of a combined preventive strategy is combined preventive strategy is currently the most practical and cost currently the most practical and cost effectiveeffective

Why can’t the diagnostic tests Why can’t the diagnostic tests give a definitive answer?give a definitive answer?

• PCR is the preferred test for PCR is the preferred test for confirmation of active clinical confirmation of active clinical infection – fish are sick and dyinginfection – fish are sick and dying

• PCR can detect some latent carriers PCR can detect some latent carriers but not all – surveillance screening of but not all – surveillance screening of “normal” populations OR fish in “normal” populations OR fish in quarantinequarantine

• PCR is the most economical and PCR is the most economical and easiest to implementeasiest to implement

What about the antibody What about the antibody tests?tests?• Fish that have antibodies against KHV mean Fish that have antibodies against KHV mean

that they have been exposed to the virus at that they have been exposed to the virus at some time in the pastsome time in the past

• Two possibilities – active infection with Two possibilities – active infection with outbreak just starting OR latent carrieroutbreak just starting OR latent carrier

• Antibody producing immune cells take time Antibody producing immune cells take time to ramp up with new infections – false to ramp up with new infections – false negativesnegatives

• Same cells may slow down or stop antibody Same cells may slow down or stop antibody production if non-lethal infection occurred a production if non-lethal infection occurred a long time ago and fish no longer sick – false long time ago and fish no longer sick – false negative on carrier statenegative on carrier state

Individual vs population Individual vs population screening:screening:

• Antibody testing is the preferred method Antibody testing is the preferred method for detecting carrier status in valuable for detecting carrier status in valuable individuals * individuals *

• But what about testing groups of 10, 100 But what about testing groups of 10, 100 or 1000 fish?or 1000 fish?

• Unless you test them all by antibody Unless you test them all by antibody detection methods and get all true detection methods and get all true negative results each time – can NOT be negative results each time – can NOT be 100% sure 100% sure

How to prevent KHV:How to prevent KHV:

• Quarantine (Q)Quarantine (Q)

• Water temperature in between 21 – Water temperature in between 21 – 27C (70 – 80F) …… 24C (75F)*27C (70 – 80F) …… 24C (75F)*

• 30 day minimum time period30 day minimum time period

• ““Reverse quarantine” after 30 days Reverse quarantine” after 30 days initial Q periodinitial Q period

• Diagnostic screening testsDiagnostic screening tests

QuarantineQuarantine

• DefinitionDefinition– Separate tank or Separate tank or

pond w/ separate pond w/ separate water & filtration water & filtration systemsystem

– Preferably located Preferably located away from the main away from the main pondpond

• Time PeriodTime Period4-6 weeks 4-6 weeks

Can KHV be treated?Can KHV be treated?

• No treatment for KHV!No treatment for KHV!

• Virus can remain viable for 3 days in Virus can remain viable for 3 days in water without fish hostswater without fish hosts

• Common disinfection protocols can Common disinfection protocols can eliminate the virus from eliminate the virus from contaminated systems and contaminated systems and equipmentequipment

Regulatory Concerns with Regulatory Concerns with KHV:KHV:

• 2007 – World Organization for Animal 2007 – World Organization for Animal Health (OIE) added KHV to the Health (OIE) added KHV to the notifiable disease list for finfishnotifiable disease list for finfish

• USDA-APHIS asks accredited USDA-APHIS asks accredited veterinarians and diagnostic labs to veterinarians and diagnostic labs to report positive cases to the AVIC of report positive cases to the AVIC of the state in which the case occurredthe state in which the case occurred

Regulatory Concerns with Regulatory Concerns with KHV:KHV:• USDA-APHIS has no mandatory USDA-APHIS has no mandatory

requirements on KHV status for koi requirements on KHV status for koi moving interstate or internationalmoving interstate or international

• No mandatory depopulation for No mandatory depopulation for positive KHV fishpositive KHV fish

• KHV now a Hawaii State reportable KHV now a Hawaii State reportable disease – positive cases must be disease – positive cases must be reported to the Hawaii State reported to the Hawaii State VeterinarianVeterinarian

Spring Viremia of Carp Spring Viremia of Carp (SVC)(SVC)• RNA virusRNA virus• Affects koi & goldfishAffects koi & goldfish• Mortality up to 70% in affected populationsMortality up to 70% in affected populations• Temperature range Temperature range

12-24°C12-24°C54-72°F54-72°F

• OIE reportable diseaseOIE reportable disease• FAD and mandatory depopulation required FAD and mandatory depopulation required

by both State and USDA-APHISby both State and USDA-APHIS

New USDA Guidelines for New USDA Guidelines for SVCSVC• 8 spp. of fish that are susceptible to SVC 8 spp. of fish that are susceptible to SVC

are now under USDA oversight for are now under USDA oversight for importation into the U.S.importation into the U.S.

• Required documentsRequired documents– USDA import permit (form VS-135)USDA import permit (form VS-135)– Veterinary health certificate from the exporting Veterinary health certificate from the exporting

countrycountry

• USDA VS Port Veterinarian must inspect USDA VS Port Veterinarian must inspect each shipment at entry porteach shipment at entry port