Assessment of Immunological Responses of Broilers Vaccinated with Newcastle Strains Using Either Spray or Water Delivery Systems

ABSTRACT

(1) Background:  Vaccine programs have been failing to provide complete immunity against the Newcastle disease virus (NDV). This study examines the immunological responses to the live LaSota and Avinew (VG/GA) vaccines given through spray or drinking water to broiler chickens;

(2) Methods:  The experimental vaccination included one unvaccinated control group and four other groups of the same size. There were three duplicates, each with 30 birds, with each group amounting to ninety birds. In addition to the status of the cellular and humoral immunity, we examined the weights and histology of the bursa and spleen;

(3) Results:  The bursa weight index (BWI) significantly increased on days 28 and 42 in the vaccinated groups, regardless of the vaccination method (p≤0.05). The spleen weight index (SWI) significantly increased on day 42 in VG/GA vaccinated birds, and on day 28 in chicks vaccinated with the LaSota strain (p≤0.05). T cell proliferation in response to stimulation with PHA was significantly higher across all vaccinated groups compared to the unvaccinated ones. Serum IgG was significantly elevated on days 21, 28, and 42 post-vaccination with VG/GA or LaSota strain for both vaccination routes (p≤0.05);

(4) Conclusions:  The data generated from this study demonstrated that significant immune responses can be generated with the two examined Newcastle vaccine strains using the spray and drinking water vaccination routes.

Keywords:
Antioxidant enzymes; Avinew; ELISA; immunity; LaSota; ross chickens; vaccine

INTRODUCTION

Evaluating vaccine efficiency is important, as vaccines induce specific immunity in poultry farms. Newcastle disease (ND) has been a problem for countries where it is endemic worldwide. (Sedeik et al., 2019). ND is still a significant obstacle to the worldwide exchange of poultry and poultry items (Landman et al., 2017). The disease is widely distributed and impacts a wide scope of birds, including both confined and wild species (Igwe et al., 2014; Igwe et al., 2018). In spite of the advances in immunization programs, ND remains a significant threat to poultry industries worldwide; however, the infection can be managed through the effective implementation of vaccination programs (Martinez et al., 2018). Traditional vaccine delivery methods may not work for small and local producers, thus suitable immunization techniques should be researched (Mebrahtu et al., 2018).

ND, a ‘notifiable disease’, is caused by infection with virulent strains of avian paramyxovirus, a strain of avian orthoavulavirus1 (Amarasinghe et al., 2019). All strains of NDV belong to the order Mononegavirales, subfamily Avulavirinae, family Paramyxoviridae, genus Orthoavulavirus (Walker et al., 2019). Five pathotypes of infection are present; viscerotropic velogenic, neurotropic velogenic, mesogenic, lentogenic, and asymptomatic. The lentogenic pathotype incorporates the nonpathogenic strains of the virus that are used in vaccine manufacture, such as La Sota and lyophilized Villegas-Glisson/University of Georgia (VG/GA). The mesogenic pathotype includes low virulent strains of the virus and is also used as a vaccine. However, because mesogenic viruses are defined as virulent by the WOAH (OIE) and increased virulence of the antigen in this vaccine type has been reported, mesogenic-strain live vaccines are banned in many countries. The velogenic pathotype causes the most pronounced type of disease, with mortalities reaching up to 100% in vulnerable chickens (Igwe et al., 2018). Broiler chickens are usually vaccinated against ND using non-virulent live viruses administered through spray, drinking water, or eye drops. Although drinking water is usually the most popular route for vaccine administration, the use of aerosol methods is also common (Landman et al., 2017). Antibody responses to vaccination are influenced by the route of vaccination, which results in different levels of protection against the disease (Khodayari & Feizi, 2017; Landman et al., 2017). Comparing the viable vaccine strains and determining the best method of vaccine delivery is essential for the prevention of ND (Janmohammadi et al., 2020). The efficacy of any vaccine is influenced by multiple factors that can be related to the vaccine itself, route of administration and those that are endogenous to animals. Even though poultry vaccination is widely adopted by poultry businesses, disease eradication programs at a national or local level to control significant poultry diseases such as ND have been rare (Ghahramani et al., 2014).

Correct vaccination protects against disease manifestations in affected flocks through the production of significant levels of humoral immune responses. There are three fundamental objectives when utilizing vaccines to control ND: I) prevent clinical manifestation of the disease; ii) decrease the spread of virulent strains; and iii) increase the viral dose required to cause the infection (Kapczynski et al., 2013). Unfortunately, only the first objective is targeted by the current control strategies, since veterinarians lack the tools to evaluate the efficacy of vaccination on the achievement of the second and third goals. The virus-neutralizing antibodies protect against the development of the clinical signs of disease but cannot prevent viral shedding. Through the production of different cytokines, T cells are involved in the host’s defense mechanisms against the infection, playing an important role in viral clearance (Kapczynski et al., 2013).

NDV infection induces both humoral and cellular immunity. The appropriate NDV live vaccine for hatching facilities and farms should be safe, while retaining strong immunity promotion under consolidated spray and drinking water immunization programs (Kim et al., 2016). The use of the live attenuated LaSota strain as a vaccine for first immunization against ND has negative impacts on the improvement of adaptive immune response in broilers (Martinez et al., 2018). Both humoral and cell-mediated immune responses have been reported to be influenced by the genetic background of chickens after vaccination (Okoroafor et al., 2019). However, immune responses to La Sota vaccination among Ross-breed chickens have been the focus of few studies. Different serologic tests have been used for measuring NDV-specific antibodies in infected flocks, including hemagglutination inhibition (HI) and ELISA (Trombetta et al., 2018).

Current poultry operations expose birds to various immunological challenges, starting at the hatchery. A fruitful immunization program should produce strong, constant, and durable immunity. The present study, therefore, aimed to compare the antibody titers and cell-mediated immune response produced by VG/GA (Avinew) and LaSota live vaccines administered via two different routes (spray and drinking water) in the Ross breed of broiler chickens, using two different assays for antibody quantification, HI and ELISA. Performance traits such as weights, gains, feed consumption, and conversion will also be evaluated. In addition, leukogram, in vitro phagocytic index, the histopathology of spleen and bursa of Fabrica (BF), and the BF and spleen weight indexes (BWI, SWI) were examined. Serum levels of anti-oxidative enzymes such as superoxide dismutase, catalase, and total antioxidant capacity were taken into consideration. The indicator parameters were assessed for broilers from day one up to 42 days of age, in a carefully planned experiment conducted in the chicken houses at the Agricultural and Veterinary Research Station of Qassim University.

MATERIALS AND METHODS

Institutional Review Board Statement

The care and handling of the broiler chicks followed the regulations of the Animal Care Committee of Qassim University (QU - No.5322-cavm-2017-1-16-S).

Experimental chickens and management

A total of 450 unsexed one-day-old broiler chicks of the Ross© breed were kindly given by Alwatanya Company for Poultry, Qassim, Othal, Saudi Arabia. The chicks were placed in separate pens. Each pen (3m2) housed 90 chicks, divided into 3 replicates using wire mesh partitions. The average weight of each chick was calculated by dividing the total weight of the chicks by their total number. Light was provided 24 hours per day, in the form of natural light during the day and artificial during the night. Ventilation and temperature in each pen were controlled by the Dicam master unit (Farmex-UK). Each pen had a temperature sensor connected to the control unit. Air conditioning/pressure was provided by a special automatic system (U.S. Feed-std-290E). The broiler starter and grower-finisher diets in premixed form were obtained from the ARASCO company (Saudi Arabia) to provide the requirements for broiler according to National Research Council (1994). The starter ration was offered for the first three weeks, and then was replaced by the finisher ration up to the end of the experiment. The diets and water were provided ad libitum till the end of the experiment (42 days). The feed was based on soybean and corn - free of antibiotics and growth hormones.

Experimental design

The experimental vaccination consisted of a total of four equal major groups plus an unvaccinated control group. Each group contained 90 birds with three replicates (30 birds each). The first group was unvaccinated and considered as a control group. Chicks of the second and third groups were vaccinated with lyophilized Villegas-Glisson/University of Georgia (VG/GA) lentogenic vaccine (Avinew NeO, Boehringer Ingelheim, Lyon, France) via drinking water and spray at days 7 and 21 respectively. Chicks of the fourth and fifth groups were vaccinated with the NDV-La-Sota vaccine (Intervet, Holland) via drinking water and spray, following the same regimen of the second and third groups. For the oral route via drinking water (DW), vials of the lyophilized VG/GA and NDV-La-Sota vaccines containing 2000 and 1000 doses, respectively, were diluted and reconstituted with distilled water for application, following the instructions of the manufacturers (1 dose per 1 ml of distilled water). They were then given to experimental chickens, except for the unvaccinated group. Water was withheld for a few hours before vaccination. The spray route of vaccination was administered by using a hand-held spray flask. Hand pressing was performed constantly and each press yielded one ml. Chicks were forced to gather in a corner of the pen, and ventilation was closed for 30 minutes during the spraying process. The unvaccinated group was treated with a spray flask with diluent only.

Broiler growth measurements

Multiple parameters were used to assess broiler performance. Chicks in each group were weighed on an individual basis and weekly until the end of the experiment, using a second decimal scale. Weekly weight gain (BWG) was calculated as body weight - initial weight. Daily feed records for each group were kept using an automatic weighing device. At the end of each week, feed leftovers were weighed. For each replicate within each group, feed consumption and feed conversion ratio (FCR = Total feed intake / Total body weight growth) were computed.

For the calculation of Bursa of Fabricius (BF) and spleen weight indexes (BWI, SWI), three birds from each replicate were sacrificed at days 14, 28, and 42 of age. The chicks were slaughtered by a single quick cutting of the throat to minimize pain. The birds were dissected, and the BF and spleen were carefully removed, and weighed separately. The percentage of live wet organ weight (mg) 100 /BW (g) was used to compute the weight indices of the examined organs (Arafat et al., 2018).

Blood sampling and hematological analysis

On days 14, 28, and 42, wing veins were used to obtain blood samples and smears from three birds per replication, stored in both plain and heparinized tubes. After 10 minutes of centrifugation at 300 xg, the separated serum was collected and stored at -20°C until further analysis. According to Cray & Zaias (2004), the cross-sectional approach was used to determine the percentages of heterophils (H), lymphocytes (L), and monocytes (M), as well as the calculated heterophil/lymphocyte ratio (H/L) . Leukocytes were counted at a rate of 100/slide, and the percentages of the cells were determined. According to Campbell & Ellis (2007), total leukocyte count (TLC) was measured in blood collected in heparinized tubes using Natt and Herrick solution (1:200 dilution).

In vitro carbon clearance assay

Phagocytic activity was determined based on the removal of injected carbon particles (Spinu & Degen, 1993). At the end of the experiment, 1.5 ml of heparinized blood from each group was mixed with 6 ul of India ink (Pelikan AG D-3000, Hanover, Germany). The samples were divided into three portions of 0.5 ml each, and they were incubated at 37 °C for 0, 20, and 40 min. 150 ul of each sample was further diluted by adding 2 ml of saline and centrifuged for 5 minutes at a low speed. To determine optical densities, supernatants were collected and tested at 535 nm using a spectrophotometer. The measurements were scaled to a log base 2 scale. The slope of the optical density (log2) on time (h) regression was used to determine the phagocytic index. The phagocytic index is calculated as follows: logOD1 - logOD2 / t2 - t1, where t1 and t2 are 40 and 20 minutes, respectively.

Glucose consumption assay

At the end of the experiment, the glucose consumption assay was performed. To eliminate or lyse RBCs to isolate leukocytes, an autoclaved red blood cells (RBC) lysis buffer made up of NH4Cl (0.155M) 90 g, KHCO3 (0.01M) 10 g, and EDTA (0.1mM) 370 mg was used. Before use, the lysis buffer was filtered through a 0.22 m filter and diluted at a proportion of 1:10 in ddH2O. 200 ul of whole blood were combined with 2 ml of lysis buffer and incubated at room temperature for 5 minutes. The lysis buffer was then removed by centrifuging the mixture at 300 x g. The glucose consumption assay, which was first described by Kosti et al. (2010), was used to measure lymphocyte proliferation . In this test, phytohemagglutinin (PHA), a T-cell mitogen, was utilized. Cells were plated onto a 24-well plate in triplicate with or without 5 g/ml PHA. There were 2 x 106 cells in 200 ul of culture media (RPMI, Sigma Aldrich) in each well. During the course of three days, the plate was incubated in a CO2 incubator. The amount of glucose present in the medium was measured using commercial assay kits (Human GmbH, 65205 Wiesbaden, Germany), by tracking changes in optical density at 500 nm. The quantity of glucose that was ingested (in mg/dl) minus the quantity of glucose in the non-stimulated cell culture was used to calculate lymphocyte proliferation.

Serum antioxidant enzymes

Utilizing ELISA Kits and following the manufacturer’s instructions, the serum levels of catalase (CAT), superoxide dismutase (SOD), and total antioxidant capacity (TAC) were measured (Biodiagnostic kits, Giza, Egypt).

Antibody quantification by Enzyme-Linked Immune Sorbent Assay (ELISA)

IgM and IgG concentrations were determined on days 14, 21, 28, and 42 using commercial kits specific for the chicken immunoglobulin isotypes, according to the specifications provided by the manufacturer (SunLong Biotech Co., LTD).

Antibodies quantification by Haemagglutination (HA) and Haemagglutination Inhibition (HI) test

Antibody titers against VG/GA and La-Sota were determined by the HI test as described by Roudi et al. (2018) at d 14, and 28 and 42. The data were expressed as the 2 logarithm base. Sheep red blood cells SRBC were used to quantify the antibody response.

HA test

This test was performed before the HI test to evaluate the virus strains NDV-La-Sota and VG/GA HA unit. The titration was recorded to the maximum dilution generating no streaming (complete HA), equivalent to one HA unit. The unit can also be calculated from the original series of dilutions.

HI test

This test was conducted after HA to measure the antibodies against virus strains produced by the NDV-La-Sota and VG/GA vaccines in the serum samples of chickens in different groups. Titers were calculated as the logarithm of the peak dilution point that caused full agglutination. The reading of HI was considered as total anti-ND virus antibodies (total Igs). To measure IgM (mercaptoethanol-sensitive) and IgG (mercaptoethanol-resistant) anti-ND virus antibodies, mercaptoethanol was added to each well, and the procedure continued as previously to obtain IgG readings. The difference between the total Igs and the IgG reading was recorded as IgM. The antibody titers were expressed as logarithms to the base two (log2).

Histopathology of spleen and BF

Immediately after being obtained, spleen and BF tissue samples were fixed in 10% neutral buffered formalin and then packed in paraffin wax. Following the procedure outlined by Guo et al. (2018), thin slices of 4-5 μm thickness were treated with hematoxylin and eosin (H&E) stain to microscopically investigate the alterations.

Statistical Analysis

GraphPad Prism version 5 software was used to allocate the obtained results for statistical analysis. The use of ANOVA (One-way) allowed for the identification of significant group differences. The Student-Newman-Kuels test was used in cases where there were significant differences between unvaccinated and vaccinated groups. When p≤0.05, differences were regarded to be significant. All information was entered separately.

RESULTS

Broilers’ General Performance

The final body weight (BW) and the body weight gain (BWG) in the VG/GA strain-vaccinated chicks were significantly higher (p≤0.05) than those in the control (unvaccinated) group. Compared to the unvaccinated group, chicks demonstrated a significant elevation in the final BW and BWG following immunization with the LaSota strain when using either the water (p≤0.05) or spray (p≤0.01) vaccination routes. The food conversion rate (FCR), which was 1.7 in the unvaccinated control chickens, was observed to be 1.3 and 1.4, respectively, in the groups that received the VG/GA strain immunization through drinking water or spray. Regardless of the method of vaccination, the FCR in the chickens given the LaSota strain vaccine was 1.5, as compared to 1.7 in the unvaccinated control group (Table 1).

Table 1
BW, BWG, individual feed consumption, and FCR of Ross chickens with two routes of vaccination with Avinew (VG/GA) or LaSota strain vaccines.

Groups	Initial BW (g)	Final BW (d 42)	BWG (g)	Consumption (g)	FCR
Unvaccinated	42.61±2.55	2990±4.31	2947.46±4.44	4882.4±3.46	1.7±0.45
Water route VG/GA	43.85±3.16	3289±5.44a	3245.31±3.46a	4294.1±2.84	1.3±0.29
Spray route VG/GA	44.86±2.74	3315±3.87a	3270.22±4.72a	4611.8±3.44	1.4±0.84
Water route LaSota	45.71±1.56	2984±5.31a	2938.48±3.88a	4352.9±5.46	1.5±0.21
Spray route LaSota	44.48±2.53	3052±6.66b	3007.63±4.31b	4597.1±4.44	1.5±0.24

• Values with the letter a are significantly different from the control values at p≤0.05. Mean ± standard error.

Spleen and Bursa Weight Indices

The spleen weight index (SWI) only showed significant increases in chickens receiving the VG/GA strain on day 42. When compared to the control group, the bursa weight index (BWI) was significantly increased (p≤0.05) on days 28 and 42 post-vaccination via either vaccination route. In the LaSota-strain-vaccinated chickens, the SWI was significantly increased on day 28 post-vaccination, while the BWI showed significant elevation on days 28 and 42 post-vaccination through the drinking water or spray routes (p≤0.05) compared to the control group (Table 2).

Table 2
Spleen and BF weight index (SWI, BWI) of Ross chickens vaccinated with either Avinew (VG/GA) or LaSota strain vaccines.

Groups	SWI			BWI
	day 14	day 28	day 42	day 14	day 28	day 42
Unvaccinated	0.5234 ±0.042	0.3312 ±0.052	0.1035 ±0.023	0.6333 ±0.035	0.3437 ±0.015	0.2654 ±0.017
Water route VG/GA	0.4154 ±0.071	0.3521 ±0.079	0.2854 ±0.033a	0.6845 ±0.041	0.4621 ±0.044a	0.3124 ±0.015a
Spray route VG/GA	0.5512 ±0.012	0.3540 ±0.086	0.2214 ±0.018a	0.5782 ±0.024	0.4852 ±0.021a	0.3898 ±0.012a
Water route LaSota	0.4699 ±0.042	0.3333 ±0.013a	0.2655 ±0.027	0.6542 ±0.011	0.4324 ±0.021a	0.3222 ±0.010a
Spray route LaSota	0.5821 ±0.030	0.3245 ±0.041a	0.1185 ±0.098	0.6645 ±0.014	0.4612 ±0.011a	0.3180 ±0.015a

• Values with the letter a are significantly different from control value at p≤0.05. Mean ± standard error.

Cell-Mediated Immunity (CMI)

In vitro phagocytic index, glucose consumption by T-lymphocytes stimulated with PHA, and leukogram profile are the three methods that were used to evaluate the CMI in the vaccinated birds. In vitro phagocytic index, calculated at the end of the experiment, was significantly increased (p≤0.05) in the groups vaccinated through the drinking water route in both VG/GA and LaSota strains as compared to the control unvaccinated group (Table 3). T-lymphocyte proliferation was measured by glucose consumption in PHA-stimulated PBMC cultures. As shown in Table 4, as compared to the control, there were significant elevations in glucose consumption in PBMC of vaccinated chickens, regardless of the vaccine strain and vaccination route (p≤0.01).

Table 3
In vitro phagocytic index of Ross chickens vaccinated with either Avinew (VG/GA) or LaSota strain vaccines.

Groups	logOD1	logOD2	K = logOD1 - logOD2 / t2 -t1 (Phagocytic index)
Unvaccinated	1.331±0.222	1.291±0.152	0.0082±0.001546
Water route VG/GA	1.394±0.185	1.135±0.188	0.0142±0.004611a
Spray route VG/GA	1.428±0.223	1.345±0.245	0.0072±0.000244
Water route LaSota	1.483±0.191	1.208±0.103	0.0128±00032550a
Spray route LaSota	1.376±0.101	1.329±0.178	0.0016±0.000224

• Values with the letter a are significantly different from the control values at p≤0.05. Mean ± standard error.

Table 4
Glucose consumed (mg/dl) by PHA-stimulated lymphocytes of Ross chickens vaccinated with either Avinew (VG/GA) or LaSota strain vaccines.

Groups	Glucose in the medium (mg/dl) after 72hrs of incubation
	Without PHA	With PHA	Glucose consumed
Unvaccinated	33.6134±1.450	28.431±1.626	5.643±1.643
Water route VG/GA	35.2221±2.781	24.167±2.885	11.098±1.088a
Spray route VG/GA	34.4625±1.554	20.646±1.637	15.555±1.115b
Water route LaSota	36.3487±1.912	26.243±1.888	11.761±0.780b
Spray route LaSota	35.6542±2.414	15.688±2.841	10.610±1.718b

• Values with the letters a and b are significantly different from the control values at p≤0.05 and p≤0.01, respectively. PHA: Phytohemagglutinin. Mean ± standard error.

As shown in Table 5, a significant elevation (p≤0.01) in monocyte percentages (M %) was observed at day 14 post-VG/GA strain vaccination in both drinking water and spray groups compared to their unvaccinated groups. However, a significant decline in lymphocyte percentages (L %) and an elevation in the ratio of heterophils to lymphocytes (H/L ratio) and M % were observed on day 14 in the chicks of the group vaccinated with the LaSota strain via the spray route (p≤0.05) compared to the control. On day 28 post-vaccination with the VG/GA strain, total leukocytes count (TLC), H %, and H/L ratio significantly declined (p≤0.05), regardless of the vaccination route (p≤0.05); while L % and M % were significantly elevated (p≤0.05) only in chicks vaccinated using the spray route, compared to their control. In the LaSota vaccinated groups, TLC, H %, and H/L ratio significantly declined (p≤0.05), while L % was significantly increased at day 28 post-vaccination using either the water or the spray route relative to their unvaccinated group. The M % was significantly elevated at day 28 post-LaSota strain vaccination only through the spray route (p≤0.01) relative to their control group (Table 5). At day 42 post-VG/GA strain vaccination, it was noticed that H % was significantly increased with both water and spray routes of vaccination, while TLC and H/L ratio significantly declined only with the spray route compared to their control. However, TLC, H %, and H/L were significantly decreased at day 42 post-LaSota strain vaccination using either vaccination route relative to the unvaccinated group (Table 5).

Table 5
Leukocytes profile of 14-, 28- and 42-days old Ross chickens vaccinated with either Avinew (VG/GA) or LaSota strain vaccines.

Groups	Day	(TLC103/μl)	H %	L%	H/L ratio	M%
Unvaccinated	14	14.17±1.44	47.45±3.77	40.79±3.44	1.11±0.09	14.46±2.11
Water route VG/GA		13.45±2.77	43.35±4.45	38.65±2.07	1.54±0.04	24.28±1.24b
Spray route VG/GA		14.64±1.24	42.22±2.57	39.25±2.45	1.24±0.06	28.82±2.25b
Water route LaSota		14.11±2.43	38.34±2.15	46.75±4.55	0.58±0.0	17.46±1.44
Spray route LaSota		13.24±2.41	44.82±4.24	34.77±2.10a	1.34±0.04a	21.12±1.56a
Unvaccinated	28	18.75±1.48	45.12±3.11	44.42±2.97	1.04±0.04	12.44±1.25
Water route VG/GA		13.22±2.46a	34.25±2.34a	50.64±3.77	0.68±0.07a	16.24±2.58
Spray route VG/GA		14.81±1.77a	35.88±1.64a	55.25±2.34a	0.55±0.06a	19.45±2.87a
Water route LaSota		14.16±1.08a	33.14±3.85a	56.75±2.34b	0.43±0.06a	14.41±3.45
Spray route LaSota		13.49±2.44a	35.09±2.21a	51.53±2.66b	0.52±0.04a	24.33±2.83b
Unvaccinated	42	15.64±1.77	48.11±2.54	42.04±3.41	1.02±0.0	11.55±1.34
Water route VG/GA		12.77±2.84	31.21±3.88b	48.34±5.1	0.71±0.0	23.64±1.20b
Spray route VG/GA		12.06±1.88a	30.25±2.46b	56.11±2.67a	0.55±0.0a	22.85±2.12b
Water route LaSota		12.57±1.64a	36.46±2.45a	58.47±2.78b	0.48±0.02b	12.73±1.54
Spray route LaSota		13.84±2.09a	38.62±1.88a	53.12±3.44	0.49±0.07a	18.25±1.33a

• Values with the letter a are significantly different from the control values at p≤0.05. Mean ± standard error.

Antioxidant Enzyme Activities

The serum antioxidant enzyme activities of Ross chickens were significantly elevated at different days post-vaccination with the VG/GA or LaSota strains using the two examined routes of vaccination (Table 6). In VG/GA strain-vaccinated chickens, the CAT serum activity was significantly increased at day 14 post-vaccination via the drinking water route (p≤0.05). However, CAT serum activity showed a significant decline at day 14 post-vaccination, followed by a significant elevation at day 28 and 42 post-vaccination through the spray route (p≤0.05), compared to the control group. Moreover, LaSota strain vaccinated chickens showed a significant elevation in CAT serum activity at days 14 and 42 post-vaccination through the water route (p≤0.05) compared to the unvaccinated group. The serum activity of TAC showed a significant elevation in VG/GA strain vaccinated chickens at day 28 post-vaccination in water and day 28 and 42 post-vaccination by the spray route (p≤0.05) as compared to the control group. In the LaSota strain vaccinated chickens, serum activity of TAC was significantly increased only at day 42 post-vaccination through the spray route (p≤0.05) compared to the unvaccinated group. SOD serum activity was significantly elevated in VG/GA strain vaccinated chickens after 14 days post-vaccination through water (p≤0.01) and at 14 (p≤0.01), 28 (p≤0.05) and 42 (p≤0.01) days post vaccination via the spray route compared to the control. However, in LaSota strain vaccinated chickens, the SOD showed significantly elevated serum activity at day 14 (p≤0.01) and 42 (p≤0.05) post-vaccination through both drinking water and spray routes compared to the control group.

Table 6
Serum antioxidant enzymes of Ross chickens vaccinated with either Avinew (VG/GA) or LaSota strain vaccines.

Groups	CAT (ul/l)			TAC (mM/l)			SOD (U/ml)
	Sampling days			Sampling days			Sampling days
	14	28	42	14	28	42	14	28	42
Unvaccinated	312 ±4.23	311 ±4.23	322 ±3.46	2.48 ±0.88	1.77 ±0.14	2.46 ±0.41	273 ±5.48	312 ±4.56	322 ±4.68
Water route VG/GA	394 ±4.85a	258 ±3.46	294 ±4.78	2.64 ±0.12	2.92 ±0.78a	2.88 ±0.11	384 ±3.78b	286 ±6.78	288 ±3.78
Spray route VG/GA	298 ±5.64a	347 ±8.11a	396 ±5.78a	2.65 ±0.11	2.65 ±0.11	3.46 ±0.52a	355 ±5.13b	384 ±4.66a	391 ±2.81b
Water route LaSota	369 ±2.79a	301 ±5.11	389 ±4.09a	2.55 ±0.46	2.24 ±0.22	1.99 ±0.114	388 ±5.78b	354 ±3.78	375 ±8.46a
Spray route LaSota	315 ±7.64	284 ±4.19	294 ±5.11	2.87 ±0.11	2.74 ±0.14	3.46 ±0.24a	298 ±5.09a	286 ±4.77	365 ±6.87a

• Values having the letters a,b are significantly different from the control values at p≤0.05 and p≤0.01 respectively. TAC= total antioxidant capacity; CAT= Catalase; SOD= Superoxide dismutase. Mean± Standard error.

Humoral Immunity

Two tools for the evaluation of humoral immune response were used in the present study, hemagglutination inhibition (HI) and ELISA. The serum HI antibody titer of Ross chickens was significantly elevated on different days post-vaccination with the VG/GA or LaSota strains using the two examined vaccination routes relative to the unvaccinated control groups (Table 7). In particular, IgM titers were significantly elevated at day 28 post-vaccination with the VG/GA strain using the water route (p≤0.05). However, serum total Ig and IgG levels were significantly elevated at day 42 post-VG/GA strain vaccination via the spray route (p≤0.05). In the LaSota strain vaccinated chickens, serum IgM was significantly elevated at day 28 post-vaccination using the spray route (p≤0.05) compared to the unvaccinated control group. However, a significant elevation in total Ig and IgG was observed at days 28 and 42 post-vaccination, respectively, using either vaccination route (p≤0.05).

Table 7
HI antibody titer (log2) of Ross chickens vaccinated with either Avinew (VG/GA) or LaSota strain vaccines.

Groups	Sampling days
	Day 28			Day 42
	Total Igs	IgM	IgG	Total Igs	IgM	IgG
Unvaccinated	3.43±0.25	2.01±0.22	1.37±0.35	4.83±0.45	1.86±0.37	3.11±0.11
Water route VG/GA	4.54±1.30	3.57±0.16a	1.23±0.33	4.16±1.11	2.83±0.28	3.23±0.21
Spray route VG/GA	4.67±1.29	3.60±0. 28	2.16±0.41	5.89±0.10a	1.89±0.10	4.16±0.10a
Water route LaSota	4.91±1.22	2.03±0.14	2.97±0.52	7.03±1.20a	2.03±0.22	5.17±0.11a
Spray route LaSota	4.50±0.37	2.83±0.10a	1.52±0.44	7.66±1.18a	2.67±0.31	6.07±1.01a

• Values within the same strain with the letters a,b are significantly different from the control value at p≤0.05 and p≤0.01 respectively. Mean ± standard error.

Serum IgM and IgG, as measured by ELISA, were significantly elevated at different ages post-vaccination with either the VG/GA or theLaSota strains using the two examined vaccine delivery routes as compared to the unvaccinated control groups (Table 8). Particularly, serum IgM was significantly elevated at days 21 and 28 post-VG/GA strain vaccination using the water or spray route (p≤0.05). However, the serum IgG was significantly elevated at days 21, 28, and 42 following VG/GA strain vaccination through the water and spray routes (p≤0.05). In the LaSota strain vaccinated chickens, the serum IgM was significantly elevated at day 21 post-vaccination using water and spray routes (p≤0.05) compared to the control unvaccinated group. However, the serum IgG was significantly elevated at days 21, 28, and 42 post vaccinations using the water and spray routes.

Table 8
Serum immunoglobulins IgM and IgG as measured by ELISA of Ross chickens vaccinated with either Avinew (VG/GA) or LaSota strain vaccines.

Groups	IgM (ug/ml)				IgG (ug/ml)
	Sampling days				Sampling days
	14	21	28	42	14	21	28	42
Unvaccinated	3.481 ±0.97	3.446 ±0.63	4.431 ±1.33	2.559 ±0.43	2.465 ±0.47	2.468 ±0.58	3.264 ±0.44	3.456 ±0.28
Water route VG/GA	3.283 ±0.63	5. 583 ±0.55a	4.186 ±0.74	3.632 ±0.21	2.856 ±0.55	4.258 ±0.34a	8.415 ±1.71b	6.879 ±1.30a
Spray route VG/GA	3.551 ±1.021	4.792 ±0.94	6.666 ±0.45a	3.741 ±0.50	2.662 ±0.51	6.482 ±1.11b	6.864 ±1.22a	6.264 ±1.21a
Water route LaSota	3.620 ±1.64	6.502 ±0.60a	3.571 ±0.17	3.753 ±0.71	2.674 ±0.61	6.266 ±1.51b	9.756 ±1.42b	9.321 ±1.37b
Spray route LaSota	5.821 ±1.11	6.28 ±0.44a	3.006 ±0.37	3.852 ±0.52	2.159 ±0.44	6.831 ±1.33b	6.263 ±1.11a	9.080 ±1.22b

• Values having the letters a,b are significantly different from the control value at p≤0.05 and p≤0.01 respectively. Mean± Standard error.

Histological examination

The histological examination of the BF and spleen was conducted using hematoxylin-eosin stains. Histological examination of the BF of chickens of the control unvaccinated group at two different ages (28, and 42) stained by hematoxylin-eosin showed numerous lymphoid follicles on day 28. Lymphoid follicles of chickens vaccinated with the VG/GA and LaSota strains showed medulla with an extensive population of lymphocytes, especially on day 42. At 28 days of age, bursal follicles appeared large and densely populated in chickens vaccinated with the VG/GA strain by the spray route. A basal membrane, which was apparent in most sections, divided the cortex from the medulla. Bursal follicles in the unvaccinated group at 42 days of age were tiny, less packed, and had scattered vacuolation. Particularly on day 42, the medulla of BF of the chicken immunized with LaSota strain in drinking water displayed a fairly high density of lymphocyte population. On day 42 of age among chicken vaccinated with the LaSota strain through drinking water, it was noticed that the medulla had an extensive population of lymphocytes, especially at day 42, indicating a rise in the number of B-lymphoblasts that leads to the formulation of antibodies producing B-lymphocytes (Figure 1).

Figure 1
Micrograph of the BF stained with H&E (×20) at day 28 (1-5) and day 42 (6-10) of age of unvaccinated control chicken (1, 6), VG/GA strain by spray route (2,7); VG/GA strain by drinking water route (3,8); LaSota strain by spray route (4,9); LaSota strain by drinking water route (5,10). At 28 days of age, bursal follicles appeared large and densely populated in chickens vaccinated with the VG/GA strain through the spray route. The cortex was separated from the medulla by a basal membrane, visible in most sections. At 42 days of age, in the unvaccinated group, bursal follicles appeared small with a low cell density and scattered vacuolation. It is noticed that the medulla of BF of chicken vaccinated with the LaSota strain showed moderate density of lymphocytes, especially at day 42 among those vaccinated through the drinking water route. At day 42 of age among chicken vaccinated with the LaSota strain through the drinking water route, it was noticed that the medulla had an extensive population of lymphocytes, especially at day 42 of age, with a specific elevation in the number of B-lymphoblasts that lead to the formulation of antibodies producing B-lymphocytes. SI: Space interfollicular, C: Cortex, M: Medulla, LF: Lymphoid Follicle, ET: Epithelial Tissues, UC: Undifferentiated Cells, BC: Blood Capillaries; BF: bursa of Fabricius.

Histological analysis of the spleen in the groups that received vaccinations with the VG/GA and LaSota strains revealed normal histological features at all ages. Different forms of splenic nodules are clearly apparent as age advances from day 28 to day 42. Histological analysis of chicks immunized with the LaSota strains showed that the white pulp and splenic nodules expanded and became more visible on day 42 compared to day 28. In the unvaccinated group, the red pulp was overloaded with the white pulp and showed many splenic arterioles (Figure 2).

Figure 2
Micrograph of the spleen stained with H&E (×20) at 28 (1-5) and 42 (6-10) days of age of unvaccinated control chicken (1, 6), VG/GA strain by the spray route (2,7); VG/GA strain by the drinking water route (3,8); LaSota strain by the spray route (4,9); LaSota strain by the drinking water route (5,10). In vaccinated chickens, graphs show normal splenic histology and architecture. As age progresses from day 28 to the advanced age of 42, different types of splenic nodules are easily visible. Moreover, different types of red pulp and white pulp were easily visible. In unvaccinated chickens, red pulp is overloaded the white pulp and shows many splenic arterioles. RP: red pulp; WP: white pulp, SN: splenic nodule.

DISCUSSION

Numerous issues impact the outcomes of vaccination programs in broiler production, such as the effectiveness of the vaccine strain, the inhibitory effects of undesired active immunity, the ability of the strain to produce antibodies, and secondary reactions that were due to unsuitable routes of vaccination (Lim, 2014). For success in ND prevention, it is necessary to compare the available vaccine strains and routes of delivery (Okoroafor et al., 2019).

Different routes and types of NDV vaccination are used in poultry farms for different reasons, such as avoiding chick stress due to excessive treatment, and reducing vaccination costs (Ball et al., 2019). The route of vaccine administration has a pronounced effect on immunization and defense consequences in chickens (Ellakany et al., 2018). Mass application of vaccines reached about 53% and 60% when the route of application was spray and drinking water, respectively (Degefa et al., 2004). Besides, the presence of different organisms as immunosuppressive agents can prevent the effectiveness of vaccination (Perozo et al., 2012). This study was conducted to compare the effect of two administration routes, drinking water and spray, on the efficacy of VG/GA and LaSota-ND vaccine in Ross chickens.

The final BW and the BWG in chickens vaccinated with the VG/GA and LaSota strains at the end of the experiment were significantly increased after water or spray vaccination routes, compared to the control unvaccinated group. The FCR of LaSota-strain-vaccinated chickens was low after water and spray vaccination, while it was high in the control unvaccinated group. There were differences in the BW of birds vaccinated and infected with NDV. Previous data showed that infection with NDV resulted in a significant decrease in BW compared to vaccination (Ellakany et al., 2018). Another study reported no differences between control and vaccinated birds (Costa-Hurtado et al., 2015). Martinez et al. (2018) noted no significant differences in FCR between vaccinated and non-vaccinated birds (Martinez et al., 2018). The data indicated that both vaccination routes of either VG/GA or LaSota strains did not affect cumulative feed consumption and conversion.

SWI only showed a significant increase in VG/GA-strain-vaccinated chickens on day 42. However, BWI was significantly increased on days 28 and 42 post-vaccination via the water and spray routes as compared to the control. In the LaSota-strain-vaccinated chickens, the SWI was significantly increased on day 28 post-vaccination, while the BWI showed significant elevation on days 28 and 42 post-vaccination for the drinking water and spray routes compared to the control group. These data are in line with the work by Martinez et al. (2018) (Martinez et al., 2018), who observed that the immune organ indices showed a significant increase after vaccination especially BWI on day 21, and the work of Igwe et al. (2020) on SWI (Igwe et al., 2020).

Control of ND is of supreme significance in countries where it is endemic. Control is usually attained through live and inactivated vaccination programs. It is essential to note that, given the complexity of the adaptive immune response, it is frequently problematic to make cause-and-effect assumptions, since many issues impact the immune profile. In the immune system, many different types of cells and their chemical mediators contribute to immune cell regulation and both innate and adaptive immune responses to a variety of antigens (Stenger & Röllinghoff, 2001). Both humoral and cell-mediated immune (CMI) responses have been shown to play significant roles in birds’ defenses against NDV infection (Reynolds & Maraqa, 2000). Old studies have reported that it is possible to distinguish CMI rapidly after vaccination when a live NDV vaccine is used (Reynolds & Maraqa, 2000). The recent results of Martinez et al. (2018) indicated that the live LaSota strain vaccine failed to promote successful and complete initiation of CMI when used as primary immunization on the first day of age in broilers. More research is needed to better understand the specific CMI pathways that aid in the immunological response against NDV. Some studies have measured CMI specific for NDV through a blastogenesis microassay, concluding that humoral immunity and not CMI was essential for the protection of birds from a lethal challenge when vaccinated with inactivated NDV (Kapczynski et al., 2013). The role of CMI parameters was to reduce the viral shedding through direct apoptosis of infected cells (Russell et al., 1997). CMI is a part of adaptive immunity mediated through T lymphocytes, and has been proposed to be a significant agent for promoting protective immunity in chickens vaccinated against NDV (Sultan et al., 2016). T lymphocytes of the bursa express Th2 cytokines to clear the virus infection (Feau et al., 2011). They are detected 2-3 days post-infection or vaccination (Kapczynski et al., 2013). Herein, three methods were used to evaluate the CMI in birds vaccinated with two routes and strains of NDV, in vitro phagocytic index, glucose consumption by T-lymphocytes stimulated with PHA, and leukogram profile. In vitro phagocytic index was significantly increased after vaccination through the drinking water route with both the VG/GA and LaSota strains compared to the control unvaccinated group. The results of glucose consumption by PHA-stimulated lymphocytes showed a significant elevation in glucose consumption in vaccinated chickens regardless of the vaccine strain and vaccination route when compared to the control unvaccinated group. The phagocytic index is significantly affected by the state of neutrophils (Dey et al., 2015).

The results of the leukocyte profile are similar to the work of Ismail (2017) and Okorie-Kanu et al. (2018). Viremia due to vaccination also caused elevation in TLC and migration of M and H from vasculatures and bone marrow pools (Fry & McGavin, 2007). The reduction in the L% may be due to the depletion of the lymphoid tissues (Okorie-Kanu et al., 2018). The continuous period without vaccines causes continued depletion and leads to decreased immunity (Okoroafor et al., 2019). The significantly lower H/L ratio in vaccinated birds may be due to exposure to stress among the unvaccinated control, as previous studies have observed that increased H/L ratios are an important indicator of stress in birds (Gana et al., 2018).

Serum antioxidant enzyme activities of Ross chickens were significantly elevated at different days post-vaccination with two routes of vaccination of VG/GA and LaSota strains. In VG/GA strain-vaccinated chickens, the CAT serum activity was significantly increased at day 14 post-vaccination via the drinking water route. However, CAT serum activity showed a significant decline at day 14 post-vaccination, followed by a significant elevation at day 28 and 42 post-vaccination through the spray route. Moreover, LaSota-strain-vaccinated chickens showed a significant elevation in CAT serum activity at days 14 and 42 post-vaccination through the water route. The serum activity of TAC showed a significant elevation in VG/GA-strain-vaccinated chickens at day 28 post-vaccination in water and day 28 and 42 post-vaccination via the spray route. However, in LaSota-strain-vaccinated chickens, serum activity of TAC was significantly increased only at day 42 post-vaccination through the spray route. SOD serum activity was significantly elevated in VG/GA-strain-vaccinated chickens 14 days post-vaccination through water and at 14, 28- and 42-days post-spray vaccination route. However, in LaSota-strain-vaccinated chickens, the SOD showed significantly elevated serum activity at day 14 and 42 post-vaccination through both the drinking water and spray routes. The route of vaccination and the vaccine itself have previously been shown to impact the stress condition of birds. Wang et al. (2015) indicated that ND vaccination was considered a stress factor and could cause a drop in body weight gain and food conversion efficiency of chicks aged up to three weeks (Wang et al., 2015). Generally, stress may result from high ROS and/or low antioxidant levels (Han et al., 2006). Low levels of antioxidant enzymes such as SOD, CAT, and GPx were observed in 7 days old chicks after two doses of vaccination (Du et al., 2017). They concluded that stress might be due to two doses of vaccination at 1 and 3 days of age. Consequently, adaptation manifested by rising activities of antioxidant enzymes at the age of 15 days was observed. The time of adaptation was observed at the end of 42 days, as activities returned to their original levels and body mechanisms recovered from detrimental effects of the vaccine and birds adapted with time to the stress of vaccination (Mustafa, 2018).

According to Makkar et al. (2015), the serum levels of both IgM and IgG in poultry are a sign of humoral immunity being modulated and their adaptive immune responses being stimulated (Makkar et al., 2015). Chicken IgM is the predominant isotype produced following initial exposure to a novel antigen, physically and functionally mimicking its mammalian equivalent (Grönwall & Gregg 2014). After the initial interaction with an antigen, specific IgM responses rise, subsequently declining with a subsequent exposure (Martinez et al., 2018). The predominant isotype of serum is avian IgG. It is produced following the generation of the IgM isotype in the initial humoral reaction, and it serves as the primary isotype in the follow-up humoral response (Tirziu & Şereş, 2010). Two tools for the evaluation of humoral immune response were used in the present study, HI and ELISA. The serum HI antibody titer of Ross chickens was significantly elevated in comparison to the control group on different days post-vaccination with VG/GA and LaSota strains through two vaccination routes. In particular, IgM titers were significantly elevated at day 21 post-VG/GA strain vaccination via the water route. However, serum total Ig and IgG levels were significantly elevated at day 42 of VG/GA strain vaccination through the spray route. In LaSota-strain-vaccinated chickens, serum IgM was significantly elevated at day 21 post-vaccination through the spray route. However, a significant elevation of total Igs and IgG was observed at days 21 and 42 post-vaccination, with respectively the water and spray routes. In chickens, IgM and IgY (equivalent to IgG) antibodies are produced as a part of the immune reaction (Jeurissen et al., 2000). HI antibody titers were routinely used to settle defense against NDV challenges, and the mean values obtained here were similar to the data obtained previously ( Jeong et al., 2013; Awad et al., 2015). Antibodies are formed against hemagglutinin, viral proteins, and fusion glycoproteins of the virus to limit the spread of the virus (Sedeik et al., 2019). Peaks of antibodies were reached 21-28 days post-vaccination (Al-Garib et al., 2003). The detection of antibodies before elevation due to vaccination might be due to maternal antibodies following a natural decline in the unvaccinated group, as previously mentioned (Martinez et al., 2018). The use of the live LaSota strain NDV vaccine after the decline in the level of maternal antibodies resulted in a strong antigen-specific humoral immune response (Martinez et al., 2018). Therefore, in the present study, vaccination was done at one week of age.

Serum immunoglobulins IgM and IgG, as measured by ELISA, were significantly elevated at different ages post-vaccination with either VG/GA or LaSota strains through either of the utilized delivery routes compared to the control unvaccinated groups. Particularly, serum IgM was significantly elevated at days 21 and 28 post-VG/GA strain vaccination using the water and spray routes, respectively. However, serum IgG was significantly elevated at days 21, 28, and 42 following VG/GA strain vaccination using either vaccination routes. In LaSota-strain-vaccinated chickens, serum IgM was significantly elevated at day 21 post-vaccination using the water and spray routes compared to the control unvaccinated group. However, serum IgG was significantly elevated at days 21, 28, and 42 post vaccinations in both vaccination routes. The results were in agreement with Landman et al. (2017), who observed high antibody titers with the spray route due to the presence of virus-loaded respirable droplets/dry particles in the spray (Landman et al., 2017). VG/GA strain spray vaccination was done previously and seems to be most effective if there were no interference factors such as bacterial infections (Khodayari & Feizi, 2017). Measurement of serum IgM and IgG by both ELISA and HI was recently done with the same parallel results (Attia et al., 2020).

Hematoxylin-eosin stains were used in the current study for histological examination of the BF and spleen. These organs are widely recognized as being crucial avian lymphoid organs, and serving as primary immunological sites for induction of immunological responses against antigens (Abdul-Aziz, 2016). The study of the BF and spleen aimed to guarantee that the different strains and routes of vaccine had no severe hostile impact on the immune responses. The degree of distribution of white and red pulps, as well as the number of splenic nodules, are used for assessing the spleen to determine its immunological status. B cells multiply and undergo differentiation in the splenic nodules. Histological analysis of the spleen in the groups that received vaccinations with the VG/GA and LaSota strains revealed normal histological features at all ages. Different forms of splenic nodules are clearly apparent as age advances from day 28 to day 42. Histological analysis of chicks immunized with LaSota strains showed that the white pulp and splenic nodules expanded and became more visible on day 42 compared to day 28. In the unvaccinated group, the red pulp is overloaded with the white pulp and shows many splenic arterioles.

To regulate humoral immunity, chicken BF grows rapidly after hatching and achieves a massive size at the age of one to four months (Abdul-Aziz, 2016). A large number of lymphoid or bursal follicles make up the BF. Each follicle is made up of a cortex and medulla that are divided by a corticomedullary border of blood capillaries and undifferentiated epithelial cells (UC) that develop into the B lymphocytes that produce antibodies (Abdul-Aziz, 2016; Cooper, 2015). Connective tissue, epithelial cells, and blood capillaries cover the cortex of the lymphoid follicles. In addition to the B-lymphocytes’ production of antibodies, the lymphoid or bursal follicles are the site of B-lymphocytes synthesis, proliferation, and ultimate differentiation. Taylor & McCorkle (2009) reviewed the development of the concept that the lymphoid follicles are a key organ in immunology (Taylor Jr & McCorkle Jr, 2009). Histological examination of BF of chickens of the control unvaccinated group at two different ages (28, and 42) stained with hematoxylin-eosin showed numerous lymphoid follicles on day 28. Lymphoid follicles of chickens vaccinated with the VG/GA and LaSota strains showed medulla with an extensive population of lymphocytes, especially on day 42. At 28 days of age, bursal follicles appeared large and densely populated in chickens vaccinated with VG/GA strain via the spray route. A basal membrane, which was apparent in most sections, divided the cortex from the medulla. Bursal follicles in the unvaccinated group at 42 days old were tiny, less packed, and had scattered vacuolation. Particularly on day 42, the medulla of the BF of the chicken immunized with the LaSota strain in drinking water displayed a fairly high density of lymphocyte population. On day 42 of chicken vaccination with the LaSota strain through drinking water, it was noticed that the medulla had an extensive population of lymphocytes, especially at day 42, indicating a rise in the number of B-lymphoblasts that leads to the formulation of antibodies producing B-lymphocytes. This demonstrated how the immune system of birds is complicated and influenced by various factors including age, immunization route, and stress (Attia et al., 2017; Luskin & DeAngelo, 2019). At the age of 28 days, in the vaccinated groups, some follicles appeared with mildly reduced lymphocytes, which might trigger the immune response, as previously reported by (Cooper, 2015).

CONCLUSION

In conclusion, humoral immune response, represented by immunoglobulins, and cell-mediated immunity, represented by T cell activity, were stimulated by the two vaccine strains used in this study. Histologically, the bursa and spleen kept their structure, and it could be shown that all the doses and routes used produced minor differences with no adverse effects on the organs. This was expected, as the two vaccine virus strains are lentogenic pathotypes. Data generated from this work can be used for monitoring current vaccination schedules that require special monitoring assays.

ACKNOWLEDGEMENTS

The researchers would like to thank the Deanship of Scientific Research, Qassim University, for the support of this project.

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Edited by

• Section Editor:
  Maria Fernanda Burbarelli

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Data will be available upon request.

Publication Dates

• Publication in this collection
  31 Mar 2025
• Date of issue
  2025

History

• Received
  22 Nov 2023
• Accepted
  31 Jan 2025