Jacquinet  C.¹, Gardin Y.²
¹,²,CEVA Santé Animale, La Ballastière, BP 126, 33501 Libourne Cedex, France

Introduction:

For many years in France, the vaccination of broiler chickens against infectious bursal disease (IBD) has most frequently occurred by using attenuated live vaccines through the drinking water route. They are described as “intermediate” or “intermediate plus”, depending on their ability to prevent mildly or highly virulent field viruses, and depending on their ability to break through the maternally-derived antibodies (MDA) in young chicks.

In practice, the administration and the success of the vaccination are strongly jeopardized by the following issues:

• Interference of the vaccination with passive immunity and variability in MDAs. It is therefore compulsory to determine the optimum vaccination time (OVT) by when the vaccine can break through to induce an active immunity. Due to cost and previous experience, OVT determination by assessing the MDA titer in day-old chicks is not regularly done.
• Issues related to the drinking water vaccine administration route. Numerous factors can partly or fully inactivate the vaccine virus after solubilization in drinking water: the presence of residual chlorine or metallic ions, for example.
• Finally, a major obstacle to full vaccination success is the inability to ensure every chicken did take in the proper amount of vaccine solution within a short period of time (2 hours) before the vaccine’s activity drops.

Despite the efforts of poultry production technical staff with regular information and training updates regarding the correct vaccination technique (audits, use of dye, use of chlorine neutralizing agents, etc.), it is noteworthy to notice that IBD vaccination failures are common.

A new generation of IBD vaccines allows poultry producers to overcome these issues using a live attenuated vaccine of immune-complex type (Cevac Transmune^®). It is made of a vaccine virus suspension mixed to a specific antiserum in well-defined proportions. This antigen-antibody complex suspension should be individually administered in the hatchery, either by in ovo injection of 18-day-old embryos, or by subcutaneous route in day-old chicks.

The antigen-antibody complexes are not destroyed by the immune system of the bird, but persist most likely because they are “trapped” by the follicular dendritic cells of the spleen and of other lymphoid organs (unpublished Ceva studies). The antigens (vaccine viruses) are progressively released due to the permanent degradation of the antibodies of the complex. As soon as the MDAs, also submitted to degradation, reach a moderate to low level, the released vaccine viruses are not neutralized anymore. They can reach the bursa of Fabricius (BF), replicate there and induce an active immunity. This “vaccine take” relies upon the initial MDA status of the chick, and therefore takes place at variable, but always optimal, times depending on the chick.

In practice, when poultry are vaccinated against IBD, this phenomenon occurs between approximately 3 and 5 weeks of age.

Modern diagnostic tools make monitoring the vaccine take of classical vaccines by drinking water or through immune complex vaccine in the hatchery possible. They enable confirmation and the comparison of the actual protection rate of chicken flocks in the field.

Materials and Method:

The replication of the vaccine virus in the BF is evidence of the vaccine take of a live attenuated IBD vaccine. The current laboratory tools enable assessment of the following events:

• Onset of lesions in the BF follicles: detection by histopathology;
• Presence of the vaccine virus in the BF: detection by RT-PCR;
• Induction of an active immunity including production of specific antibodies: detection by serology.

Type of samples:

5  to 10 broilers per farm were sampled as follows:

• 5 half BF kept in formalin for histopathology analysis;
• 5 half BF kept frozen for PCR;
• 5 to 10 serum samples for serology.

 Histopathology:

The 5 BF samples kept in formalin were submitted to histopathology analysis. Lesions measurement is performed individually in each BF; it enables detection of virus replication and characterization of the nature and extent of the lesions, if necessary.

Virology:

To start, IBD virus presence was checked in the 5 frozen BF samples by RT-PCR. A drawback of this technique is the possibility, although rare, of false negative results due to poor storage or transportation conditions, the presence of inhibiting substances (traces of intestinal content?), or a too-early replication of the virus (dilution in the pool of 5 BF).

In a second step, enzyme restriction analysis by RFLP is the way to distinguish vaccine and field virus. Regarding Cevac Transmune, this technique enables a “reasonable” detection of the presence of the Winterfield 2512 vaccine strain.

Lastly, the VP2 hypervariable gene sequencing of IBD virus (nucleotide sequence 721-1128 bp) is a way to identify the IBD virus strain very precisely.

Serology:

The serum samples were assayed by Gumboro ELISA (Biochek kit). This technique prevents false positive results, and is able to detect low antibody levels, which are expected when the active immunity starts. A positivity threshold of 500 is the evidence of the onset of an active immunity.

Age at sampling and interpretation of the results:

The chickens were sampled at around 30 days of age (minimum 27 days – maximum 40 days). Hence, whichever classical vaccine was used (“intermediate” vaccine administered around 15-18 days, or “intermediate plus” vaccine administered around 12-14 days), there should be evidence of vaccine intake by any technique, because the interval of time is sufficient. The vaccine virus replication is expected to occur 5 days post-vaccination at the latest, and the onset of antibody response 15 days post-vaccination at the latest. At 30 days of age, the BF should show some lesions, an IBD virus identical to the vaccine strain should be detected, and the antibody response should be either well-established or should begin from 15 days post-vaccination onwards.

Regarding the immune-complex vaccine, due to a vaccine antigen release that is customized to every chick, 4 situations can be met according to the vaccine virus replication stage:

1)  Onset of vaccine virus replication:

• Positive W2512 RT-PCR, positive histopathology and negative serology: the replication of the vaccine virus starts, the antibodies are not detected yet.
• Negative RT-PCR W2512, positive histopathology, and negative serology: the replication of the vaccine virus has started, as evidenced by the lesions, but no virus could be detected or identified (escaping from the primers? virus dilution in the 5 BF?); an infection by a wild virus may be possible.

2)  The vaccine virus replication is well established:

• Positive W2512 RT-PCR, positive histopathology and clear seroconversion (ELISA titer >500) in at least one sampled bird.

3)  “Advanced” vaccine virus replication:

• Positive RT-PCR (or negative for reasons that are still poorly explained, see above), positive histopathology, clear and homogeneous seroconversion.

4)  Lack of vaccine intake:

• All criteria are negative.

As a consequence, farms were stamped as “Positive” (confirmed vaccine intake), or “Negative” (no sign of vaccine intake). In a few cases, because there was no histopathology analysis, the samplings were done too early, or the results could not be interpreted, no conclusion could be drawn and the farm was stamped as “Doubtful”.

Sampled farms:

4 companies of standard broilers and one company of free-range broilers were monitored in this study. In each company, a similar number of farms were using either conventional vaccines (intermediate or intermediate plus) by drinking water (DWV), or immune-complex vaccine in the hatchery (ICV). Altogether, 114 farms were included in this study.

Results:

Table 1: Summary of Results (in number of farms and in overall percentage):

Org.*	Vaccine Group	Positive	Negative	Doubtful
1	DWV	2	12	2
	ICV	8	2	1
2	DWV	0	5	5
	ICV	8	0	4
3	DWV	1	8	5
	ICV	13	3	0
4	DWV	6	4	1
	ICV	3	0	0
5	DWV	6	1	0
	ICV	14	0	0
Total	DWV 58 farms	15 (26%)	30 (52%)	13 (22%)
	ICV 56 farms	46 (82%)	5 (9%)	5 (9%)

* Org. = organization (company)

Conclusion:

This study confirmed the actual issues related to Gumboro vaccination in broiler farms; it also showed that in half of the cases vaccination in the farm was ineffective. The absence of vaccine take explains why it is typical to detect unexpected farm isolates (either wild or vaccine strains) in the BF. This is due to the fact that intermediate type vaccines, widely used in this study, are very sensitive even to low MDA titers and have a poor BF invasion capability.

Significantly higher rate of vaccine take was recorded in farms where an immune-complex vaccine was used in the hatchery, since this vaccine is not sensitive to the MDA and the hatchery is a controlled environment. Contrary to mass vaccination by drinking water, it is an individual vaccination that adapts to every chicken.

The available diagnostic tools (histopathology, virology, serology) are reliable and can be used to monitor vaccine intake in one sampling session at around 30 days of age. Despite the limited number of sampled birds per farm (5), the obtained results could be interpreted in the majority of cases (80% to 90% according to the vaccination type). In this study, 12% (7 out of 56) false negative results were obtained by RT-PCR, when samplings were done at an early replication stage. A larger sampling would probably reduce the rate of doubtful results.

Note: this is an English translation of a paper that was first issued in French, and published in the 9^th French Days of Poultry Research (Neuviemes Journees de Recherche Avicole), held in Tours, France, 29-30 March 2011. The translation was made by C. Cazaban and kindly reviewed by A. Zlatic.

References

C.E.Whitfill et al. Determination of optimum formulation of a novel infectious bursal disease virus (IBDV) vaccine constructed by mixing bursal disease antibody with IBDV. Avian Diseases 39: 687-699, 1995.

E.E.Haddad et al. Efficacy of a novel infectious bursal disease virus immune complex vaccine in broiler chicken. Avian Diseases 41: 882-889, 1997.

M.Kelemen et al. Pathological and immunological study of an in ovo complex vaccine againts IBD. Acta Veterinaria Hungarica 48: 443-454, 2000