Triple La Sota re-vaccinations can protect laying chickens for 3 months against drop in egg production caused by velogenic viscerotropic Newcastle disease virus infection.

One hundred and ten Isa Brown layers were vaccinated with La Sota, once at point of lay at 18 weeks and three times at peak of lay which occurred at 27-29 weeks of age. Thereafter, they were weekly monitored for haemagglutination inhibition (HI) antibody decline. The first batch A of the layers were challenged with velogenic viscerotropic Newcastle disease (vvND) virus (vvNDV) on day 24 post-vaccination (PV), when the geometric mean titre (GMT) was 84.4, batch B were challenged on day 48 PV at GMT of 42.2, while batch C were challenged on day 97 PV at GMT of 21.1. The individual chicken HI antibody titres of the 10 layers in batch C at the day of challenge were: 7 layers had HI titres of 16, 2 layers had HI titres of 32 and 1 layer had HI titres of 64. Each challenge in the three batches produced no clinical signs including drop in egg production. But there was initial swelling of the spleen followed by atrophy with high antibody responses. The virus was recovered in all the cloacal swabs on days 3-9 post-challenge (PC) at low titres. On days 145 PV and 48, post-Batch C challenge the remaining hyperimmunized unchallenged layers demonstrated a drop in total % egg production (p < .05) and changes in egg quality. The HI GMT was 256. The virus was recovered in all the cloacal swabs on days 3-9 following appearance of clinical signs. There was no mortality in the experiment. Based on the above observations, it is concluded that triple La Sota re-vaccination can protect layers against a drop in egg production in areas where vvNDV infection is enzootic.

INTRODUCTION

Newcastle disease (ND) is one of the most important diseases of poultry (Czegledi et al., 2006). It affects virtually all avian species producing a wide range of clinical signs which can be inapparent, mild, moderate and severe. Chickens are most susceptible, turkeys are less susceptible while geese and ducks are resistant. (Eze et al., 2014; Igwe, Ezema, Eze, & Okoye, 2014; Okorie‐Kanu, Okorie‐Kanu, & Okoye, 2016; Okoroafor et al., 2018; Piacenti et al., 2006) The velogenic ND (vND) is characterized by high mortality rates and can affect the digestive, respiratory, nervous, female reproductive and lymphoid systems (Cattoli, Susta, Terregino, & Brown, 2011; Ezema, Eze, Shoyinka, & Okoye, 2016; Okoroafor et al., 2018; Okpe, Ezema, Shoyinka, & Okoye, 2015; Wakamatsu, King, Kapczynski, Seal, & Brown, 2006). It is the greatest impediment to poultry production in many parts of the world and is enzootic in Africa, Middle and Far East and the Americas (Czegledi et al., 2006; Echeonwu, Ireogbu, & Emeruwa, 1993; Solomon, Aboinik, Joannis, & Bisschop, 2012). The disease is caused by Newcastle disease virus (NDV) which is an orthoavulavirus. It is a non‐segmented, single‐stranded, negative‐sense RNA virus belonging to the genus Orthoavulaviru (Absalon, Cortes‐Espinosa, Lucio, Miller, & Afonso, 2019) subfamily Avulavirinae within the family Paramyxoviridae, order Mononegavirales (Lamb et al., 2005; Mayo, 2000). Since the emergence of ND in 1926, there has been continuous emergence of new virulent genotypes from global epizootics with year to year change in genome sequence of the virus (Diel et al., 2012 and Snoeck et al., 2013). Control of ND has been difficult because of a very wide host range and many of the hosts demonstrate inapparent infections making it easy to transfer the disease to other hosts. The wide range of clinical signs of ND, which resemble those of many other poultry diseases, make early diagnosis of ND difficult in the field. Control of the disease is by vaccination and biosecurity. Vaccination can protect against some clinical signs but not against multiplication and shedding of the virus (Bwala, Clift, Duncan, Bisschop, & Oludayo, 2012; Cattoli et al., 2011; Miller et al., 2013; Miller, Estevez, Yu, Suarez, & King, 2009; Okoroafor et al., 2018; Okwor et al., 2016; Sa e Silva, 2016) and some lesions associated with the disease (Bwala et al., 2012; Ezema et al., 2016; Ezema, Okoye, & Nwanta, 2009; Okoroafor et al., 2018).

One of the major clinical signs of ND in layer chickens is a drop in egg production. This is a serious problem in those countries where vvND is enzootic. At the peak of the risk period each year, many vaccinated layer chickens manifest drop in egg production with or without other clinical signs (Ezema et al., 2009). Some show complete cessation of egg production. These situations can last for several weeks leading to huge economic losses to egg producers. Furthermore, Bwala et al. (2012) and Igwe, Ihedioha, and Okoye (2018) in a study on the pathology of velogenic viscerotropic NDV (vvNDV) challenge in the reproductive system of layer chickens, reported a drastic drop in egg production in ND vaccinated layers. The aim of this study was to develop a vaccination schedule that will provide adequate protection against a drop in egg production caused by vvNDV infection in areas where the disease is enzootic.

MATERIALS AND METHODS

Experimental animals

One hundred and ten Isa brown pullets were obtained at the age of 12 weeks from a commercial hatchery (Zartech Farms, Ibadan). The pullets were vaccinated against ND using HB1, La Sota and Komarov strains, Marek's disease, infectious bursal disease (IBD) and fowl pox prior to delivery (Table 1). The Marek's and HB1 vaccines were given for 1 day old, La Sota for 3 weeks and Komarov for 6 weeks of age. The IBD vaccine was administered at 2 and 4 weeks, respectively, and pox at 7 weeks of age. All the vaccines were live vaccines produced by the National Veterinary Research Institute, (NVRI), (Vom, Plateau State, Nigeria) with the exception of the Marek's disease vaccine (Israel) which was foreign. The birds were housed in isolation at the experimental poultry unit of the Department of Veterinary Pathology and Microbiology, University of Nigeria, Nsukka. The pullets were kept in deep litter, fed Growers Mash (Topfeeds®) from time of arrival till 18 weeks of age and then Layers Mash (Topfeeds®) till the end of the experiment. Water and feed were provided ad libitum. General care of the birds was provided in accordance with the Institutional Animal Care and Use Committee, as outlined in the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching. At 18 weeks of age, the pullets were vaccinated with La Sota to boost their immunity at point of lay. At 27 weeks of age, the layers were re‐vaccinated with La Sota. The re‐vaccination was repeated at 28 and 29 weeks of age which corresponded with the peak of lay at weeks 5 and 6 of lay, respectively (Table 1).

Table 1

Experimental design

Pre‐challenge vaccine administration

The La Sota vaccine was manufactured by the National Veterinary Research Institute (NVRI), (Vom, Nigeria) and had a median embryo infective dose (EID50) of 106.8 per ml. Vaccination was by oral drench each time. Each bird received 1 ml of the diluted vaccine.

Pre‐challenge serology

The antibody responses against NDV in 10 of the vaccinated layers were determined two weeks post‐vaccination and thereafter monitored weekly using the HI test by OIE (Animal Health) (2012) At a substantial or marked decline of the titre, 20 layers were randomly selected, taken to a distant location and challenged with vvNDV. The first challenge (batch A) took place on day 24 post‐vaccination (PV) when the geometrical mean titre (GMT) of the layers was 84.4, batch B layers were challenged on day 48 PV with a GMT of 42.2, batch C on day 97 PV with a GMT of 21.1 and batch D who were not challenged, but suffered a natural outbreak of vvND on day 145 PV (Table 1). The layers that were challenged in batches A, B and C, which showed no clinical signs, became the control for batch D.

vvNDV inoculum

A Nigerian strain of velogenic viscerotropic NDV (vvNDV) known as duck/Nigeria/903/KUDU–113/1992 was used. It was isolated from apparently healthy ducks, purified and characterized by Echeonwu et al. (1993). The strain belongs to NDV class II, genotype XVII (Shittu et al., 2016). The inoculum had a median embryo effective dose (EID50) of 106.4/ml and the challenged layers were each given 0.2 ml of the inoculum intramuscularly (IM). The unchallenged layers served as the control in batches A, B and C. A natural vvNDV infection of the unchallenged layers occurred later and was taken as the last challenge group, D. The layers that were earlier challenged and showed no clinical signs in batches A, B and C were used as controls.

Clinical signs and lesions

After each challenge, the layers were monitored 21 days post‐challenge (PC) for clinical signs with special attention to egg production and quality. Three layers each were sacrificed from the control and challenged groups on day 3 PC and examined for gross lesions. Samples of the oviduct and spleen were fixed and processed for histopathology.

Post‐challenge serology

At each challenge, blood samples were collected from 10 layers each from the challenged and control groups on days 0, 7, 14 and 21 PC. The serum samples were assayed for HI antibody using HA/HI method of OIE (Animal Health) (2012). The antibody geometrical mean titre (GMT) was calculated using the Tube Method and Table provided by Villegas and Purchase (1989).

Virus isolation

Cloacal swabs were collected from three layers each in the challenged and control groups on days 3, 6 and 9 PC for NDV isolation and confirmation in embryonating chicken eggs using the method of OIE (Animal Health) (2012).

Identification and confirmation of allantoic fluid isolate

The presence of hemagglutinating virus and confirmation of the agent to be NDV in allantoic fluids harvested were demonstrated using the HA and HI tests (OIE, 2012). The NDV‐monospecific antiserum used in the HI test was prepared in chickens following the methods of Grimes (2002).

Statistical analysis

The percentage egg production was compared between challenge and control groups using the Student's t test. The HI results are presented as geometrical mean titres (GMT) of the batches.

RESULTS

Clinical signs

Clinical signs were not observed in batches A, B and C layers which were challenged at antibody GMTs of 84.4, 42.2 and 21.1 respectively. There was no difference (p > .05) between the challenged and their controls in the weekly % egg production in all the groups (Table 2). The total % egg production was (p < .05) less in the challenged than the control layers in group D only (Table 2). In batch D that were naturally infected, about 10% of the layers had a slight cough in the first week of infection. Clinical signs included a reduction in feed and water consumption and voiding of greenish faeces. Production of misshapen, small and white coloured eggs for brown laying type and thin shelled and shell‐less eggs were observed. No deaths were recorded in any of the batches.

Table 2

Percentage egg production of the various batches post‐challenge/infection

Batches	Mean percentage egg production, ± standard deviation.
	Chall/infected	Control	Probability value
Batch A	94.00 ± 4.58	94.00 ± 4.58	p = .830
Batch B	91.00 ± 5.20	97.33 ± 2.08	p = .158
Batch C	93.67 ± 3.06	91.33 ± 2.31	p = .355
Batch D*	73.00 ± 6.25	90.00 ± 2.00	p = .032

Asterisk indicates significant difference between the Challenged/Infected and Control (p < .05).

Lesions

The spleen was enlarged on the day 3 PC, but atrophic on day 7 PC in all the batches (Figure 1). The ovarian follicles were atretic in batch D only. Sections of the spleen showed lymphocytic necrosis and depletion in all the batches. Sections of the oviduct showed deciliation, oedema, necrosis of the epithelium and glands, infiltration by mononuclear cells, fibrosis and later, hyperplasia of epithelial and glandular cells in batch D only (Figures 2, 3, 4). There was lymphocytic necrosis and depletion around the sheathed arterioles with fibrin deposition in the sections of the spleen (Figure 5).

Figure 1

Atrophy of the spleen in challenged Batch C layers

Figure 2

Infundibulum showing hyperaemia, oedema, infiltration by mononuclear cells and hyperplasia of the epithelial cells in infected Batch D layer. Bar=50 micrometer

Figure 3

Magnum showing oedema and necrosis of the glands in infected Batch D layer. Bar = 50 micrometer

Figure 4

Uterus showing severe necrosis of the glands, oedema and infiltration by mononuclear cells in infected Batch D layer. Bar = 50 micrometer

Figure 5

Spleen showing lymphocytic depletion and fibrin deposition around the sheathed arterioles in infected Batch D layer. Bar = 500 micrometer

Serology

The challenge with the vvNDV each time led to steady rise in antibody titre from days 7 to 21 PC (Table 3). The control groups showed a continuous decline from GMT 84.4 in Batch A to 13.9 in Batch C. On the day of challenge of batch C layers, the GMT was 21.1, but the individual HI titres in the layers were 16 in 7 layers, 32 in 2 and 64 in 1 layer (Table 3). In batch D, there was tremendous rise on day 21 post‐infection that was much higher than was observed in other batches.

Table 3

Haemaglutination inhibition (HI) antibody titres (GMT) in all the challenge/infected and control groups

Batches	Means of HI titre, ±standard deviation
	Chal/infected	Control	Probability value
Batch A*	107.40 ± 21.91	68.70 ± 15.01	p = .031
Batch B	67.50 ± 25.02	40.15 ± 2.59	p = .116
Batch C*	36.33 ± 10.77	17.03 ± 3.02	p = .033
Batch D	150.65 ± 121.67	24.50 ± 3.93	p = .130

Asterisk indicates significant difference between the Challenged/infected and Control groups (p < .05).

ND virus isolation

Virus was isolated from all the cloacal swabs in all the batches (Table 4) at HA titres of 2–8. All the isolates were confirmed in a HI test using known NDV antiserum.

Table 4

Virus isolation (HA tires) from cloacal swabs in vvNDV challenge/infected groups

Days PC	Batches
	A	B	C	D
3	3a/3b (4)	3/3 (4)	3/3 (4)	3/3 (4)
6	3/3 (4)	3/3 (4)	3/3 (4)	3/3 (8)
9	3/3 (4)	3/3 (4)	3/3 (2)	3/3 (4)

Number of swabs positive for NDV antigen.

Total number of swabs tested.

DISCUSSION

This study showed that chicken layers could be well protected against all of the clinical signs including drop in egg production for a minimum of three months by triple La Sota revaccinations. It should be highlighted that this is contrary to the current practice of one La Sota revaccination at 3‐ to 6‐month intervals practiced by most of the producers. But it should be pointed out that this vaccination programme may not be adequate in other locations where other strains of the vvNDV are enzootic. The drop in egg production in batch D was observed on day 145 PV and day 48 post‐Batch C challenge. The antibody titre of the layers was not known at the time of the field infection. But the last experimental challenge of the layers in batch C was on day 97 PV at individual titres of 16 (24) and above and that produced no clinical signs. Layers may thus be fully protected against the clinical signs of vvNDV challenge at HI titres of 16 and above. This is also in agreement with the reports of others who worked on the antibody titres required to protect young chickens that did not have additional stress of egg production against clinical signs and mortalities (Allan & Gough, 1974; Allan, Lancaster, & Toth, 1978; Nagy, Krell, Dulac, & Derbyshire, 1991 ; Nasser et al., 2000; Phillips, 1973). The group A layers were challenged 2 weeks after the last revaccination at 29 weeks of age at the peak of egg production. This age was chosen because egg production causes a lot of stress which makes the layers susceptible to many infections. In this experiment, the layers were vaccinated four times with La Sota vaccine. One at point of lay and three at peak of lay and protection lasted 97 days. The triple revaccinations at the peak of egg production produced high antibody titres which gave protection for longer durations than single revaccination. This is not the vaccination practice in the commercial farms even in those countries including Nigeria where vvND is enzootic. Usually, the pullets are given Komarov vaccine intramuscularly at point of lay. But the use of this vaccine is being phased out as the poultry production grows into large industries where thousands and millions of birds are kept, at the same time, in one farm and handling of the birds individually for vaccination is not possible. Specifically, in Nigeria, laying chickens are given Komarov vaccine at point of lay and La Sota vaccine before the peak period of vvND outbreaks which is December to March each year. During this time, millions of local or indigenous chickens which are not vaccinated against any disease are wiped out in fulminating epizootics. Many commercial laying chickens show a drastic fall in or complete cessation of egg production with or without other clinical signs even after the single La Sota revaccination. This is the reason for the triple La Sota revaccinations before challenge in this experiment. The double revaccination will be tried in another experiment. Our results are not in agreement with those of Bwala et al. (2012) and Igwe et al. (2018) who reported drastic drop in egg production at challenge of vaccinated layers with vvNDV. The later vaccinated their layers with Komarov at 9 weeks of age and point of lay before challenge at peak of egg production. The former also administered single La Sota vaccination before challenge.

At each challenge, in this experiment, the virus was isolated in all the cloacal swabs from days 3 to 9 PC in all the batches confirming infection with the vvNDV. Virus shedding was at low titres. The low titres of the virus shedding may be due to the earlier observation that at high levels of vaccine antibodies virus shedding is low at challenge because a large fraction of the viral inoculum is neutralized by the antibodies (Miller et al., 2013, 2009). The atrophy, necrosis and depletion of lymphocytes in the spleen observed at each challenge is in agreement with the observation of some workers who reported that, at challenge, vaccine antibodies could protect chickens against developing clinical signs of vND but not against atrophy, necrosis and depletion of lymphocytes in the lymphoid organs and shedding of the virus. The bursa and thymus had regressed in the layers at the time when this experiment was carried out. While the antibody titres were steadily waning in the control groups, the challenged birds showed an appreciable rise of titres in all the batches but the rise was highest in the Batch D that had natural infection and where infection took place when the layers had the lowest antibody titres. The drop in egg production that occurs in many farms at the seasonal peak periods of vvND outbreaks in Nigeria and other tropical countries, where this disease is enzootic, is most likely to be due to low unprotective HI antibody concentrations even in some recently vaccinated flocks and the high virulence of vvNDV. Frequent power outages can make it difficult to maintain the cold chain required for safe storage of vaccines. Even when the vaccines are potent, many immunosuppressive diseases can lead to poor antibody response. These include infectious bursal disease, chicken infectious anaemia, Marek's disease, aflatoxicosis and many other mycotoxicoses, avitaminosis A and ammonia gas intoxication (Hoerr, 2010). Environmental temperatures, nutrition and some medications also affect antibody response. These factors can be worsened by poor biosecurity measures found in developing counties. These are facts that the egg producers have to bear in mind and guide against for effective vaccination of their layers.

In conclusion, egg producers in areas where vvND is enzootic are advised to boost the resistance of their layers against vvNDV infection by using triple La Sota vaccinations every 3 months to avoid losses in egg production.

Statement of animal rights

The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. All animal studies were approved by the University of Nigeria Committee on Medical and Scientific Research Ethics. This project was therefore performed in accordance with the ethical standards laid down in the US National Research Council's guidelines for the Care and Use of Laboratory Animals.

CONFLICT OF INTEREST

The authors do not have any conflict of interest to report.