1
Immune responses and interactions following simultaneous application of live
2
Newcastle disease, infectious bronchitis and avian metapneumovirus vaccines
3
in specific-pathogen-free chicks
4
5
6
7
8
9
10
11
Faez Awad a,b, Anne Forrester a, Matthew Baylis a, Stephane Lemierec, Richard Jones a &
12
Kannan Ganapathy a,*
13
14
15
16
17
18
a
University of Liverpool, Leahurst Campus Neston, South Wirral, CH64 7TE, UK
19
b
University of Omar Al-Mukhtar, Faculty of Veterinary Medicine, Al-Bayda, Libya
20
c
Merial S.A.S., 29 avenue Tony Garnier, 69348 Lyon cedex 07, France
21
22
23
24
25
26
27
28
29
30
* Corresponding author Tel.: +44 151 7946019; fax: +44 151 7946005.
31
E-mail address: gana@liv.ac.uk
32
33
34
35
1
36
ABSTRACT
37
38
Interactions between live Newcastle disease virus (NDV), avian metapneumovirus (aMPV)
39
and infectious bronchitis virus (IBV) vaccines following simultaneous vaccination of day old
40
specific pathogen free (SPF) chicks were evaluated. The chicks were divided into eight
41
groups. One group served as unvaccinated controls, whereas the other seven groups were
42
vaccinated against NDV, aMPV and IBV (single, dual or triple). Haemagglutination
43
inhibition (HI) NDV antibody titres were similar in single, dual or triple-vaccinated groups.
44
Levels of aMPV enzyme-linked immunosorbent assay (ELISA) antibodies were suppressed
45
when the aMPV vaccine was given with other live vaccines. The cellular and local immunity
46
induced by administration of live NDV, aMPV or IBV vaccines (individually or together)
47
showed a significant increase in the expression of CD4+, CD8+ and IgA bearing B-cells in
48
the trachea compared to the unvaccinated group. There were no significant differences
49
between the vaccinated groups. Despite lowering the serological antibody response to aMPV,
50
simultaneous vaccination with live NDV (VG/GA strain), aMPV (subtype B), and IBV
51
(H120) did not affect protection against aMPV or IBV. NDV challenge was not included due
52
to local restrictions; however, HI titres in the NDV vaccinated groups reached above the
53
protective titre. It appears that the efficacy of each of the live vaccines were not compromised
54
when given simultaneously (dual or triple) in young SPF chicks.
55
56
Key words: chick, vaccines, interactions, immune responses, NDV, aMPV, IBV
57
58
59
60
61
2
62
1. Introduction
63
Newcastle disease virus (NDV), avian metapneumovirus (aMPV) and infectious bronchitis
64
virus (IBV) are important and common causes of respiratory diseases in chickens. The losses
65
due to these pathogens are significantly reduced by the use of live and inactivated vaccines
66
worldwide, including the use of combined live vaccines. All three viruses initially replicate
67
in the epithelium of the respiratory tract (Alexander, 2000; Cavanagh and Gelb, 2008; Gough
68
and Jones, 2008), and their ability to induce protective immunity may therefore be reduced if
69
it is necessary to apply all three live vaccines simultaneously. Previous in vivo studies,
70
demonstrated that IBV interfered with the replication of virulent NDV (Hanson et al., 1956),
71
live NDV vaccines (Thornton and Muskett, 1975) and aMPV vaccines in chickens (Cook et
72
al., 2001). Ganapathy et al. (2005) demonstrated that NDV vaccination delayed the
73
replication of the live aMPV vaccine, and also reduced the humoral antibody responses.
74
These studies relied on serum antibody titres, either measured by ELISA or HI test to
75
demonstrate the impact of simultaneous vaccine application in chicks. Systemic antibody
76
responses have been shown to have a poor correlation with protection against IBV (Pensaert
77
and Lambrechts, 1994) and aMPV infections (Cook et al., 1989). Working with live
78
NDV+IBV and aMPV vaccine viruses (Cook et al., 2001; Ganapathy et al., 2005; Tarpey et
79
al., 2007), the authors suggested that the protection against these viruses was likely induced
80
by cell-mediated and local immune responses, however this was not investigated.
81
It has been reported that cellular and local immunity plays a critical role in the protection of
82
chicks from NDV, aMPV and IBV infections (Takada and Kida, 1996; Seo and Collisson,
83
1997; Rautenschlein et al., 2011). A number of studies have examined the induction and role
84
of cell mediated immunity after vaccination with live NDV (Russell et al., 1997; Rauw et al.,
85
2009) or IBV vaccines (Okino et al., 2013) alone. In a simultaneous vaccination study,
86
Ganapathy et al. (2005) reported that IgA levels in the tear of chicks that were given NDV
3
87
alone or dually with aMPV were not affected. Despite the widespread application of
88
combined live respiratory viral vaccines in young chicks, very little is known about the
89
cellular and local immune responses following simultaneous application of live NDV, aMPV
90
or IBV vaccine viruses. The objective of this study was to evaluate the interactions between
91
live NDV, aMPV and IBV vaccine viruses in young SPF chicks. In addition to humoral
92
antibody responses, the cell-mediated and local antibody responses in the trachea were also
93
assessed. Furthermore, the protection conferred against virulent IBV and aMPV was
94
evaluated.
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
4
112
2. Materials and Methods
113
114
2.1. Eggs and chicks
115
Fertile eggs from SPF White Leghorn chickens (Lohmann Animal Health, Cuxhaven,
116
Germany), were incubated and hatched in our facilities (University of Liverpool). Chicks
117
were placed in separate isolation units and maintained according to animal welfare guidelines
118
and under strict biosecurity measures. Food and drinking water were provided ad libitum.
119
2.2. Vaccines
120
NDV vaccine (strain VG/GA), aMPV vaccine (subtype B), IBV vaccine (strain H120) were
121
kindly provided by Merial SAS, (Lyon France).Vaccines were reconstituted as recommended
122
by the manufacturer. For NDV and aMPV vaccines, each 1,000 dose vial was reconstituted in
123
2 ml of chilled sterile water (SW) and thoroughly mixed with 100 ml of SW. For IBV
124
vaccine, a 5,000 dose vial was reconstituted in 5 ml of SW and then 1 ml was mixed with 100
125
ml of SW. For dual-vaccination, NDV vaccine was first reconstituted and then followed by
126
aMPV or IBV vials as previously described. Each pair was then mixed in 100 ml SW. For
127
triple vaccination, NDV, aMPV and IBV were reconstituted as above and all three were
128
diluted in 100 ml of SW.
129
130
2.3. Challenge viruses
131
132
2.3.1. IBV
133
The Massachusetts (M41) strain (Raj and Jones, 1997) was grown in embryonated chicken
134
eggs (ECE) and titrated in trachea organ cultures (TOC) as described previously (Cook et al.,
135
1976). Titres were expressed as median ciliostatic doses (CD50) and calculated by the method
136
of (Reed and Muench, 1938). The titre obtained was 105 CD50/ml.
5
137
138
2.3.2. aMPV
139
aMPV subtype B (Ganapathy et al., 2007) virus was propagated and titrated in TOC as
140
previously described (Cook et al., 1976). The median ciliostatic dose was calculated as
141
previously described (Reed and Muench, 1938). The titre obtained was 104.5 CD50/ml.
142
143
3. Experimental designs
144
Three hundred and twenty day-old chicks were randomly distributed in eight groups; each
145
group consisted of 40 chicks per isolation unit. The vaccination treatments consisted of either
146
a single NDV, aMPV or IBV vaccine dose, or a combination (dual or triple) of the three
147
(Table 1). Each chick was vaccinated via oculo (50 µl) nasal (50 µl) route. Dosages received
148
by each bird were as recommended by the manufacturers (Table 1). Following vaccination,
149
the chicks were observed daily for clinical signs. Oropharyngeal (OP) swabs were randomly
150
collected prior to vaccination from ten chicks and at 3, 7, 14, 21, 26 and 35 days post
151
vaccination (dpv) from ten chicks per groups for reverse-transcriptase polymerase-chain
152
reaction (RT-PCR) analysis. For serology, blood was collected prior to vaccination and at 21
153
and 35 dpv from eight chicks in each group. At 21 dpv, five chicks from each group were
154
humanely killed and trachea samples were collected from each bird, immediately placed in
155
aluminium foil cups containing cryo embedding compound (OCT) and frozen in liquid
156
nitrogen (-190°C). The trachea was used for detection of CD4+, CD8+ and IgA-bearing B
157
cells by immunohistochemistry. All samples were stored at -70oC until processing. At 21 dpv,
158
ten birds from each group were transferred to a different isolation room and one was
159
challenged with 0.1 ml virulent aMPV subtype B (103.5 CD50/bird) by the oculo-nasal route.
160
An additional ten birds from each groups were challenged with 0.1 ml virulent IBV M41 (104
6
161
CD50/bird) via the same route. The remaining 15 chickens in each group were left
162
unchallenged.
163
164
3.1. Detection of vaccine viruses by RT-PCR
165
Pooled daily swabs were dipped as a single sample per group, per day in a labelled bijou
166
containing 1.5 ml of guanidium isothiocyanate (solution D) (Chomczynski and Sacchi, 2006).
167
RNA was extracted using the guanidinium thiocyanate-phenol chloroform method
168
(Chomczynski and Sacchi, 2006). RT-PCR was performed as previously described for NDV
169
(Aldous and Alexander, 2001), aMPV (Cavanagh et al., 1999) and IBV (Worthington et al.,
170
2008) .
171
172
3.2. Serology
173
NDV antibodies were detected by a haemagglutination inhibition test (HI) (Allan and
174
Gough, 1974). For aMPV and IBV, sera were tested by commercial enzyme-linked
175
immunosorbent (ELISA) assays (BioChek, Gouda, The Netherlands).
176
177
3.3. Immunohistochemical analysis of cell-mediated responses in tracheal sections
178
Indirect immunohistochemical staining was conducted to quantify immune cell populations
179
of CD4+, CD8+ or IgA-bearing B-cells as previously described (Rautenschlein et al., 2011),
180
with some modification. Briefly, tracheal samples were sectioned using a cryostat at 5 μm
181
and mounted on poly-L-lysine-coated glass slides. Sections were immersed in ice-cold
182
acetone for 10 min and air dried at room temperature.
183
Slides were incubated for 20 min in 0.03% hydrogen peroxide (H2O2) diluted in PBS, and
184
washed three times in PBS. Following incubation with horse serum for 20 min, tissue
185
sections were incubated with specific monoclonal antibodies (Mabs) [Mouse anti-chicken
7
186
CD4 (Clone CT-4) at 0.5 µg/ml; 1:1000, mouse anti CD8 alpha (Clone CT-8) at 0. 25 µg/ml,
187
1:2000, mouse anti-chicken IgA (Clone A-1) at 0.5 µg/ml, 1:1000]. Slides were incubated
188
overnight at 4ºC. After primary antibody incubation, sections were washed in PBS and
189
incubated with the secondary antibody (anti-mouse IgG) for 30 minutes. After washing three
190
times with PBS, sections were incubated with 1% Avidin DH and 1% biotinylated
191
horseradish peroxidase (ABC reagent). Colour development was achieved by addition of 4%
192
3,3-diaminobenzidine (DAB) substrate containing 2% hydrogen peroxide and 2% buffer
193
stock solution. After a further wash in super quality water, tissue sections were counter
194
stained with hematoxylin for one minute. Finally, sections were dehydrated, mounted with
195
aquatic mounting medium and covered with a coverslip. All immunostained CD4+, CD8+
196
and IgA bearing B-cell cells were counted in five randomly selected microscopic fields
197
(400X magnification). The average number of positive cells per 400X microscopic field was
198
calculated for each cell type and sample.
199
3.4. Assessment of protection against aMPV challenge
200
Following aMPV infection, chicks were observed daily up to 13 dpc to record clinical signs.
201
Chicks in each group were examined individually; their beaks were squeezed gently behind
202
the nostrils to show the presence of nasal exudate, and clinical signs were scored as described
203
previously (Jones et al., 1992); No clinical signs = 0, clear nasal exudates = 1, turbid nasal
204
exudates = 2, frothy eyes and/or swollen intraorbital sinuses in conjunction with nasal
205
exudates = 3. Scores for each group were calculated based on the mean observed per total
206
number of chickens at each day.
207
208
3.5. Assessment of protection against IBV challenge
209
Following IBV M41 challenge, all chicks were observed daily for clinical signs attributable
210
to IBV infection. At 5dpc, the chicks were humanely killed for gross lesions and determine
8
211
level of protection of the trachea using the ciliostasis test, as previously described (Cook et
212
al., 1999). Ciliary activity was scored as follows; All cilia beating =0, 75% cilia beating =1,
213
50% cilia beating = 2, 25% cilia beating =3, none beating (100% ciliostasis) = 4. The lower
214
the percentage of ciliary beating (and hence the higher the calculated score), the higher the
215
level of protection afforded by the vaccination.
216
217
3.6. Statistical analyses
218
The data of the serological responses, cell-mediated immune responses (CMI) and aMPV
219
clinical signs were analysed statistically using analysis of variance (ANOVA), followed by
220
Tukey’s test. Differences were considered to be significant when p ≤ 0.05. All analyses were
221
conducted using the GraphPad Prism software, 6.0.1.
222
223
224
225
226
227
228
229
230
231
232
233
234
235
9
236
4. Results
237
238
4.1. Clinical signs post-vaccination
239
No clinical signs were observed in any of the vaccinated groups.
240
241
4.2. Detection of vaccine viruses by RT-PCR
242
None of the viruses was detected in pooled swabs from any group prior to the vaccination.
243
NDV was detected at 3 dpv only in the single and dual-vaccinated NDV groups, and up to 7
244
dpv in the triple vaccinated group (Table 2). aMPV was detected up to 7 dpv in birds that
245
received single vaccine and 14 dpv in those given NDV and aMPV vaccines. In contrast,
246
aMPV was detected for a much longer duration (up to 21 dpv) when administered with IBV
247
or with NDV and IBV vaccines. In all IBV-vaccinated groups, IBV was detected to the end
248
of the experiment.
249
250
4.3. Detection of NDV HI antibodies
251
HI titres in groups not vaccinated with NDV vaccine remained below the detectable levels.
252
Chicks vaccinated with NDV vaccine either alone or in combination had significantly higher
253
antibody titres than chicks not vaccinated with NDV vaccine (Fig. 1). There were no
254
significant differences in the level of HI antibody titres between groups that received the
255
NDV vaccine.
256
257
4.4. Detection of aMPV antibodies by ELISA
258
Sera collected prior to vaccination, or from aMPV unvaccinated groups, did not have
259
detectable levels of aMPV antibodies. At 21 dpv, chickens vaccinated solely with the aMPV
260
vaccine showed significantly higher levels of antibody titre than chickens vaccinated with the
261
aMPV vaccine combined with either NDV and/or IBV (Fig. 2). At 35 dpv, antibody titres in
10
262
the single aMPV vaccinated chickens remained higher than in the dual or triple vaccinated
263
chickens (Fig. 2).
264
265
4.5. Detection of IBV antibodies by ELISA
266
No IBV antibodies were detected in sera collected prior to vaccination, or from groups not
267
vaccinated with IBV vaccine. Chicks vaccinated with H120 vaccine alone or in combination
268
with other vaccines showed high level of antibody titre against IBV (Fig. 3). However, there
269
were no significant differences in the level of antibody titres between the groups that received
270
H120 vaccine.
271
272
4.6. CD4+, CD8+ and IgA-bearing B-cells in the trachea
273
The average counts of CD4+, CD8+ and IgA bearing B-cells in the trachea were evaluated by
274
immunohistochemistry. Single, dual or triple vaccinated groups showed significantly greater
275
expression of CD4+, CD8+ and IgA bearing B-cells (Table 3) in the trachea compared to the
276
unvaccinated control groups. However, there were no significant differences between
277
vaccinated groups at 21 dpv.
278
279
4.7. Effects of NDV and IBV vaccinations on protection against aMPV challenge
280
281
4.7.1. Clinical signs
282
Following virulent aMPV challenge, no clinical signs were observed in the groups vaccinated
283
with aMPV alone or co-delivered with NDV and/or IBV (Fig. 4). In contrast, chickens not
284
vaccinated against aMPV showed clinical signs, such as clear to turbid nasal discharges at 3
285
dpc and had completely recovered by 11 dpc. There were no significant differences in clinical
286
signs seen in the groups that did not receive the aMPV vaccine.
287
11
288
4.8. Effect of NDV and aMPV vaccination on protection against IBV challenge
289
290
4.8.1. Clinical signs and gross lesions
291
Following IBV challenge, no clinical signs were observed in the group vaccinated with H120
292
vaccine alone or in combination with the other vaccines. Chickens not vaccinated with H120
293
vaccine showed respiratory clinical signs at 1 dpc which included tracheal râles, coughing,
294
sneezing and head shaking. At necropsy at 5 dpc, chicks vaccinated with H120 vaccine were
295
found to be free of lesions, while unvaccinated chicks had slight congestion in the trachea and
296
pale and mild swelling of the kidneys.
297
298
4.8. 2. TOC assessment for protection against IBV challenge
299
Virulent IBV M41 caused substantial damage to the tracheal epithelium of chickens not
300
vaccinated with the H120 vaccine. Groups that were given the H120 vaccine showed 95-98%
301
protection against IBV M41. In the unchallenged groups, almost 100% of ciliary protection
302
was recorded.
303
304
305
306
307
308
309
310
311
312
12
313
5. Discussion
314
315
We evaluated the effect of co-delivering live NDV, aMPV and IBV vaccine viruses in
316
combination in SPF chicks. Previous studies have reported the simultaneous application of
317
NDV and aMPV (Ganapathy et al., 2005; Ganapathy et al., 2006), aMPV and IBV (Cook et
318
al., 2001), or NDV, aMPV and IBV (Tarpey et al., 2007) in chicks. Those studies reported
319
that simultaneous application of live NDV or IBV vaccines reduces humoral antibody
320
responses to virulent aMPV, but the chicks were protected against disease. The authors
321
strongly suggested that cell-mediated immunity may have played an important role in
322
conferring the protection against challenge viruses of NDV, aMPV or IBV but this was not
323
investigated. In the current comprehensive study, in order to generate comparative data, the
324
same strains of live NDV, aMPV and IBV vaccine viruses were given to young SPF chicks in
325
single, dual or triple combinations. In addition to humoral antibody responses, we monitored
326
the cell-mediated and local immune responses in the trachea of the birds using
327
immunohistochemistry.
328
329
Following single and simultaneous (dual and triple) application of the live vaccine viruses, to
330
one day old SPF chicks no respiratory or any other clinical signs were found. Similar
331
observations were reported when live NDV and aMPV (Ganapathy et al., 2007), aMPV and
332
IBV (Cook et al., 2001) and NDV+IBV and aMPV (Tarpey et al., 2007) were concurrently
333
administered to SPF chicks. In the previous studies (Beaudette and Hudson., 1937; Cavanagh
334
et al., 1999; Cook et al., 2001; Cook and Cavanagh, 2002; Ganapathy et al., 2006; Ganapathy
335
et al., 2007; Gelb et al., 2007; Wakamatsu et al., 2007), the distribution and persistence of the
336
live vaccine viruses in various tissues and OP swabs were monitored. In our study, we used
337
RT-PCR to trace the vaccine viruses in OP swabs and it was noted that the live IBV vaccine
338
virus was detected throughout the experimental duration. This demonstrates the persistence
13
339
of IBV, which also could be related to its ability to dominate the other live respiratory
340
vaccines. Cook et al. (2001) reported that this is likely due the rapid replication of IBV. In
341
our experience, under experimental and field conditions, we are able to detect IBV vaccine
342
viruses by RT-PCR for many weeks following vaccination.
343
344
For the NDV vaccine, it appears that the virus is cleared rapidly when given alone or
345
simultaneously with IBV or aMPV, but lasted slightly longer (up to 7 dpv) when all three
346
vaccine viruses were given together. The aMPV vaccine virus could be detected in OP swabs
347
for a much longer (up to 21 dpv) duration when administered with IBV or in combination
348
with IBV and NDV. Prolonged detection of aMPV after dual vaccination with NDV has been
349
reported before (Ganapathy et al., 2005). There appears to be a delayed detection of NDV or
350
aMPV when co-delivered with live IBV vaccine virus, potentially indicating the dominance
351
of IBV virus. Despite this, it is evident that the NDV or IBV vaccine viruses are not
352
completely eliminated, and instead allowed to replicate at a later time when co-administered
353
with IBV.
354
355
An important indication of successful vaccine-take is a serological response, as this indicates
356
the ability of the vaccine to attach, replicate and induce immune responses, including
357
humoral antibodies. In this study, IBV and aMPV humoral antibodies were measured using
358
commercial ELISA kits and HI was used for detection of NDV antibodies. For IBV and
359
NDV, the results demonstrate that simultaneous dual or triple vaccinations did not affect the
360
levels of the antibodies induced. This is particularly important for NDV, as HI antibody
361
levels are commonly used as an indicator of protection (Goddard et al., 1988; Reynolds and
362
Maraqa, 2000). For aMPV, the lower humoral antibody titres in groups that were given dual
14
363
or triple vaccinations were at the same level as previously reported (Cook et al., 2001;
364
Ganapathy et al., 2005; Tarpey et al., 2007).
365
366
There are currently no published data on the cell-mediated responses to dual or triple
367
vaccinations, even though such programmes are increasingly practiced in the poultry industry
368
worldwide. We attempted to measure the CD4+ and CD8+ responses in the trachea following
369
single, dual or triple vaccinations. In addition, the trachea was also assessed for IgA-bearing
370
B-cells as an indicator of the local immune response. Responses were evaluated at 21 dpv
371
before the flocks were separated for challenge against IBV or aMPV. Our findings showed
372
significant increases in the expression of CD4+, CD8+ or IgA bearing B-cell in the trachea
373
from vaccinated compared to unvaccinated groups. The absence of significant differences
374
between the vaccinated groups demonstrates that at the tracheal level, similar intensities of
375
cell-mediated responses was induced by the single, dual and triple vaccinations.
376
In this study the CD8α was examined thought the other T cell subset might play an important
377
role following the challenge viruses. Following NDV vaccination, it was detected vigorous
378
proliferation in the CD8α and TCRγδ population both in immune and in naïve chickens upon
379
antigen stimulation (Dalgaard eta l., 2010). A previous study investigated lymphocytic
380
responses in the chicken trachea to Mycoplasma gallisepticum infection. By staining for a
381
variety of cell-surface markers (CD4, CD8, TCRγδ, TCRαβ1 and TCRαβ2), the distribution
382
of the different T-cell populations in the tracheal mucosa of infected birds played an
383
important role against the infection (Gaunson et al., 2000). In addition, it demonstrated the
384
importance of γδ T cells, after Salmonella immunization (Berndt,et al., 2006).
385
386
As part of the study, the efficacy of live vaccines when given simultaneously (dual or triple)
387
against single application were compared. For IBV and aMPV, the protections conferred
388
against challenge viruses were evaluated. For NDV, due to local regulations, no challenge
15
389
was carried out; instead NDV HI titres following vaccination were used. Our results showed
390
that irrespective of live vaccine combinations, protection against IBV was not affected, as
391
ciliary protection of 95-98% was achieved in all IBV-vaccinated groups. Similar findings
392
have previously been shown in other in vivo studies (Cook et al., 1986; Cook et al., 2001;
393
Gelb et al., 2007). For NDV, mean HI titres of 2-5 log2 and above were considered to provide
394
clinical protection (Reynolds and Maraqa, 2000; Ganapathy et al., 2007; van Boven et al.,
395
2008; Shim et al., 2011; Jeong et al., 2013). In this study, it appears that simultaneous
396
application of live NDV vaccine with aMPV, IBV or both does not compromise the immune
397
response or protection conferred against NDV.
398
399
With increasing losses due to aMPV infection in broiler, layer and breeder chickens, live
400
vaccines against aMPV are applied, often along with live IBV, NDV or both. Previous work
401
has shown that protection against aMPV was not compromised when the live aMPV vaccine
402
was given with live IBV (Cook et al., 2001), NDV (Ganapathy et al., 2005) and NDV+IBV
403
combined vaccine (Tarpey et al., 2007). Results from this study demonstrate no loss of
404
protection when the aMPV vaccine was given either alone, or in combination.
405
protection was achieved irrespective of the humoral antibody levels. This finding supports
406
previous reports contending that humoral antibodies do not have a major role in protection
407
against aMPV challenge (Jones et al., 1992; Naylor et al., 1997). In a long-term experimental
408
study, turkey hens given a live aMPV vaccine at 12 days of age were protected against
409
challenge at 22 weeks, even with no significant levels of ELISA antibody at the time of
410
challenge (Williams et al., 1991). This demonstrates that antibody detection is an insufficient
411
parameter for estimating the degree of protection in aMPV vaccinated poultry flocks
412
(Rubbenstroth and Rautenschlein, 2009). Our study confirms previous suggestions that the
16
Such
413
protection against aMPV was due to cell-mediated and local immune responses, as the levels
414
of cell-mediated and IgA-bearing B-cells were similar in all aMPV-vaccinated groups.
415
416
417
418
6. Conclusions
419
Based on the findings presented in this study, it appears that the efficacy of the live NDV,
420
aMPV and IBV vaccine viruses were not compromised when given simultaneously in dual or
421
triple combinations to young SPF chicks. There was a suppression of humoral antibody
422
responses to aMPV, but responses to IBV and NDV were not affected. At the tracheal level,
423
no variations in the levels of cell-mediated or IgA-bearing B-cells were seen between
424
vaccinated groups.
425
426
427
428
429
430
431
432
433
434
435
436
437
17
438
References
439
440
441
Aldous, E.W., Alexander, D.J., 2001. Detection and differentiation of Newcastle disease
virus (avian paramyxovirus type 1). Avian Pathology 30, 117-128.
442
443
Alexander, D.J., 2000. Newcastle disease and other avian paramyxoviruses. Revue
Scientifique et Technique 19, 443-462.
444
445
446
Allan, W.H., Gough, R.E., 1974. A standard haemagglutination inhibition test for Newcastle
disease. (1). A comparison of macro and micro methods. Veterinary Record 95, 120123.
447
448
449
Beaudette, E.R., Hudson., C.B., 1937. Cultivation of the virus of infectious bronchitis.
Journal of the American Veterinary Medical Association 90, 51-58.
450
451
452
Berndt, A., Pieper, J., Methner, U., 2006. Circulating gamma delta T cells in response to
Salmonella enterica serovar enteritidis exposure in chickens. Infection and immunity
74, 3967-3978.
453
454
455
Cavanagh, D., Gelb, J., 2008. Infectious bronchitis. In: Y. M. Saif, A.M. Fadly, J.R. Glisson ,
L.R. McDougald, Nolan, L.K., Swayne, D.E. (Eds.), Diseases of poultry Blackwell
Publishing, Ames, 117-133.
456
457
458
Cavanagh, D., Mawditt, K., Britton, P., Naylor, C.J., 1999. Longitudinal field studies of
infectious bronchitis virus and avian pneumovirus in broilers using type-specific
polymerase chain reactions. Avian Pathology 28, 593-605.
459
460
461
Chomczynski, P., Sacchi, N., 2006. The single-step method of RNA isolation by acid
guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on.
Nature Protocol 1, 581-585.
462
463
464
Cook, J.K., Huggins, M.B., Orbell, S.J., Mawditt, K., Cavanagh, D., 2001. Infectious
bronchitis virus vaccine interferes with the replication of avian pneumovirus vaccine
in domestic fowl. Avian Pathology 30, 233-242.
465
466
467
468
Cook, J.K., Orbell, S.J., Woods, M.A., Huggins, M.B., 1999. Breadth of protection of the
respiratory tract provided by different live-attenuated infectious bronchitis vaccines
against challenge with infectious bronchitis viruses of heterologous serotypes. Avian
Pathology 28, 477 - 485.
469
470
471
Cook, J.K., Smith, H.W., Huggins, M.B., 1986. Infectious bronchitis immunity: its study in
chickens experimentally infected with mixtures of infectious bronchitis virus and
Escherichia coli. The Journal of General Virology 67 1427-1434.
472
473
Cook, J.K.A., Cavanagh, D., 2002. Detection and differentiation of avian pneumoviruses
(metapneumoviruses). Avian Pathology 31, 117-132.
474
475
476
Cook, J.K.A., Darbyshire, J.H., Peters, R.W., 1976. The use of chicken tracheal organ
cultures for the isolation and assay of avian infectious bronchitis virus. Archives of
Virology 50, 109-118.
18
477
478
479
Cook, J.K.A., Holmes, H.C., Finney, P.M., Dolby, C.A., Ellis, M.M., Huggins, M.B., 1989.
A live attenuated turkey rhinotracheitis virus vaccine. 2. The use of the attenuated
strain as an experimental vaccine. Avian Pathology 18, 523-534.
480
481
482
483
Dalgaard, T.S., Norup, L.R., Rubbenstroth, D., Wattrang, E., Juul-Madsen, H.R., 2010. Flow
cytometric assessment of antigen-specific proliferation in peripheral chicken T cells
by CFSE dilution. Veterinary immunology and immunopathology 138, 85-94.
484
485
486
Ganapathy, K., Cargill, P., Montiel, E., Jones, R.C., 2005. Interaction between live avian
pneumovirus and Newcastle disease virus vaccines in specific pathogen free chickens.
Avian Pathology 34, 297-302.
487
488
489
490
Ganapathy, K., Cox, W.J., Gough, R.E., Cargill, P., Montiel, E., Jones, R.C., 2007. Protection
in specific pathogen free chickens with live avian metapneumovirus and Newcastle
disease virus vaccines applied singly or in combination. Avian Pathology 36, 313317.
491
492
493
494
Ganapathy, K., Todd, V., Cargill, P., Montiel, E., Jones, R.C., 2006. Interaction between a
live avian pneumovirus vaccine and two different Newcastle disease virus vaccines in
broiler chickens with maternal antibodies to Newcastle disease virus. Avian
Pathology 35, 429-434.
495
496
497
498
Gaunson, J.E., Philip, C.J., Whithear, K.G., Browning, G.F., 2000. Lymphocytic infiltration
in the chicken trachea in response to Mycoplasma gallisepticum infection.
Microbiology 146, 1223-1229.
499
500
501
502
Gelb, J.J., Ladman, B.S., Licata, M.J., Shapiro, M.H., Campion, L.R., 2007. Evaluating viral
interference between infectious bronchitis virus and Newcastle disease virus vaccine
strains using quantitative reverse transcription-polymerase chain reaction. Avian
Diseases 51, 924-934.
503
504
Goddard, R.D., Nicholas, R.A.J., Luff, P.R., 1988. Serology-based potency test for
inactivated Newcastle disease vaccines. Vaccine 6, 530-532.
505
506
507
Gough, E.R., Jones, R.C., 2008. Avian Metapneumovirus. In: Y. M. Saif, A.M. Fadly, J.R.
Glisson , L.R. McDougald, Nolan, L.K., Swayne, D.E. (Eds.), Diseases of poultry.
Blackwell, Ames, Iowa 101-110.
508
509
Hanson, L.E., White, F.H., Alberts, J.O., 1956. Interference between Newcastle disease and
infectious bronchitis viruses. American Journal ofVeterinary Research 17, 294-298.
510
511
512
513
Jeong, S.H., Lee, D.H., Kim, B.Y., Choi, S.W., Lee, J.B., Park, S.Y., Choi, I.S., Song, C.S.,
2013. Immunization with a thermostable Newcastle disease virus K148/08 strain
originated from wild mallard duck confers protection against lethal viscerotropic
velogenic Newcastle disease virus infection in chickens. PloS one 8, e83161.
514
515
516
Jones, R.C., Naylor, C.J., Al-Afaleq, A., Worthington, K.J., Jones, R., 1992. Effect of
cyclophosphamide immunosuppression on the immunity of turkeys to viral
rhinotracheitis. Research in Veterinary Science 53, 38-41.
19
517
518
519
520
Naylor, C.J., Worthington, K.J., Jones, R.C., 1997. Failure of maternal antibodies to protect
young turkey poults against challenge with turkey rhinotracheitis virus. Avian
Diseases 41, 968-971.
521
522
523
Okino, C.H., Alessi, A.C., Montassier Mde, F., Rosa, A.J., Wang, X., Montassier, H.J., 2013.
Humoral and cell-mediated immune responses to different doses of attenuated vaccine
against avian infectious bronchitis virus. Viral immunology 26, 259-267.
524
525
526
Pensaert, M., Lambrechts, C., 1994. Vaccination of chickens against a Belgian
nephropathogenic strain of infectious bronchitis virus B1648 using attenuated
homologous and heterologous strains. Avian Pathology 23, 631-641.
527
528
529
Raj, G.D., Jones, R.C., 1997. Effect of T-cell suppression by cyclosporin on primary and
persistent infections of infectious bronchitis virus in chickens. Avian Pathology 26,
257-276.
530
531
532
Rautenschlein, S., Aung, Y.H., Haase, C., 2011. Local and systemic immune responses
following infection of broiler-type chickens with avian metapneumovirus subtypes A
and B. Veterinary immunology and immunopathology 140, 10-22.
533
534
535
536
Rauw, F., Gardin, Y., Palya, V., van Borm, S., Gonze, M., Lemaire, S., van den Berg, T.,
Lambrecht, B., 2009. Humoral, cell-mediated and mucosal immunity induced by
oculo-nasal vaccination of one-day-old SPF and conventional layer chicks with two
different live Newcastle disease vaccines. Vaccine 27, 3631-3642.
537
538
Reed, L.J., Muench, H., 1938. A simple method of estimating fifty per cent endpoints.
American Journal of Epidemiology 27, 493-497.
539
540
541
Reynolds, D.L., Maraqa, A.D., 2000. Protective immunity against Newcastle disease: The
role of antibodies specific to Newcastle disease virus polypeptides. Avian Diseases
44, 138-144.
542
543
544
545
Rubbenstroth, D., Rautenschlein, S., 2009. Investigations on the protective role of passively
transferred antibodies against avian metapneumovirus infection in turkeys. Avian
Pathology 38, 427-436.
546
547
548
549
Russell, P.H., Dwivedi, P.N., Davison, T.F., 1997. The effects of cyclosporin A and
cyclophosphamide on the populations of B and T cells and virus in the Harderian
gland of chickens vaccinated with the Hitchner B1 strain of Newcastle disease virus.
Veterinary immunology and immunopathology 60, 171-185.
550
551
Seo, S.H., Collisson, E.W., 1997. Specific cytotoxic T lymphocytes are involved in in vivo
clearance of infectious bronchitis virus. Journal of Virology 71, 5173-5177.
552
553
554
555
Seo, S.H., Pei, J., Briles, W.E., Dzielawa, J., Collisson, E.W., 2000. Adoptive transfer of
infectious bronchitis virus primed αβ T cells bearing CD8 antigen protects chicks
from acute infection. Virology 269, 183-189.
20
556
557
558
Shim, J.-B., So, H.-H., Won, H.-K., Mo, I.-P., 2011. Characterization of avian paramyxovirus
type 1 from migratory wild birds in chickens. Avian Pathology 40, 565-572.
559
560
561
Takada, A., Kida, H., 1996. Protective immune response of chickens against Newcastle
disease, induced by the intranasal vaccination with inactivated virus. Veterinary
Microbiology 50, 17-25.
562
563
564
Tarpey, I., Huggins, M.B., Orbell, S.J., 2007. The efficacy of an avian metapneumovirus
vaccine applied simultaneously with infectious bronchitis and Newcastle disease virus
vaccines to specific-pathogen-free chickens Avian Diseases 51, 594-596.
565
566
Thornton, D.H., Muskett, J.C., 1975. Effect of infectious bronchitis vaccination on the
performance of live Newcastle disease vaccine. Veterinary Record 96, 467-468.
567
568
569
van Boven, M., Bouma, A., Fabri, T.H., Katsma, E., Hartog, L., Koch, G., 2008. Herd
immunity to Newcastle disease virus in poultry by vaccination. Avian Pathology 37,
1-5.
570
571
572
573
Wakamatsu, N., King, D.J., Seal, B.S., Brown, C.C., 2007. Detection of Newcastle disease
virus RNA by reverse transcription-polymerase chain reaction using formalin-fixed,
paraffin-embedded tissue and comparison with immunohistochemistry and in situ
hybridization. Journal of Veterinary Diagnostic Investigation 19, 396-400.
574
575
Williams, R.A., Savage, C.E., Jones, R.C., 1991. Development of a live attenuated vaccine
against Turkey rhinotracheitis. Avian Pathology 20, 45-55.
576
577
578
579
Worthington, K.J., Currie, R.J.W., Jones, R.C., 2008. A reverse transcriptase polymerase
chain reaction survey of infectious bronchitis virus genotypes in Western Europe from
2002 to 2006. Avian Pathology 37, 247 - 257.
580
581
582
583
584
585
586
587
588
589
590
591
21
592
593
594
595
596
597
598
599
600
601
602
Fig.1. NDV HI antibody titres in chickens administered with live NDV vaccine alone or with other live
603
vaccines. Absent of letters indicate no significant difference between vaccinated groups. Vertical bars represent
604
standard error of the means. n= 8.
605
606
Fig. 2. aMPV ELISA antibodies in chickens vaccinated with aMPV or with NDV and/or IBV. Different
607
superscripts represent significant differences in the antibody response. Vertical bars represent standard error of
608
the means (n= 8).
609
610
Fig. 3. IBV ELISA antibodies in chickens vaccinated with IBV alone or with NDV and/or aMPV. Absent of
611
letters indicate no significant difference between vaccinated chickens. Vertical bars represent standard error of
612
the means (n= 8).
613
614
Fig. 4. Mean clinical scores in the vaccinated and unvaccinated chickens after aMPV challenge. No clinical
615
signs were observed in the chickens vaccinated with aMPV vaccine alone or together with NDV or IBV or both.
616
617
618
Table 1
619
Experimental design for the single and simultaneous application of dual or triple live NDV,
620
aMPV and IBV vaccines in SPF chicks
621
622
Table 2
22
623
RT-PCR detection of vaccine viruses following vaccination of chickens with NDV, aMPV
624
and IBV vaccines
625
626
Table 3
627
Immunostaining of trachea sections at 21dpv using Mab for presence of CD4+ and CD8+ T-
628
cells and for IgA-bearing B cells
629
630
631
632
633
634
635
636
637
23