doi:10.5455/vetworld.2013.134-138 Evaluation of an inactivated vaccine for nephropathogenic infectious bronchitis virus

Aim: To evaluate an inactivated vaccine for nephropathogenic infectious bronchitis in broiler with special reference to its ability for passing maternal antibodies to broiler chicks. Materials and Methods: An inactivated vaccine against nephropathogenic infectious bronchitis (NIB), prepared using an isolate obtained from natural outbreak of NIB was administered to broiler parents at the point of lay, leaving the control birds unvaccinated. Eggs laid below the desired weight (> 52 g) by vaccinated hens were utilized for yolk serology. Chicks obtained from hens of both group were subjected for serology and challenge with wild type of nephropathogenic IB isolates. Serology of the yolk and serum was carried out using haemagglutination (HI) test and ELISA. Results: Yolk serology revealed a geometric mean titre of 415.9 and 15188±768 in HI test and ELISA respectively on 28 days post vaccination (dpv) as against 16.0 and 1881±86 in yolk from unvaccinated hens. The HI test and ELISA indicated that the level of maternal antibody (MAb) in the chicks obtained from vaccinated hens was significantly (P< 0.01) higher on seven days of age than that of chicks from unvaccinated hens. However, the level of Mab of the chicks obtained from vaccinated hens decreased to below the level of protection at two weeks of age. Wild isolate and another isolate obtained from different geographical area were used for challenge dividing the chicks from vaccinated and unvaccinated hens equally. Mortality was observed in the challenged chicks from vaccinated (one in heterologus challenge) and unvaccinated (two) hens. Examination of kidney specimens collected from dead chicks revealed mottling and severe congestion grossly and inflammatory, degenerative and necrotic changes microscopically. Conclusion: The partial cross-protection against heterologous challenge and incomplete protection against homologous challenge with wild isolates were noticed. Keyowrds: infectious bronchitis virus, nephropathogenicity, protection, vaccine


Introduction
Narrow range of available vaccine strains and minimal cross-protection among serotypes complicate In India, infectious bronchitis (IB) is one of the the process of prevention of IB.Furthermore the rate at important poultry diseases and only vaccines prepared which alteration of amino acid sequence in S1 subunit from Massachusetts strain 41 (M41) is being used.
of spike glycoprotein occurs is high, leading to emergence Despite the use of this live attenuated infectious of many serotypes of IBV.Despite vaccination with bronchitis virus (IBV) vaccine, it is not uncommon to Mass serotype for prevention of IB, high mortality due find the disease problem.The variant strains of IBV to nephropathy occurs in broiler chickens and in layer that are antigenically different from vaccine strain could chickens during growing stage.be circulating among chickens in India and recently An inactivated vaccine prepared from the isolate nephropathogenic IB was reported to be present in obtained from nephropathogenic IB outbreak was evaluated nephropathogenic IBV in India [1,2] and China [3].In with respect to its ability to prevent the disease.Southeast Asia, appearance of a new variant of nephro-

Materials and Methods
pathogenic IBV is also reported [4,5] whereas in Middle East there was variant IBV causing respiratory symptoms Preparation of oil-adjuvant killed vaccine: The isolate [6].Thus, determining the genotypic or serotypic identity Ind/KA/07/6 was utilized for the preparation of oilof a field strain is important for selecting an appropriate adjuvant killed vaccine as per the recommendations of vaccine.Serotyping requires antisera to all the IBV FAO (1993) [7] and as follows: isolates circulating throughout the world to actually Virus : 12.5 per cent (v/v) assign a specific serotype to the particular isolate.In Liquid paraffin : 86.0 per cent (v/v) the absence of World reference laboratory for IB, this is Spam 80 : 1.25 per cent (v/v) highly impracticable.
Tween 20 : 0.25 per cent (v/v) The adjuvant components were first mixed chicks in vaccine group and six chicks were challenged The GMT and mean (± SE) ELISA level of antibody to IBV.
titres between the groups were subjected for paired 't' After 21 days of post vaccination, all the birds in 3.0 test and the comparison of the titre was done at 1 per both groups were inoculated oculonasally with 10 cent level.EID of field isolate Ind/KA/07/6 per bird.After 14 50 days, birds were humanely sacrificed to observe gross

lesions.
Safety: All the ten embryos in ECE inoculated with the Healthy broiler parent birds before administration vaccine were found to be alive.Six days after of inactivated IBV vaccine were obtained from a inoculation, the embryos showed normal growth private hatchery in Namakkal, Tamil Nadu.Ideal without any type of lesion comparable with the management with ad libitum sanitized water and embryos of control ECE.broiler parent feed were provided to the birds till the completion of trial, which was approved by Institutional Efficacy: The GMT of the chicks vaccinated with Animal Ethical Committee.
inactivated Ind/KA/07/6 before vaccination, 7, 14, 21 Commercially available Newcastle disease killed and 28 dpv was 13.0, 16.0, 39.4, 157.7 and 512.0 vaccine was administered subcutaneously at the rate of respectively which differed significantly at < 0.05 level th 0.5 ml per bird to all the birds during17 week of age.from control group as the GMT of the chicks in control th On 19 week of age, six female and one male parent group was lower than vaccine group.birds were separated to form vaccine group.Inactivated Assessment of immune response by HI test and vaccine prepared from field isolate Ind/KA/07/6 was ELISA: The GMT of the yolk prepared from eggs administered at the rate of 0.5 ml per bird subcutanobtained from vaccine group differed significantly eously to all the birds in vaccine group.Two female and from control group (P< 0.01).GMT reached a peak of one male parent birds which were not vaccinated 415.9 on 28 dpv and decreased thereafter to 104.0 on 42 against IB and kept as control group.
dpv.Egg yolk prepared on 28 dpv showed 415.9 and Hens started laying from 21 weeks of age.Eggs 15188±768 in HI test and ELISA respectively which o were collected and stored after labeling at 4 C for was the highest (Table 1).collection of yolk to assess the level of antibody present The level of antibody in parent birds of vaccinated in the yolk by ELISA (M/s Synbiotic Corp., USA) and group was ascertained by HI test and ELISA before HI test.Eggs weighing 52 g and above were collected vaccination, 14 and 21 dpv.Similarly, antibody titre separately from vaccinated and unvaccinated hens and was also assessed in control group.It showed an set for hatching.Chicks hatched out from respective increase in GMT (294.1) and ELISA titre (9921±614) groups were used for the challenge study.Twenty four during 21 dpv which was significantly (P>0.01)higher chicks from vaccinated hens and 12 chicks from than that of control group (Table 2).unvaccinated hens were utilized for the trial.Twelve The HI test and ELISA indicated level of MAb in the vaccine group was significantly (P< 0.01) higher completion of trial.on seven days of age than the titre observed in control Pathology of kidney: Grossly, mottling of kidney and group.However, the MAb level in vaccine group nephritis were observed in three dead chicks of control decreased to below the level of protection during second group (9 and 13 dpc) and mottling and severe week of age.Chicks in vaccine group challenged with congestion of kidney (Fig. 1) in a dead chick of vaccine two isolates showed sharp fall in MAb titre during group (13 dpc).Two chicks each from all the groups on second week and increase in antibody to IBV during 14 dpc were sacrificed to recover the kidney samples third week of age in both the groups.There is for virus isolation and histopathology.Kidney significant difference (P<0.01)noticed in GMT and specimens from dead and sacrificed chicks were mean ELISA titre on 7, 14, 21 and 28 days of age subjected for virus isolation in ECE.On third passage, between the vaccinated and control groups.Increase in dwarfing and curling of toes of embryo noticed in ECE antibody titre was also evident in the control group inoculated with kidney samples from the dead chicks.during third week of age (Table 3).
All other kidney samples including those of control th group did not produce embryo lesions in ECE.Protection study: On 6 day of age, 12 chicks obtained Microscopically, inflammatory, degenerative and from vaccinated hens and six chicks from unvaccinated necrotic changes were observed in kidney from dead hens were challenged with wild isolate Ind/KA/07 and chicks.Necrosis of tubular epithelial cells, infiltration 12 chicks of vaccinated hens and six chicks of of lymphocytes and plasma cells in the interstitium unvaccinated hens were challenged with wild isolate were seen in kidney specimen of chick died on 9 dpc in Ind/TN/07/2.Two chicks on 9 dpc with isolate the control group (Fig. 2).In the kidney specimen from Ind/KA/07/1 and another on 13 dpc with isolate the chick died on 13 dpc in control group, severe Ind/TN/07/2 died in control group.One chick from necrosis of tubular epithelial cells and accumulation of vaccine group died due to nephropathy on 13 dpc with RBC in the interstitium were noticed (Fig. 3).The Ind/TN/07/2.No other clinical signs or mortality was kidney from the chick died on 13 dpc in vaccine group noticed in rest of the chicks of all the groups till the

tion of tubular epithelial cells and accumulation of
The broiler chickens with MAb against IBV degenerated cells within the tubules.In the kidney developed protective immunity by 21 dpv with H120 sections recovered from control group on 14 dpc vaccine administered at one day of age [16] and the hydrophic degeneration of tubular epithelial cells (Fig. same cannot be corroborated with field scenario where 4) was observed.
there was incidence of nephropathy in broiler chicks even after vaccination with H120 or Mass serotypes.

Discussion
This can be attributed to poor cross-protection and Satisfactory results of efficacy trial of the occurrence of infection at very early age.For protection inactivated vaccine indicate that the vaccine is quite against nephritis, vaccination with homologous attenuated enough to induce protective immune response.Neverstrain in broiler chicks with MAb is necessary [17].In theless, the results might not be true indicator as the contrast, the inactivated vaccine developed utilizing challenge was carried out with the isolates which may nephropathogenic IBV strain was found to protect NIB not be virulent enough.
caused by different variants [18,19].Earlier workers suggested vaccination programmes Cross-protection evinced in this trial can be against IB are based on determination of IBV strains attributed to the fact that antibodies produced might causing the disease in the field and best protection neutralize the heterologous strain where challenges against challenge is achieved by a vaccine containing with four European serotypes in vaccinated and the homologous strain and the one containing unvaccinated broilers yielded 100 per cent serotypeheterologous strains may not induce enough crossspecific VNT [13].A live attenuated nephropathogenic protection [12].
IB virus vaccine offered high level of cross-protection In the present study, GMT differed significantly for variants [18].However, especially after challenge on 21, 28 and 35 dpv whereas ELISA titre differed of the vaccinated broilers with a heterologous serotype, significantly on 7, 14, 21, 28 and 35 dpv in yolk antibodies were detected against IBV serotypes to serology between the vaccine and control hens.which the birds had not been exposed.On the contrary, Comparable results in both the tests show that HI test it was showed that VNT for different serotypes alone is sufficient for checking the immune response performed on chicken embryo kidney cells, was highly since elevated titre was evident only after 21 dpv in HI serotype-specific after one inoculation [14].test.Serology of these hens also revealed that the Death due to nephritis of two chicks inoculated vaccine prepared is able to induce high level of immune with isolates on 9 and 13 dpc in control group and death response as the titres of unvaccinated hens was of a chick due to nephropathy in vaccine group on 13 significantly lower (P<0.01).This observation correlates dpc with Ind/TN/07/2 indicates that the presence of with the findings of previous work [13] where HI test MAb in the vaccine group offers protection to the detected antibodies between one and two week post chicks against challenge with the isolate (Ind/KA/07/1) infection.However, Gelb and Killian (1987) [14] procured from similar location as that of isolate used found that although HI test was serotype-specific when for inactivated vaccine preparation.There is some level used to detect antibodies after single inoculation with of cross-protection as only one chick died out of 12 IBV, specificity of the test lower than that of virus chicks inoculated with Ind/TN/07/2 as against two out neutralization test (VNT).
of 12 in control group.The immune response in the chicks from vaccinated Mottling of kidney and nephritis observed in the hens was better than those from unvaccinated hens as dead chicks of control group denoted that the virus was assessed by HI test and ELISA on one week of age.
able to replicate in kidney and caused damage.Similar However, the MAb level in vaccine group decreased to lesion was observed in dead chick inoculated with below the level of protection during second week of age.
Ind/TN/07/2 which indicated that presence of strain-Chicks in vaccine group challenged with isolates specific antibodies is necessary to prevent nephroshowed sharp fall in MAb titre during second week and pathogenicity.increase in antibody to IBV during third week of age in Severe renal lesions such as necrosis of tubular both the groups.Increase in antibody titre was also epithelium, infiltration of lymphocytes and plasma evident in the control group during third week of age.cells in the interstitium noticed in control group chicks The rise in antibody titre might be attributed to indicated that great invasiveness of both isolates.response of the chicks to the infection caused by isolate Similar finding was reported in SPF chicks [20].used for challenge.
Observation of moderate renal lesions such as haemo-It was reported that a high level of virus neutralising rrhage in between tubules, degeneration of tubular antibodies against D388 in breeders correlated with epithelium and accumulation of degenerated cells higher level of protection in progeny against early within the tubules in a chick of vaccine group died on challenge.The local protection at 10 days of age was 13 dpc denoted that nephropathogenicity is minimized.not much higher than in unvaccinated progeny.This Similar finding was reported where there was penetration indicated that a (heterologous) vaccination at day one and destruction of the tubular epithelium and the does not provide a sufficient protection against invasion of the surrounding interstitium by heterophils

Figure- 1 .
Figure-1.Kidney showing mottling and severe congestion in chick of vaccine group 14 dpc.

Figure- 2 .Figure- 3 .Figure- 4 .
Figure-2.Kidney exhibiting necrosis of tubular epithelial cells, interstitial infiltration of lymphocytes and plasma cells from chick died on nine dpc in the control group (H&E 1000X) Figure-3.Kidney showing severe necrosis of tubular epithelial cells and congestion in the interstitium from the chick died 13 dpc in control group (H&E 1000X) Figure-4.Kidney from control group on 14 dpc showing hydropic degeneration of tubular epithelial cells (400X) The oil-adjuvant inactivated vaccine was injected at group and six chicks were challenged with 10 EID of 50 the rate of 0.2 ml per bird intramuscularly.wild field isolate Ind/TN/07/2 on the same day of age.Chicks were observed for abnormalities and death.
o serum samples were stored at -20 C until tested for the Statsitical analysis:

mean titre HI and mean ELISA titre ± SE Table-1.
Comparison of GMT and mean ELISA titre of the egg yolk prepared from vaccinated and control groups Table-2.Geometric mean HI titre of vaccinated and unvaccinated parent birds before and after vaccination

Table - 3
. Geometric mean HI titre and mean ELISA titre of chicks obtained from vaccinated and unvaccinated hens GMT with different superscripts differ significantly at P<0.01 level on 21 to 35 dpv in the vaccine group * -Both titres differ significantly (P<0.01) between vaccinated and control groups on 7, 14 and 21 days of age