Understanding Immunity Against New Castle Disease in Broiler Chickens

Dr Susim Mukul Ray

M.V.Sc (Gold Medalist)

Head - Technical & Promotion

Zydus AHL

Is Broiler Immunity Fragile ?

Talk outline

• Principles of immunity in broiler chickens

• Susceptible period/Immunity gap

• Correlates of protection

• Ideal vaccination program

Talk outline

• Principles of immunity in broiler chickens

• Susceptible period/Immunity gap

• Correlates of protection

• Ideal vaccination program

Principles of immunity in broiler chickens�

1. Passive –– Maternal derived antibodies (MDA)

2. Active –– Through vaccination or exposure or infection

Principles of immunity in broiler chickens�

Active Immunity

Principles of immunity in broiler chickens�

Innate immunity is the first line of defense --- triggers inflammation !

Comes into action after the first exposure of antigens (pathogens)

Non-specific (anything foreign) and has no memory !

Principles of immunity in broiler chickens�

After innate immune response is mounted, the adaptive immune response initiates which creates memory and prepares for the second exposure !

Specific to particular antigen (pathogen)

Principles of immunity in broiler chickens�

The memory/adaptive immunity fades if there’s no second exposure within few weeks interval

Principles of immunity in broiler chickens�

1. Mucosal immunity (e.g. sIgA in tracheal washing, lachrymal fluid/tears, intestinal mucosa)

2. Cellular immunity (e.g. Tracheal epithelial cell, Enterocytes)

Sites:

3. Humoral immunity (e.g. Blood)

Principles of immunity in broiler chickens�

1. First exposure of antigen through vaccination will stimulate production of IgM by B cells

2. Second exposure of antigen would result in gradually switching of IgM to IgY by B cells

Class switching in humoral immune response:

3. IgY is significantly effective than IgM in pathogen neutralisation

Principles of immunity in broiler chickens�

1. Route of vaccination governs site of mucosal immune response, e.g. Eye drop or nasal route elicits mucosal immune response in respiratory tract

2. Compartmentalisation of mucosal immunity governed by live vaccine replication characteristics

Key facts on mucosal immune response:

Principles of immunity in broiler chickens�

1. First exposure of antigen through vaccination will stimulate production of IgM by B cells

2. Second exposure of antigen would result in gradually switching of IgM to IgY by B cells

Class switching in humoral immune response:

3. IgY is significantly effective than IgM in pathogen neutralization

Principles of immunity in broiler chickens�

1. Live ND vaccines = Cellular & mucosal mostly + less humoral

2. Inactivated vaccine = 100% humoral + No cellular/mucosal

Key facts on vaccines:

Talk outline

• Principles of immunity in broiler chickens

• Susceptible period/Immunity gap

• Correlates of protection

• Ideal vaccination program

Poll 1 –– Correlates of protection against ND in broilers

Q1. Which arm of immunity is having higher role in protection against ND in broiler chickens –– descending order ?

• Cellular immunity > Local/Mucosal immunity (sIgA) > Humoral immunity (IgM/IgY)
• Local/Mucosal immunity (sIgA) > Cellular immunity > Humoral immunity (IgM/IgY)
• Humoral immunity (IgY/IgM) > Cellular immunity > Local/Mucosal immunity (sIgA)
• Local/Mucosal immunity (sIgA) > Humoral immunity > Cellular immunity

Q2. Which arm of immunity plays early role in protection against ND in broiler chickens ?

• 1st –– Maternal derived antibodies (MDA), 2nd –– Cellular immunity, 3rd –– Local/Mucosal immunity (sIgA), 4th –– Humoral immunity
• 1st –– Maternal derived antibodies (MDA), 2nd –– Local/Mucosal immunity (sIgA), 3rd –– Cellular, 4th –– Humoral immunity
• 1st –– Maternal derived antibodies (MDA), 2nd –– Humoral, 3rd –– Cellular, 4th –– Local/Mucosal immunity (sIgA)
• 1st –– Humoral immunity, 2nd ––Maternal derived antibodies (MDA), 3rd –– Cellular, 4th –– Local/Mucosal immunity (sIgA)

(Pathogenesis: Course of NDV infection)

• Viral replication at Mucosal surfaces of respiratory tract and GI tract

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• NDV affinity for erythrocytes allowing the virus to spread systemically via blood to other organs

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• F0 cleavage by host proteases will determine the virulence and different tissue tropism

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• HN characteristics as well as other proteins contributes to the virulence

Correlates of protection

What are we trying to do with NDV Vaccines?

1. Protect the mucosal surfaces at the port of entry with CMI and IgA

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• Prevent the invasion of the virus to internal organs with humoral Ab and other immune components

Correlates of protection

ND Control – Biosecurity and immunity

Biosecurity

Mucosal immunity

Humoral immunity

Correlates of protection

ND Control - Biosecurity & Vaccines

Biosecurity

Live vaccines

Live and inactivated

Mucosal protection

Ab+

Correlates of protection

• Priming with live vaccine for mucosal/cellular immunity

Correlates of protection

What should be our targets ?

• Vaccinating chicks with VH clone live via eye drop (1^st application) & drinking water (subsequent application)

• Higher sIgA level in tracheal washing, lachrymal secretion & GI tract – marker for mucosal immune response

• Boosting with inactivated vaccine

• Vaccination with high potency inactivated vaccine
• For broilers – HI titer > 4 log2
• For layers & breeders – HI titer > 8 log2 or > 14000 (IDEXX ELISA)

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Correlates of protection

• Single stranded RNA genome, ~15000 Nucleotides

• The genome codes for 6 proteins:

1. N - Nucleoprotein
2. P - Phosphoprotein
3. M- Matrix
4. F - Fusion Surface protein
5. HN - Hemagglutinin - Neuraminidase surface protein
6. L - RNA polymerase

4

5

3

6

2

1

Why humoral immunity is so important ?

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Step1

Step 2

Correlates of protection

Why humoral immunity is so important ?

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Step1

Step 2

• HN protein on the surface of NDV helps in attachment process i.e. Step 1

• HN protein binds with sialic acid receptor on tracheal epithelial cell of chicken

• Following attachment process by HN protein in step 1, conformational change of F protein occurs

• In step 2, F protein helps in fusion of viral envelope with chicken cell membrane

• Finally, viral RNA enters chicken cell resulting in infection

Correlates of protection

Correlates of protection

Why humoral immunity is so important ?

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Correlates of protection

Why humoral immunity is so important ?

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Correlates of protection

• Genetic classification of NDV is based on fusion protein (F) sequencing

• NDV genotypes circulating in India are - 4, 6, 7 & 13 (new)

• There is minor (0.1 -10%) variation in HN gene sequence

Why humoral immunity is so important ?

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Correlates of protection

HN protein

Fusion protein

Minor (0.1 -10%) variation in HN gene sequence

• Strong anti-HN IgY antibodies prevents attachment process
• 90 – 99.9% cross protection against all NDV isolates

• Successful blocking of HN protein makes F protein non-functional/redundant
• Infection process is blocked
• Genotype mismatch i.e. differences in F protein sequence of field & vaccine strain only lead to shedding of NDV

There are variations in F protein sequence across NDV genotypes

Therefore, high anti HN IgY levels are extremely critical for 100% protection against NDV infection

Why humoral immunity is so important ?

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Talk outline

• Principles of immunity in broiler chickens

• Susceptible period/Immunity gap

• Correlates of protection

• Ideal vaccination program

Poll 2 –– Broiler susceptibility to ND infection

Q1. Which is the most susceptible period for NDV incubation (age) in broiler chickens ?

• Day 1 – 7
• Day 7 – 14
• Day 14 – 21
• Day 21 – 28
• Day 28 – 35

Fragility of immunity in broiler chickens ?

Susceptible period/Immunity gap

ND live

ND live

ND live

Critical HI titer

Susceptible period/Immunity gap

ND live

ND live

ND live

Critical HI titer

Neutralised by MDA, stimulates sIgA & cellular immunity in respiratory tract, no role in humoral immunity

Fragility of immunity in broiler chickens ?

Susceptible period/Immunity gap

ND live

ND live

ND live

Critical HI titer

Role in mucosal & cellular immunity; to some extent humoral immunity

Fragility of immunity in broiler chickens ?

Susceptible period/Immunity gap

ND live

ND live

ND live

Critical HI titer

Mucosal & Cellular (mostly) + humoral (less)

Fragility of immunity in broiler chickens ?

Susceptible period/Immunity gap

ND live

ND live

ND live

Critical HI titer

--IgY (MDA)---

Fragility of immunity in broiler chickens ?

Challenges of class switching !!

Susceptible period/Immunity gap

ND live

ND live

ND live

Critical HI titer

--IgY (MDA)---

---------IgM--------

Challenges of class switching !!

Fragility of immunity in broiler chickens ?

Susceptible period/Immunity gap

ND live

ND live

ND live

Critical HI titer

--IgY (MDA)---

------Slow class switching to IgY-------

---------IgM--------

Challenges of class switching !!

Fragility of immunity in broiler chickens ?

Susceptible period/Immunity gap

ND live

ND live

ND live

sIgA/Mucosal immunity

Critical HI titer

Fragility of immunity in broiler chickens ?

Susceptible period/Immunity gap

ND live

ND live

ND live

sIgA/Mucosal immunity

Cellular immunity

Critical HI titer

Fragility of immunity in broiler chickens ?

Susceptible period/Immunity gap

ND live

ND live

ND live

sIgA/Mucosal immunity

Cellular immunity

Critical HI titer

Huge window of susceptibility !!

Fragility of immunity in broiler chickens ?

Susceptible period/Immunity gap

ND live + ND Inactivated

ND live

ND live

Critical HI titer

The susceptibility window reduces by 4-5 days

Fragility of immunity in broiler chickens ?

Faster class switching to IgY

Susceptible period/Immunity gap

Fragility of immunity in broiler chickens ?

• Live vaccines alone create large window of susceptibility –– Can be reduced by high potency/good quality inactivated vaccine

• Class switching is slow with live vaccines –– Can be expedited by high potency/good quality inactivated vaccine

• Priming with live vaccine for mucosal/cellular immunity

What should be our targets ?

• Vaccinating chicks with VH clone live via eye drop (1^st application) & drinking water (subsequent application)

• Higher sIgA level in tracheal washing, lachrymal secretion & GI tract – marker for mucosal immune response

• Boosting with inactivated vaccine

• Vaccination with high potency inactivated vaccine
• For broilers – HI titer > 4 log2
• For layers & breeders – HI titer > 8 log2 or > 14000 (IDEXX ELISA)

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To counteract enterotropic ND challenge, mucosal immunity in GI tract is critical.

Susceptible period/Immunity gap

1. Developed from LaSota strain by 2 step cloning process
2. There is difference of 97 nucleotides (out of 15198 i.e. 0.6% difference) between original LaSota strain (AF07776) and VH clone
3. It has resulted VH clone to be able to protect chickens from both respiratory and enteric field viruses
4. It is least stressful among all commercially available ND live vaccines
5. VH clone elicits uniform immune response
6. It offers 100% protection against very virulent pathotype

Susceptible period/Immunity gap

How to achieve strong mucosal immunity?

Lasota & B1 replicates in respiratory tract only

IgA level in respiratory tract

3 days

7 days

3 days

7 days

IgA level in intestinal tract

No protection against viscerotropic velogenic/enteroptropic NDV

ND live vaccine :

Susceptible period/Immunity gap

How to achieve strong mucosal immunity?

Apathogenic enteric strain like VH replicates in both respiratory & intestinal tract.

Hence, protection at both local sites

T = Trachea

L = Lung

D =Duodenum

Ct =Cecal tonsil

Perozo et al., 2008

Therefore, apathogenic enteric strain like VH clone live is an ideal solution.

Susceptible period/Immunity gap

How to achieve strong mucosal immunity?

ND inactivated vaccine:

1. 6x more ND antigen than competition
2. Benefits of inactivated VH clone ND antigen
3. Produces protective and uniform titer over longer duration
4. 100% protection against very virulent pathotype of ND virus (vvNDV) and all genotypes viz, 4, 5, 7 & 13.
5. At 0.1 ml dose, PD50 value = 100

Susceptible period/Immunity gap

How to achieve strong humoral immunity?

more ND antigen

concentration than competition

6X

0.1 ml

0.2 ml

0.3 ml

0.4 ml

0.5 ml

10^7.3

10^8.1

10^8.4

10^7.6

10^7.78

10^8.58

10^7.91

10^8.70

10^8.00

10^8.80

Nectiv Forte = 10^8.8 EID₅₀/0.5 ml; Competitor = 10⁸ EID₅₀/0.5 ml

ND inactivated vaccine:

Susceptible period/Immunity gap

How to achieve strong humoral immunity?

released slowly by stable oil emulsion adjuvant

EID₅₀ and PD₅₀ : What are these parameters and why do we use PD_{50 ?}

• Inactivated ND vaccines can be compared by their antigen content - namely EID₅₀

• EID₅₀ is determined BEFORE the inactivation and therefore does not represent the actual efficacy parameter – PROTECTION.

• PD₅₀ (Protective Dose 50) is based on vaccinating SPF birds and then challenging them. Therefore this parameter is based on injection volume PROTECTION value.

ND inactivated vaccine:

Susceptible period/Immunity gap

How to achieve strong humoral immunity?

EID₅₀ and PD₅₀ : What are these parameters and why do we use PD_{50 ?}

• PD₅₀ parameter was adopted by the EU pharmacopeia as the parameter to evaluate the vaccine efficacy

• Usually, the volume of injection is linearly correlated to the PD₅₀ value so if it is 50 at 0.1 it will be around 100 in 0.2.

• Limits – PD50 of a given volume should not be less than 50

ND inactivated vaccine:

How to achieve strong humoral immunity?

Susceptible period/Immunity gap

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*Data based on SR-Nectiv Forte E-537 (Phibro file); ** Data is based on other producers official information

Highest PD₅₀

even at lowest volume of injection

ND inactivated vaccine:

Susceptible period/Immunity gap

How to achieve strong humoral immunity?

Talk outline

• Principles of immunity in broiler chickens

• Susceptible period/Immunity gap

• Correlates of protection

• Ideal vaccination program

Poll 3 –– Vaccination program of broiler chickens followed in Nepal

Q1. Which vaccination program do you follow in broiler chickens ?

• Day 1 –– ND inactivated vaccine + ND live (eye drop), Day 9 –– ND live (eye drop), Day 19 –– ND live (dw) Day 7 – 14
• Day 1 –– ND live (eye drop), Day 18 –– ND live (dw) Day 21 – 28
• Day 1 –– ND live (eye drop), Day 18 –– ND live (dw)
• Day 1 –– ND live (eye drop), Day 18 –– ND live (dw), Day 28 –– ND live (dw)
• Day 5 –– ND live (eye drop), Day 18 –– ND live (dw)
• Day 5 – ND live (eye drop), Day 18 –– ND live (dw), Day 28 –– ND live (dw)

Ideal vaccination program

*MB-1 is hatchery adapted IBD live and can be used with Nectiv Forte. Normal saline solution (NSS) should be used as diluent for MB-1. The dose can be adjusted either to 0.1 or 0.2 ml per chick based on volume of diluent.

Instead of MB-1, IBD MB live should be done in d/w at 12d

#Day 9 vaccine should be postponed to day 10 if chicks ND-MDA level is > 4000 (IDEXX). The goal is to achieve titer > 3 log2 HI during 15 – 18 day.

Achieving titer close to 4log2 HI titer during 15 – 20 d is the key factor for protection of commercial broilers from NDV challenge

Thanks for your attention !