Koi Herpes Virus (KHV) Allen C. Riggs DVM, MS Aquaculture Development Program- Disease Prevention (ADP-DP) Hawaii Department of Agriculture

What is KHV? Cyprinid herpesvirus-3 or CyHV-3 Cyprinid herpesvirus-3 or CyHV-3 DNA virus – family Herpesviridae DNA virus – family Herpesviridae Susceptible species – Common carp (Cyprinus carpio carpio), Koi (Cyprinus carpio koi) and Ghost carp (C. carpio goi) Susceptible species – Common carp (Cyprinus carpio carpio), Koi (Cyprinus carpio koi) and Ghost carp (C. carpio goi) Resistant species – common goldfish (Carassius auratus) and grass carp (Ctenopharyngodon idella) Resistant species – common goldfish (Carassius auratus) and grass carp (Ctenopharyngodon idella)

KHV mortality patterns: 80 – 90% mortality in susceptible populations 80 – 90% mortality in susceptible populations Clinical disease occurs must commonly when water temperatures are between 22 – 27C (72 – 81F) Clinical disease occurs must commonly when water temperatures are between 22 – 27C (72 – 81F) However new cases have been reported in lower water temperatures down to 18C (64F) However new cases have been reported in lower water temperatures down to 18C (64F)

Signs of KHV: Non-specific Non-specific Gill mottling with white necrotic patches dispersed between normal red gill tissue Gill mottling with white necrotic patches dispersed between normal red gill tissue Bleeding gills Bleeding gills Sunken eyes Sunken eyes Skin lesions Skin lesions Notched nose Notched nose

Methods of transmission: Direct contact with infected fish Direct contact with infected fish Direct contact with fluids from infected fish Direct contact with fluids from infected fish Direct contact with water or mud which infected fish had been exposed to Direct contact with water or mud which infected fish had been exposed to Fomites/vectors from contaminated systems Fomites/vectors from contaminated systems

How do fish become infected? Infective virus enters susceptible fish through gills, skin and possibly gut Infective virus enters susceptible fish through gills, skin and possibly gut Depending upon water temperature, susceptible fish that get exposed may become infected and die Depending upon water temperature, susceptible fish that get exposed may become infected and die OR may survive the initial outbreak and become carriers OR may survive the initial outbreak and become carriers

Latent carriers Survivors of initial infection typically never show clinical signs again Survivors of initial infection typically never show clinical signs again However they are still infected and can spread disease to susceptible fish However they are still infected and can spread disease to susceptible fish This latent carrier possibility is what makes KHV especially problematic to commercial dealers * This latent carrier possibility is what makes KHV especially problematic to commercial dealers *

Water temperature and KHV: Clinical KHV disease has an average incubation period of 14 days following naïve fish exposed to infected or carrier fish Clinical KHV disease has an average incubation period of 14 days following naïve fish exposed to infected or carrier fish Most mortalities occur between 22 and 25.5C (72 -78F) Most mortalities occur between 22 and 25.5C (72 -78F) Virtually no mortalities above 30C (86F) Virtually no mortalities above 30C (86F) New cases have shown clinical outbreaks in temperatures below 21C (70F) New cases have shown clinical outbreaks in temperatures below 21C (70F)

How to confirm KHV infection: No one can tell by just looking at the fish! No one can tell by just looking at the fish! Laboratory tests are required Laboratory tests are required Direct methods – actually detect the virus or “pieces” of its genetic material Direct methods – actually detect the virus or “pieces” of its genetic material Indirect methods detect antibodies produced by the fish after exposure to the virus Indirect methods detect antibodies produced by the fish after exposure to the virus

Direct methods Virus isolation (growing the virus) on KoiFin (KF) cell lines Virus isolation (growing the virus) on KoiFin (KF) cell lines Polymerase chain reaction (PCR) Polymerase chain reaction (PCR) Best samples from alive then euthanized fish – posterior kidney Best samples from alive then euthanized fish – posterior kidney Non-lethal samples of blood, feces or gill clips can be used but results are often less reliable Non-lethal samples of blood, feces or gill clips can be used but results are often less reliable

Indirect methods: Enzyme-linked immunosorbent assay (ELISA) Enzyme-linked immunosorbent assay (ELISA) Virus neutralization (VN) Virus neutralization (VN) Non-lethal sample using blood Non-lethal sample using blood A positive ELISA or VN indicates that a fish has antibodies ( protective immune product) against KHV virus* A positive ELISA or VN indicates that a fish has antibodies ( protective immune product) against KHV virus*

No test answers all KHV questions! Negative tests by either direct or indirect methods do NOT mean fish are not carriers Negative tests by either direct or indirect methods do NOT mean fish are not carriers No test definitely detects all carriers or survivors of outbreaks No test definitely detects all carriers or survivors of outbreaks Using diagnostic tests as a part of a combined preventive strategy is currently the most practical and cost effective Using diagnostic tests as a part of a combined preventive strategy is currently the most practical and cost effective

Why can’t the diagnostic tests give a definitive answer? PCR is the preferred test for confirmation of active clinical infection – fish are sick and dying PCR is the preferred test for confirmation of active clinical infection – fish are sick and dying PCR can detect some latent carriers but not all – surveillance screening of “normal” populations OR fish in quarantine PCR can detect some latent carriers but not all – surveillance screening of “normal” populations OR fish in quarantine PCR is the most economical and easiest to implement PCR is the most economical and easiest to implement

What about the antibody tests? Fish that have antibodies against KHV mean that they have been exposed to the virus at some time in the past Fish that have antibodies against KHV mean that they have been exposed to the virus at some time in the past Two possibilities – active infection with outbreak just starting OR latent carrier Two possibilities – active infection with outbreak just starting OR latent carrier Antibody producing immune cells take time to ramp up with new infections – false negatives Antibody producing immune cells take time to ramp up with new infections – false negatives Same cells may slow down or stop antibody production if non-lethal infection occurred a long time ago and fish no longer sick – false negative on carrier state Same cells may slow down or stop antibody production if non-lethal infection occurred a long time ago and fish no longer sick – false negative on carrier state

Individual vs population screening: Antibody testing is the preferred method for detecting carrier status in valuable individuals * Antibody testing is the preferred method for detecting carrier status in valuable individuals * But what about testing groups of 10, 100 or 1000 fish? But what about testing groups of 10, 100 or 1000 fish? Unless you test them all by antibody detection methods and get all true negative results each time – can NOT be 100% sure Unless you test them all by antibody detection methods and get all true negative results each time – can NOT be 100% sure

How to prevent KHV: Quarantine (Q) Quarantine (Q) Water temperature in between 21 – 27C (70 – 80F) …… 24C (75F)* Water temperature in between 21 – 27C (70 – 80F) …… 24C (75F)* 30 day minimum time period 30 day minimum time period “Reverse quarantine” after 30 days initial Q period “Reverse quarantine” after 30 days initial Q period Diagnostic screening tests Diagnostic screening tests

Quarantine Definition Definition –Separate tank or pond w/ separate water & filtration system –Preferably located away from the main pond Time Period Time Period 4-6 weeks

Can KHV be treated? No treatment for KHV! No treatment for KHV! Virus can remain viable for 3 days in water without fish hosts Virus can remain viable for 3 days in water without fish hosts Common disinfection protocols can eliminate the virus from contaminated systems and equipment Common disinfection protocols can eliminate the virus from contaminated systems and equipment

Regulatory Concerns with KHV: 2007 – World Organization for Animal Health (OIE) added KHV to the notifiable disease list for finfish 2007 – World Organization for Animal Health (OIE) added KHV to the notifiable disease list for finfish USDA-APHIS asks accredited veterinarians and diagnostic labs to report positive cases to the AVIC of the state in which the case occurred USDA-APHIS asks accredited veterinarians and diagnostic labs to report positive cases to the AVIC of the state in which the case occurred

Regulatory Concerns with KHV: USDA-APHIS has no mandatory requirements on KHV status for koi moving interstate or international USDA-APHIS has no mandatory requirements on KHV status for koi moving interstate or international No mandatory depopulation for positive KHV fish No mandatory depopulation for positive KHV fish KHV now a Hawaii State reportable disease – positive cases must be reported to the Hawaii State Veterinarian KHV now a Hawaii State reportable disease – positive cases must be reported to the Hawaii State Veterinarian

Spring Viremia of Carp (SVC) RNA virus RNA virus Affects koi & goldfish Affects koi & goldfish Mortality up to 70% in affected populations Mortality up to 70% in affected populations Temperature range Temperature range12-24°C54-72°F OIE reportable disease OIE reportable disease FAD and mandatory depopulation required by both State and USDA-APHIS FAD and mandatory depopulation required by both State and USDA-APHIS

New USDA Guidelines for SVC 8 spp. of fish that are susceptible to SVC are now under USDA oversight for importation into the U.S. 8 spp. of fish that are susceptible to SVC are now under USDA oversight for importation into the U.S. Required documents Required documents –USDA import permit (form VS-135) –Veterinary health certificate from the exporting country USDA VS Port Veterinarian must inspect each shipment at entry port USDA VS Port Veterinarian must inspect each shipment at entry port