ANTIBODY TITERS AGAINST NEWCASTLE DISEASE AND INFECTIOUS BRONCHITIS VIRUSES IN BROILER BREEDERS RAISED UNDERFLOOR VERSUS CAGE SYSTEM

: The experiment was evaluated to find out the serum antibody titer of broiler breeders raised under different rearing systems (cage vs. floor) against Newcastle disease virus and infectious bronchitis disease virus. Two hundred and forty-day-old broiler breeders were equally divided into six groups Floor (V+CH), (V+Not CH) and (Not V+CH) and cage (V+CH), (V+Not CH), and (Not V+CH). Birds were vaccinated with Mukteswar, La Sota, and Massachusetts M41 in 1 st week, 3 rd week, and 4 th week. Blood /serum samples were collected at 42 days of age. Collected data were analyzed by using a complete randomized design. The geometric mean titer (GMT) for Newcastle disease virus (NDV) of broiler breeders reared under floor versus cage housing system was measured by using the hem agglutination inhibition test and the defensive potentiality of vaccines was measured by determining the survivability rate of chickens by challenge infection. Birds vaccinated and virus-challenged showed a protective antibody titer against Newcastle disease virus in both housing systems. Significantly higher GMT was recorded for the non-vaccinated group having virus-challenged in cage female (9.76 ±0.34) and floor female (9.94 ±0.23). Protective antibody titer was calculated for broiler breeders who were regularly vaccinated. There were no significant differences (P≥0.05) observed in the cage versus floor housing system. Higher serum antibody titer in caged females (9.22±0.36) and floor (8.98±0.32) was calculated due to infection challenges. Morbidity and mortality were significantly affected by the cage and floor-rearing system. Frequencies of diseased birds and mortality were found higher on the floor compared to cages in the non-vaccinated group. Despite the regular vaccination sick female broiler breeders were found higher compared to cage broiler breeders. In the non-vaccinated group floor reared birds have a higher percentage of morbidity and mortality compared to cages. In conclusion cage and floor-rearing systems have no adverse effect on serum antibody titer and survivability of broiler breeders.


INTRODUCTION
Poultry production is one of the most vital livestock-producing activities because it provides affordable animal protein to humans (Abbas, 2020).There are many factors, which directly or indirectly affect poultry production, their welfare, and economics (Abbas et al., 2020).Infectious disease is one of the most important factors, which can adversely affect poultry production (Abbas et al., 2017).Several viruses can infect and abruptly change the shape of poultry production.Two such viruses are Newcastle disease virus (NDV) and infectious bronchitis disease virus (IBV), which cause Newcastle disease and infectious bronchitis, respectively.These viruses are highly contagious and are often fatal leading to adverse economic impacts on the poultry industry (Ashraf and Shah, 2014, Afonso et al., 2013, Miller et al., 2013, Aldous and Alexander, 2008, Aldous et al., 2003).
The World Organization for Animal Health (OIE) has categorized Newcastle disease as a notifiable disease.This disease was first reported from Java (Indonesia) and Newcastle (England) in 1926, but now occurs worldwide (Alexander, 2001) and is caused by NDV, negative sense, linear, single-stranded, nonsegmented, enveloped RNA virus belonging to the family of Paramyxoviridae, genus avulavirus, species avulavirus 1, NDV (Mayo 2002).Chicken and turkeys are more susceptible to NDV than ducks and geese (Liu et al., 2008).Strains of NDV are classified based on pathogenicity as lentogenic (low), mesogenic (intermediate), and velogenic (highly virulent) (Afonso et al., 2013).The Newcastle disease affects the respiratory system, nervous system, and digestive system of birds and is transmissible in poultry through direct contact with feces and respiratory discharge or by contaminated food, water, equipment, and human clothing (Boven et al., 2008).The disease in humans is characterized by conjunctivitis, fever, headache, and malaise.
Infectious bronchitis is an acute, highly contagious viral disease.In addition to the respiratory tract, IBV can also infect the kidneys, gonads, and oviducts (Lee et al., 2004).The disease was first reported in the U.S. in the 1930s.Coronaviruses are nonsegmented, positive-sense viruses containing a singlestranded RNA genome.Chickens of all ages are affected by IBV (Cavanagh et al, 2007), which results in severe damage to the respiratory system, complete cessation of egg laying, or production of misshapen thin-walled eggs.Damage to kidneys may result in nephritis and death.The IBV is transmissible through direct contact or utensils, egg-packing tools, manure, etc.).
Both Newcastle disease and infectious bronchitis can occur even after regular vaccination of the flock.The hemagglutination inhibition (HI) test is used to check the chicken's immune status, measuring the level of antibodies against these viruses in the field, and assessing the efficacy of the vaccine used.Despite regular vaccination, the occurrence of NDV and IBV outbreaks causing high mortalities and economic losses indicate that vaccination strategies are not able to completely stop these outbreaks.One of the reasons could be that the outbreaks are caused by viral strains that are different from those contained in the vaccine.Vaccine failures represent major problems for poultry production and hence determining specific virus strains and providing the best hygiene could control viral diseases and enhance profitability.This study was undertaken to provide the idea of the best rearing system to enhance the profitability and welfare of the birds.

MATERIALS AND METHODS
Study Site: The experiment was conducted in the control shed of the Poultry Complex unit, Department of Poultry Science the University of Agriculture, Peshawar, Pakistan.
Centrifugation, serum collection, haemagglutination, and haemagglutination inhibition were tests performed at the histopathology laboratory of the same university.The study was financially supported by the Higher Education Commission of Pakistan.Broiler breeders were vaccinated according to Hubbard guidelines.
Serum Collection: Blood samples (approximately 3 ml/bird) were collected from the wing veins of broiler breeders on day 42 of post-vaccination.The blood was collected in Lavender-top serum separator tubes.Centrifuge the tube containing blood @ 2000×g for 10 minutes to separate serum from blood cells.The serum was transferred to an Eppendorf tube and tested for antibody titer against Newcastle disease virus and infectious bronchitis disease virus by hem agglutination inhibition (HI) test.

Preparation of Chicken Red Blood Cell (1% v/v) suspension:
Chicken blood was collected from a live bird in a poultry shed Blood was collected in a 15 ml falcon tube containing 2 ml EDTA solution as an anticoagulant.Tubes were kept in the ice box and brought to the lab within 15 minutes.blood was then centrifuged at 1500 rpm for 5-7 minutes and the supernatant was poured off.PBS (1X) was added into the falcon tube containing blood and centrifuged at 1500 rpm for 5-7 minutes.This step is repeated 4-5 times for washing chicken blood and 1% v/v suspension of chicken RBC was prepared by adding PBS.

Haemagglutination test (HA):
This test is used for getting 4HA or 8 HA units which are further used in the haemagglutination inhibition test.A 96-well plastic plate was used for the HA test.A total of 50 l of PBS was dispensed into each well of each row.A total of 50l of NDV and IBV antigen suspension was placed into the first well of each plate and two-fold dilutions by transferring 50 l of fluid from each well to the next and lastly discarding 50 l from the last well.A negative control well was prepared by using only PBS.Then 50 l of 1% chicken RBC suspension was added and allowed to stand for 40 minutes.The result was recorded, the last well to show hemagglutination, which indicates 1 HA unit.The 4HA unit was measured by dividing the 1HA suspension by 4.
Haemagglutination inhibition (HI) test: PBS (50l) was dispended into 12 wells of the plastic v-bottomed 96well microtiter plate.Two-fold serial dilution of field serum (50l) was made up to the 11 th well.An equal volume (50l) of the 4HA unit of ND virus was added into each well up to the 12 th well.Then the mixture was kept for 30 min at room temperature then 50 l of 1 % (v/v) chicken RBC suspension was added to all wells.The RBC is allowed to settle down for 40 minutes.Agglutination was assessed by tilting the plates.The samples showing peculiar central button-shaped settling of RBCs were recorded as positive.The last well which had complete inhibition of hemagglutination was considered as the HI antibody titer.

RESULTS
Antibody Titre of broiler breeders on floor versus cage housing system: The geometric mean titer (GMT) for Newcastle disease virus (NDV) of broiler breeders reared under floor versus cage housing system is shown in Table (3).Hubbard's guideline of vaccination was followed.The level of NDV antibody titers of broiler breeders on the floor and in cages was measured by using the hem agglutination inhibition test and the defensive potentiality of vaccines was measured by determining the survivability rate of chickens by challenge infection.The majority of the analyzed samples having been vaccinated and virus-challenged at different intervals of time showed a protective antibody titer against the Newcastle disease virus in both housing systems.There was no significant difference (P≥0.05)observed in the geometric mean titer for both housing systems.Numerically cage-reared broiler breeders have higher GMT values for males (7.31±0.54)and females (7.65 ±0.38) compared to floorreared males (6.97 ±0.67) and females (6.85 ±0.71) in a group having regular vaccinated and virus challenged.There were no observable variations in GMT value having not been challenged by the virus.Nonsignificantly higher GMT was recorded for the nonvaccinated group having virus challenges for cage females (9.76 ±0.34) and floor females (9.94 ±0.23).Protective antibody titer was calculated for broiler breeders who were regularly vaccinated.There were no significant differences (P≥0.05)observed in the cage versus floor housing system.Numerically antibody titer for the cage housing system in all groups was found better compared to the floor housing system of rearing.Titer for cage reared male (7.61±0.98)and for floor was (6.64±0.56).Higher antibody titer for caged females (9.22±0.36)and floor (8.98±0.32)was calculated due to infection in virus-challenged birds not vaccinated.

Morbidity of broiler breeders under floor versus cage
housing system: Broiler breeders on the floor and cagehoused systems were observed regularly.Diseased birds were shifted to separate groups where proper treatment and cure were provided.There was no significant difference found in the vaccinated male broiler breeders in cages compared to the floor.Frequencies of diseased birds were found higher for males on the floor (37.84± 1.22) than males in cages (33.78 ± 0.82) in the nonvaccinated group.Despite the regular vaccination sick female broiler breeders (24.02 ± 1.5) were found higher compared to cage broiler breeders (6.37 ± 0.34).In the non-vaccinated group floor reared females (43.60±0.99)have a higher percentage of morbid birds compared to cages (32.10±1.73).

Mortality of broiler breeders under floor versus cage housing system:
Routinely dead birds from each group and its concerned isolated unit were collected.Significantly lowest mortality in males was recorded in vaccinated groups.The numbers of dead birds were found higher in non-vaccinated groups, the highest percent mortality of male broiler breeders (28.08± 0.82) was found on the floor and (20.95± 0.73) were found in cages.Non-significantly higher mortality was recorded for floor females (21.87± 0.57) compared to cage females (5.53± 0.06) in vaccinated groups.A similar result was obtained from the non-vaccinated group, the number of dead birds for floor females (27.84±1.05) was higher compared to cage birds (22.73±0.45).

DISCUSSION
In Pakistan, the velogenic Newcastle disease and infectious bronchitis disease have been enzootic in local and commercial poultry for years.Despite regular vaccination, the occurrence of NDV and IBV outbreaks, high mortalities, and economic losses (Absalon et al., 2019) indicate that vaccination strategies were not able to completely stop outbreaks of Newcastle disease virus and infectious bronchitis virus.Such evidence represents major problems for poultry production, therefore determining specific virus strains and providing the best hygiene facility in the form of cages to control viral diseases and enhance the profitability of farmers.(OIE 2004).(Alexander et al., 2012,).
Determination of Antibody titers is the basic method of immunology to analyze and predict a disease and health status of the chicken.The housing system has a great influence on plasma antibody titer value (Buijs et al., 2009;Arbona et al., 2011).Stress has a direct effect on the immunity of chickens and reducing stress under an open environment was speculated to improve antibody titer (Rehman et al., 2017).In addition to biosecurity, welfare and the removal of infected birds, vaccinations and environmental enrichment are a critical element to control Newcastle disease (Marangon andBusani, 2006, Seal et al., 2000).These viruses shed into the environment from infected birds, and increase the probability of infection in vaccinated birds (Marangon andBusani, 2006, Miller et al., 2009).Managemental control of these viruses is a need of the profitable poultry industries.In the present study, broiler breeders were compared on the floor versus cage housing system for measuring their immune level at the brooding stage by haemagglutination inhibition test.
In the present study antibody titers were at normal range and clinical signs were absent in the chickens having been vaccinated properly and uninfected with the virus.The bird's antibody titers were improved in the group that had been vaccinated and challenged by antigen and the antibody titers of the birds remained at peak levels up to 40 weeks (Ghahramani et al., 2014).The present results are in line with the result of (Oberländer et al., 2020) who concluded that the antibody titer of the birds against ND has been improved by vaccinating and stimulating by antigen.The current finding is parallel with the finding of (Abdi, et al., 2016 andOsadci et al., 2011).The present results of the study for vaccinated broiler breeders were log 2 5 to log 2 8 , which means protective antibody titer, supported by ( van Boven et al., 2008).(Kapczynski and King, 2005) and (Allan et al., 1978) found a higher survival rate of birds having HI titer not less than log 2 5 and not more than log 2 8 .There were no significant (P≥0.05)changes observed for antibody titers in both housing systems, although there was a numerical increase found for cage-reared birds than floor-reared birds.In contrast, Matur et al., (2015) found non-significantly lower antibody titers in the cage housing system due to social stress and improved titers level by furnishing cages.Mench et al. (1986) Guesdon et al. (2004), Moe et al. (2004), andGuémené et al. (2004) found that there was no remarkable difference observed in the immunity level of different housing systems.Similar to our finding, it was previously found that improvement in the environment increased antibody production in the layer (El-Lethey et al., 2000).It has also been reported by (Zulkifli et al., 2002) that environmental enrichment, improves antibody levels in broilers.(Mahmoud et al 2020, Ahmad et al., 2019) found no alteration in the antibody titer level of the birds in different housing systems.
The unvaccinated-challenged broiler breeders on the floor and in cages resulted in higher antibody titers compared to the vaccinated groups.The results of the study are in line with the result of Macpherson & A. Feest (1978) who found higher antibody titer for birds due to severe infection.Morbidity and mortality were found higher in these birds due to the attacks of NDV and IBV viruses. Sarcheshme et al., (2015) found higher mortality and gross lesion in unvaccinated chicken.

Conclusion:
Cage and floor-rearing systems have no adverse effect on serum antibody titer and survivability of broiler breeders.