Troubleshooting a Marek’s Disease Outbreak

Enrique Montiel, DVM, MSc., ACPVMerial Avian Global Enterprise

Gainesville, GA©2005 Merial Limited. All rights reserved

Marek’s Disease

•Marek’s Disease was first reported in 1917 by Dr. Jozsef Marek in Budapest, Hungary

•Three serotypes described

•Causes malignant tumorsin the chicken’s skin and internal organs

Marek’s Disease

• The field virus tissue tropism and severity of the lesions may vary

• Causes mortality & condemnations atthe processing plant

• Causes immunosuppression

Marek’s Disease Virus Serotypes

Low Pathogenicitystrains- CVI988 (Rispens)- Clone C- CR-6

HVTSB1

301B

Oncogenic strains- GA- MD11- MD5- TK - RB1B- etc.

Serotype 3Serotype 2Serotype 1

• The virus replicates to the fully infectious form in the feather follicles

• The route of infection is the respiratory system by inhaling infectious viral particles

• Tumors (lesions) occur due to the malignant transformation of the lymphocytes

Marek’s Disease

Marek’s Disease

• The first wave of virus infection targets the bursa and thymus

• The infection may produce immunosuppression in a transient or permanent form

• The disease may show various clinical signs

Marek’s Disease•Very virulent strains (vvMDV+) have been reported in a number of countries around the world and have affected broilers, breeders and commercial layers

•Great economic impact

•Marek’s can be prevented by implementing a vaccination program

Clinical Signs

Marek’s Disease in Broiler Breeders

Marek’s Disease in Broiler BreedersParalysis & depressionParalysis & depression

ParalysisParalysis

Marek’s Disease in Broiler Breeders

ParalysisParalysis

Marek’s Disease in Broiler Breeders

Leg tumors ( Alabama red leg)Leg tumors ( Alabama red leg)

Marek’s Disease in Broiler Breeders

SkinTumorsSkinTumors

Marek’s Disease in Broiler Breeders

Spleen TumorsSpleen Tumors

Marek’s Disease in Broiler Breeders

Intestinal TumorsIntestinal Tumors

Marek’s Disease in Broiler Breeders

Marek’s Disease in Broilers

Leg tumors ( Alabama red leg)Leg tumors ( Alabama red leg)

Marek’s Disease in Broilers

Leg tumorsLeg tumors

Marek’s Disease in Broilers

Skin TumorsSkin Tumors

Marek’s Disease in Broilers

Liver TumorsLiver Tumors

Marek’s Disease in Broilers

Liver Tumors with necrotic centersLiver Tumors with necrotic centers

Marek’s Disease in Broilers

Control BursaControl Bursa MDV-Infected bursaMDV-Infected bursa

Marek’s Disease in Broilers

Marek’s Disease in Commercial Layers

Liver Tumors in LayersLiver Tumors in Layers

Marek’s Disease in Commercial Layers

Marek’s DiseaseThe only known methods of preventing Marek’s Disease are:

•Vaccination•Good flock management•Cleaning, disinfection and biosecurity

Marek’s Vaccination FailureMD outbreaks may appear in vaccinated flocks because:

•High field challenge by very virulent Marek’s disease virus strains (vvMDV or vvMDV+) •Interval vaccination – Challenge•Presence of immunosuppressive agents•Inadequate vaccination programs•Poor vaccine preparation and administration

•High Field challenge by Very Virulent Marek’s Disease Virus (vvMDV or vvMDV+) Strains

- If the field challenge is too high, it can overcome the vaccine immunity and produce an outbreak even in older birds

- Previous reports indicate the incidence of Marek’s outbreak in layers and broiler breeders over 40 weeks of age

Marek’s Vaccination Failure

•The Presence of other immunosuppressive agents may interfere with the development of protection against Marek’s disease

•Infectious Bursal Disease Virus (IBDV)•Chicken Anemia Virus (CIAV)•Avian Leukosis subtype J•Mycotoxins

Marek’s Vaccination Failure

•Interval Vaccination – Challenge

•Good cleaning and disinfection should delay the exposure of baby chicks to field challenge

•This delay of the exposure will allow the proper development of vaccine-provided protection

•Day old chicks should be placed in an environment as clean as possible

Marek’s Vaccination Failure

Interval

Vaccination-Challenge

Chart 1. Percent Total Affected (Lesions Plus Mortality) 2 Through 8 Weeks After Challenge With

MDV T.K. (Respiratory Route Using a Shedder System) at One Day of Age or When 5 Days Old

010203040

CVI988-A CVI988-A +HVT

CVI988-A +HVT+SB1

HVT+SB1

Vaccines

Perc

ent A

ffect

ed

1 Day Old5 days of age

Montiel, 1995Montiel, 1995

Marek’s Vaccination Failure

•Inadequate Vaccination Programs

•Vaccination programs must be designed according to the type of bird and field challenge

•Re-vaccination may be considered under high challenge conditions

Most commonly Used MD Vaccination Programs

Rispens & Combinations

HVT+SB1

HVT

Broilers

HVT+SB1

HVT+Rispens+SB1

HVT+Rispens

Layers

HVT+SB1

Rispens

HVT+Rispens

BroilerBreeders

3

2

1

Program

• The best program in meat-type birds is the HVT+Rispens combination

• More recent publications suggest that the best program for commercial layers is the HVT+Rispens+SB1 combination

Commonly Used MD Vaccination Programs

70 80 90 100

Vac

cine

s

Rispens 2+HVTRispens 1+HVTRispens+HVT+SB1

Percent Protection in Commercial Female Broilers Challenged with the MDV TK Isolate* by intra-abdominal injection 5 days post vaccination

*Challenge dose: 1:100 dilution of TK seedstockPercent ProtectionPercent ProtectionPercent Protection

Marek’s Vaccination Failure

•Vaccine preparation and administration

•Must follow the manufacturer’s instructions

•Improper vaccine preparation and administration techniques may seriously compromise the vaccine titer.

Marek’s Vaccination Failure

•Improvement in vaccine preparation and administration has contributed to the decrease in the incidence of Marek’s disease in a number of operations around the world

Preparation for

Vaccine Mixing

Mix vaccine in a clean, low-traffic area.

Frozen Vaccine Storage

Keep Frozen Vaccines Properly Identified.

Liquid Nitrogen Safety

ProtectiveClothing Required

Routinely monitor the level of liquid nitrogen in the tank.

Vaccine Handling

Check Frozen Vaccines Ampules

• Marek’s vaccines ampules may be stored inverted in the liquid nitrogen tank. When checking ampules, make sure you follow the quality assurance indicator before thawing.

Quality Assurance Indicator

(Product in the frozen state)

Acceptable Acceptable Unacceptable

Store Diluent at Room Temperature

Examine Each Bag for Contamination.

Examine Each Bag for Contamination.

Good diluentGood diluent ContaminatedContaminated

Fill thaw-bath with distilled

water.

Do not add bleach or sanitizer.

Most Hatcheries keep thaw-bath at 80 °F

(27 ° C).

Thaw no more than what is needed for

immediate use.

Ampules may be

thawed in water

At 27-37 °C

(80- 98.6 F)

Ampules may be

thawed in water

At 27-37 °C

(80- 98.6 F)

Preparation Checklist• Mixing Room

• clean, low traffic

• Liquid Nitrogen • safety considerations, adequate levels of liquid

nitrogen, no Newcastle contamination

• Diluent• room temperature, no contamination

• Thaw-bath• Fresh distilled water, 80 – 96 °F

Mixing the Vaccine

Always mix the vaccine following the instructions on the vaccine

insert

Clean and Prepare the Working Area

Wash hands thoroughly before mixing.

Use hand sanitizers

Adding Antibiotics

• Vaccines are not licensed for combination with these products.

• Some antibiotics can significantly reduce the Marek’s vaccine titer

Calculating the Number of Ampules Needed

Determining the Number of Ampules Needed

0.05 ml/egg

1200 ml bag ÷ 0.05 ml per chick = 24,000 eggs per bag

1 ampule = 2000 doses (check ampule)

At full dose, you would need 12 ampules (24,000 ÷ 2000) of the 2000 dose vaccine or 6

ampules of the 4000 dose vaccine

Thaw ampules by gently

swirling the ampules in the

thaw-bath.

Never Leave Ampules Unattended

Once an ampule has thawed, never refreeze the ampule

Ampules that have been thawed and re-frozen can lose up to 97% of its PFU.

Use paper towels to dry hands and ampules.

Cloth towels can become contaminated.

Merial’s Ampule Breaker

Steps in Vaccine Mixing

1*1*22 33

44 5555 66

1* dye added 5 minutes prior vaccine mixing

1. Disinfect the port of diluent bag2. Take some diluent in a sterile syringe3. Extract vaccine from ampule with the

diluent-filled syringe4. Gently inject the vaccine into the

diluent bag5. Take some diluent into the syringe

again and rinse the ampule6. Mix the bag gently to distribute the

vaccine evenly

Steps in Vaccine Mixing

By rinsing the ampules & ampule tips, additional virus

can be recovered.

Marek's Vaccine Titer Percent Difference in rinsed ampoules compared with a non-rinsed

control

-6% ---+12.8

0

2,000

4,000

6,000

8,000

10,000

12,000

14,000

None Ampule only Ampule +cap

Tit

er

Complete Process From Point A to Point B as Soon

as Possible

Point A

Take vaccine out of liquid nitrogen tank

Point B

Inject the vaccine in the diluent bag

Mix the bag gently to distribute the vaccine evenly.

The prepared vaccine should be

used within 1 to 2 hours from

the time of preparation.

Place the bag on ice for

transport to the in ovo machine

An ice pack should be used to keep the vaccine cool.

Proper disposal of biohazard material is

critical.

An alternate mixer should be trained and allowed to mix the vaccine at least

once a week.

Vaccine Mixing Checklist

• Hands washed, mixing area cleaned

• Dye or antibiotic added more than 5 minutes before vaccine

• Number of ampules per bag calculated properly

• Vaccine thawed properly

• Hands and ampules dried before opening the ampule

• Vaccine carefully added to bag

• Ampules and tips rinsed

• Bag gently squeezed to evenly mix the vaccine

• Alternate mixer evaluated

Vaccine Administration

Subcutaneous vaccination at one day of ageSubcutaneous vaccination at one day of age

Subcutaneous vaccination at one day of ageSubcutaneous vaccination at one day of age

Inspecting Subcutaneous Vaccination

• Conduct routine random checking of vaccinated chicks

• The minimum level of accuracy (properly vaccinated chicks) must be at least 97%

• If less than 97% accurate, the procedures need to be reviewed to detect where the problem is and implement correctives

In ovo Injection

Proper Procedures forin ovo Vaccination

Proper maintenance and QC is critical for in ovo vaccination.

Always adhere to scheduled maintenance of your vaccination equipment.

Proper equipment sterilization and cleaning is critical.

Properly maintained equipment will always be more dependable

Quality Control Manual

Prepare fresh sanitizing solutions daily.

Loading the Eggs

Run The Check Plate After Each Buggy to Make Sure There

Are No Plugged Needles

Check Eggs For Contamination

Summary

• Vaccination, in conjunction with the following initiatives, will greatly reduce the incidence of Marek's disease in the field:

– Good farm cleaning and disinfection – Proper reception practices– Adequate downtime between flocks– Accurate vaccination program

Summary (cntd.)

– Vaccination programs tailored to the type of bird and field situation

– Good vaccine preparation and administration practices

ReferencesBatra, P., A.K. Pruthi and J.R. Sadana. 1991. Effect of aflatoxin B1 on the efficacy

of turkey herpesvirus vaccine against Marek's disease. Avian Pathol. 51(1):115-119.

Calnek BW, Harris RW, Buscaglia C, Schat KA, Lucio B. 1998. Relationship between the immunosuppressive potential and the pathotype of Marek's disease virus isolates. Avian Dis. 1998 Jan-Mar;42(1):124-32.

Calnek, B.W. 1985b. Pathogenesis of Marek's Disease. A review. In B.W. Calnekand J.L. Spencer (eds). Proc. Int. Symp. Marek's Dis. pp. 374-390. Am. Assoc. Avian Pathol. Kennett Square, PA.

Calnek, B.W. 1985a. Genetic Resistance. In L.N. Payne (ed) Marek's Disease, pp 293-328. Martinus Nijhoff. Boston.

Cloud S.S., H. Lillehoj and J.K. Rosenberger. 1992. Immune Disfunction Following Infection with Chicken Anemia Agent and Infectious Bursal Disease Virus I. Kinetic Alterations of Avian Lymphocyte Subpopulations. Vet. Immunol. Immunopatho. 34:337-352. 1992.

Ficken, M.D., M.P. Nasisse, G.D. Boggan, J.S. Guy, D.P. Wages, R.L. Witter, J.K. Rosenberger and R.M. Nordgren. 1991. Marek's Disease Virus Isolates With Unusual Tropism and Virulence for Ocular Tissues: Clinical Findings, Challenge Studies and Pathological Features. Avian Pathol. 20:461-474.

ReferencesGimeno IM, Witter RL, Hunt HD, Reddy SM, Reed WM.2004. Biocharacteristics

shared by highly protective vaccines against Marek's disease. Avian Pathol. 2004 Feb;33(1):59-68

Gimeno IM, Witter RL, Reed WM. 1999. Four distinct neurologic syndromes in Marek's disease: effect of viral strain and pathotype. Avian Dis. 1999 Oct-Dec;43(4):721-37

Montiel, E. 1995. Master Thesis, Depart. of Animal Science & Agric. Biochem., University of Delaware, Newark, DE 19717-1303.

Montiel, E.R., J.K. Rosenberger and S.S. Cloud. 1994. The efficacy of available andexperimental vaccines for inducing protection against challenge with a potativevariant Marek's disease virus. Proc. 66th Northeastern Conf. on Avian Dis. pp. 34.

Otaki, Y., T. Nunoya, M. Tajima, A. Kato, Y. Nomura. 1988. Depression of vaccinalimmunity to Marek's disease by infection with chicken anemia agent. AvianPathol. 17:333-347.

Payne, L.N. 1972. Pathogenesis of Marek's disease - a review. In P.M. Biggs, G de Thé, and L.N. Payne (eds). Oncogenesis and Herpesviruses, pp. 21-37. IARC, Lyons, France.

Rosenberger J.K., S.S. Cloud, D. Anderson, E. Montiel and Y. Velasquez. 1993. Marek's Disease in Delmarva Broilers. Proc. 28th Natl. Meet. on Poultry Healthand Condemn. Ocean City Maryland pp. 70.

ReferencesWitter, R.L. 1987. New serotype 2 and attenuated serotype 1 Marek's disease

vaccine viruses: comparative efficacy. Avian Dis. 31:752-765.Witter, R.L. 1992. Safety and comparative efficacy of the CV1988/Rispens vaccine

strain. Proc. XIX World's Poult. Congress. Amsterdam, the Netherlands, p 315-319.

Witter, R.L. 1993. Evolution of Virulence Among Marek's Disease Virus Strains: Implications for Control. Proc. 65th Northeastern Conf.on Avian Dis. pp 6.

Witter RL, Gimeno IM, Reed WM, Bacon LD. 1999. An acute form of transient paralysis induced by highly virulent strains of Marek's disease virus. Avian Dis. 1999 Oct-Dec;43(4):704-20.