Modulation by essential oil of vaccine response and production improvement in chicken challenged with velogenic Newcastle disease virus

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O R I G I N A L A R T I C L E

Modulation by essential oil of vaccine response and production improvement in chicken challenged with velogenic Newcastle disease virus

E.K. Barbour^1,2, H. Shaib¹, E. Azhar³, T. Kumosani⁴, A. Iyer⁴, S. Harakeh³, G. Damanhouri⁵, A. Chaudary⁵and R.R. Bragg⁶

1 Department of Animal and Veterinary Sciences, Faculty of Agricultural and Food Sciences, American University of Beirut (AUB), Beirut, Lebanon 2 Adjunct to Biochemistry Department, King Abdulaziz University, Jeddah, Saudi Arabia

3 Special Infectious Agents Unit–Biosafety Level 3, King Fahad Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia 4 Biochemistry Department, Faculty of Science and Experimental Biochemistry Unit, King Fahd Medical Research Center, King Abdulaziz

University, Jeddah, Saudi Arabia

5 King Fahad Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia

6 Department of Microbial, Biochemical and Food Biotechnology, University of The Free State, Bloemfontein, South Africa

Keywords

chicken, essential oil, immune modulation, production, velogenic Newcastle disease virus.

Correspondence

Elie K. Barbour, Department of Animal and Veterinary Sciences, Faculty of Agricultural and food Sciences, American University of Beirut, PO Box 11-0236, Beirut, Lebanon.

E-mail: [email protected]

2013/1397: received 12 July 2013, revised 31 July 2013 and accepted 17 August 2013 doi:10.1111/jam.12334

Abstract

Aim: To evaluate the impact of Eucalyptus and peppermint essential oils on immune modulation and production of broiler chicken challenged with a molecularly characterized velogenic NewCastle disease virus (vNDV).

Methods and Results: The experimental design included five treatments with three replicate pens/treatment comprised of 12-day-old broilers chicks/replicate.

The five treatments included a positive challenge control (non-NDV vaccinated/

nonessential oil treated/challenged) (NNEOC), a negative challenge control (NDV vaccinated/essential oil treated/unchallenged) (VEOU), a non-NDV vaccinated/

essential oil treated/challenged (NEOC), a NDV vaccinated/nonessential oil treated/challenged (VNEOC) and a NDV vaccinated/essential oil treated/

challenged (VEOC). The lowest mean survival rate (00%) and lowest production performance were obtained by the positive challenge control, while the best mean survival (933%) and average body weight (2649 g) were obtained by the negative challenge controls (P<005). Among the three others challenged treatments, the best mean survival (792%), highest mean body weight at 42 days of age (2445 g), the lowest feed conversion ratio (160) and the highest serum conversion immunopotentiation at 35 days of age determined by ELISA and hemagglutination titres were obtained by the VEOC birds compared with respective means obtained by birds of the NEOC and VNEOC treatments (P<005).

Conclusions: The results supported the possibility of using the essential oils of Eucalyptus and Peppermint in broilers to immunopotentiate the response to vaccination against velogenic NDV, helping in significant improvement of survival and production.

Significance and Impact of the Study: This study provides information about the potential use of essential oils of eucalyptus and peppermint that can be exploited as commercial immunopotentiators for the protection of NDV- vaccinated broiler chickens against economic velogenic NDV.

Introduction

The velogenic NewCastle disease (ND) is included within the ‘List A’ ailment by the World Animal Health Organi-

zation, due to its significant economic impact on the poultry industry. The expected per cent mortality in broilers exposed naturally to the different strains of velogenic NDV ranges between 30 and 90 (CFSPH 2008).

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The velogenic ND is recently circulating vigorously in poultry as reported from different countries, including the outbreaks in the respective years of 2008 and 2010 in Nigeria and Pakistan (Salomon et al.2011; Mouniret al.

2012). More outbreaks were also reported in the year 2012 in Israel (OIE 2012a), Romania (OIE 2012b), Italy (OIE 2012c) and more recently in the year 2011 and 2012 in Lebanon (Barbour and Shaib 2011), leading into mortalities in broilers ranging from 60 to 80%.

The poultry industry in the developed countries has a common approach in controlling the velogenic NDV, relying on rapid diagnosis followed by immediate eradi- cation (USA, Europe, Canada) (Alexander et al. 2004).

However, most of the developing countries, and due to the absence of governmental compensation, are still experimenting on interceptions against velogenic NDV by different vaccines, including classical vaccines (killed and live) (Foster et al. 1998; Tu et al. 1998; FAO 2002) and recently commercialized vectored DNA vaccines (Huang et al. 2003; Loke et al. 2005). Unfortunately, neither the classical nor the vectored DNA vaccines are inducing an acceptable ≥80% protection against this disease (Sa’idu and Abdu 2008; Chonget al. 2010; Hossein et al. 2010).

This situation in the developing countries suffering from velogenic NDV requires an intensive search for immuno- modulators that could result in an acceptable protection against this ailment by the present vaccines. A blend of essential oil has been experimented on regarding its protection against Mycoplasma gallisepticum, infectious bronchitis virus and avian influenza virus (Barbouret al.

2006, 2011a,b), showing certain acceptable degree of protection and improvement in production. Other workers reported an improvement in broiler performance when essential oil is administered as a dietary supplement (Suk et al. 2003; Hernandez et al. 2004; Cross et al.

2007). In the year 2010, Timbermont et al. showed that broiler diets supplemented with essential oil of eucalyptus may contribute to the prevention of necrotic enteritis induced by interaction between Clostridium perfringens and Paracox-5^TM anticoccidial vaccine. The eucalyptus oil is proved to stimulate the immune system response by enhancing the phagocytic activity of monocytes (Serafino et al. 2008). In addition, the cineole-active ingredient in the eucalyptus essential oil controls the secretions of mucus in the epithelial layer of the respiratory system air passages (Juergenset al.2003, 2004). The virucidal activity of eucalyptus essential oil in vivoand in vitro against poultry viruses was documented earlier (Barbour et al.

2010). The peppermint essential oils had other documen- tations related to its antiviral effect (Siddiqui et al.1996;

Sivropoulou et al. 1997; Schuhmacher et al. 2003; Ocak et al. 2008). However, and to our knowledge, no work have explored the impact of a blend of essential oils of

eucalyptus and peppermint on production and immunity to velogenic NDV.

The aim of this research is to evaluate the impact of essential oil of eucalyptus and peppermint on production, survival and immunomodulation in NDV-vaccinated and unvaccinated broilers exposed to a hot challenge with a molecularly characterized velogenic NewCastle disease virus documented by our laboratory in gene bank under Accession Bank IT1593379seq1KC425723.

Materials and methods

NDV strain for challenge

The source of the velogenic NDV isolate used in this study for challenge is from trachea of broilers that suf- fered a severe outbreak leading to 60% mortality. This virus had a fusion gene amplicon size of 254 bp (Fig. 1), amplified by a previously described procedure (Viljoen et al.2005).

This fusion gene amplicon was sequenced at University of Saint Joseph in Beirut, Lebanon, confirming the presence of a cleavage site right after the amino acid positions 116R (Fig. 2), which is in agreement with previous docu- mentations in the literature (Nagai et al.1976; Clickman et al.1988). This isolate had a dibasic amino acids (113R and 115K) surrounding the glutamine (Q) that was posi- tioned at 114 of the fusion gene. The presence of the dibasic amino acids arginine (113R) and lysine (115K) in

1 2 3 4 5

254 bp

Figure 1Amplicon of the fusion gene of vNDV isolated from poultry in Bekka Valley, Lebanon. Lane 1: 100 base pairs molecular ladder.

Lane 2: Negative control: PCR mixture lacking template RNA. Lane 3:

Positive vNDV control (Fusion gene). Lane 4: amplicon resulting from amplification of fusion gene of sample 1 (pool of 3 broilers’ tracheas).

Lane 5: amplicon resulting from amplification of fusion gene of sample 2 (pool of 3 layers’ tracheas).

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the fusion protein sequence allowed for systemic spread of this velogenic NDV. This virus was propagated in 10- day-old chicken embryos and was used in challenging birds, allocated to specific treatments in this study, with a dose of 10⁴EID50ml ¹in the right pectoral muscle of the bird. Two birds were randomly chosen for a challenge in each replicate of 12 birds, and the rest were considered as contact challenge. The gene bank accession number of the strain used in the challenge is Bank It1593379 seq1 KC425723.

Essential oil blend in drinking water and spray

The essential oil blend was provided by Mr. Werner Krull, EWABO Co., Germany. The percentages of the major constituents of the blend were as follows: Cineol (422%), limonene (35%), ^L-menthol (487%), phelland- rene (05%), a-pinene (10%), b-pinene (03%), and terpineol (03%). The essential oil blend level used in drinking water was 002% (v/v), given ad libitum at assigned ages, defined under the experimental design sub- heading, shown below. However, the sprayed essential oil was administered in fine particle sprayer, at 2% (v/v) dilution, and in a volume of 2 ml per bird. The sprayed micelles were of≤3lm in diameter.

Broiler birds and NDV vaccination

One hundred and eighty days old broiler chicks (Cobb 500) were used in this study. The vaccination for NewCas- tle disease virus was accomplished intraoccularly at 1 and 14 day of age. The vaccine was Izovac clone, produced by IZO S.P.A., Brescia, Italy. The feed was formulated accord- ing to NRC, 1992 recommendation and was deliveredad libitumto all birds in the eight treatments.

Experimental design

The 180-day-old chicks were divided into five treatments (36 birds/treatment), and each treatment was composed of three replicates (12 birds per replicate). Each replicate was set in a separate pen. Strict isolation measures were put in place among the five treatments. The assigned days for NDV vaccination and essential oil treatment in drinking water and by spray, and for the challenge with the

velogenic NDV are summarized in Table 1. Blood was collected at 1, 14, 28, and 35 days of age for determining the conversion titres by age, which is specific to NDV virus by ELISA (Idexx, ME, USA), and to the haemagglutinin protein of the velogenic NDV virus by HI test.

Production and immune modulators parameters

The measured parameters used in evaluation of the essential oil treatments in water and by spray included: the determination of the mean live body weight in each of the three replicates within each of the five treatments; the observation of the mean per cent survivors of contact birds in the three replicates of each treatment; the calcu- lation of the mean feed conversion in each treatment; the determination of the ELISA- and HI-conversion titres through the 14, 28 and 35 days of age.

Statistical analyses

Statistical analysis was used to compare the means among the five treatments, using^ANOVA, followed by Tukey’s test, while the chi-square was used to compare the mean per cent survivors in the five treatments. Significant differences were reported at thePlevels of<005.

Results

Survival rate of broiler chickens

The mean per cent survivors of contact broilers at market age of 42 days (2 weeks post the velogenic NDV challenge) is shown in Table 2. The highest mean survivors of 933% was obtained by the negative challenge controls (VEOU), followed by a 792% survival in birds of the VEOC treatment (P<005).

Mean live body weight

The mean live body weight of contact birds at market age of 42 days (2 weeks post the velgenic NDV challenge) is shown in Table 3. The absence of survivors in contact birds of two treatments prevented us from recording their birds’ weight at the market age of 42 days. The mean live body weight of the birds in the three other DGRPLAAAGIVVTGDKAVNVYTSSQTGSIIVKLLPNMPKDKEACAKAPLEAYNRTLTTLLTPLG

DSIRKIQGSVATSGGRR QKR

Figure 2 Amino acid sequence of the fusion gene of velogenic NDV isolated from Broilers in Lebanon (Position 33–116).

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treatments differed significantly from each other, and they were in the following increasing order: VNEOC (1975 g), VEOC (2445 g) and negative challenged controls (VEOU) (2649 g;P<005).

Feed conversion

The ratio of consumed feed over the live body weight in the birds of the five treatments, up to the market age of 42 days, is presented in Table 4. Survivors up to market age were present in three out of the five treatments. The mean feed conversions of the survivors in decreasing order of efficiency were the following: VEOU (157), VEOC (160) and VNEOC (221). The improvement in the obtained mean feed conversions of 157 and 160 was significantly different than the 221 ratio (P<005).

ELISA-conversion titres

The mean ELISA-conversion titres in the surviving birds through the ages between 14–35 days are shown in Table 5.

The means of all the five treatments were attainable at 14 (NDV booster time) and 28 days of age (challenge day), while two means at 35 days of age (7 days postchallenge) were missing in two treatments, due to the absence of survivors. Surviving birds in only one treatment were able to have a significant rise in ELISA-conversion titres at 35 days of age (7 days postchallenge) (titre of 17016) compared with its titre at 14 days (4683) (P<005), namely the NDV vaccinated/essential oil treated/challenged.

However, the other two treatments that had survivors at 35 days of age had a decline of their mean titres at 35 days of age compared with that of the 14 days age, namely the VNEOC (nonsignificant decline from a titre of 5143 to 2103, P>005), and the negative challenged control birds (VEOU), showing a significant decline from a titre of 4729 to 850, (P< 005).

Hemagglutination-inhibition-conversion titres

The mean HI-conversion titres of antibodies, specific to the haemagglutinin protein of the velogenic NDV used in the test, are shown in Table 6. The pattern of the seroconversions deduced from the ELISA agreed well with that observed in the HI-seroconversion titres. Actually, the same treatment (VEOC) that had a significant rise in ELISA-seroconversion had also a significant rise in HI- seroconversion at 35 days of age (titre of 537) compared with that of the 14 days of age (titre of 00) (P<005).

In addition, the same two treatments that showed a decline in ELISA-conversion titres at 35 days of age compared with that at 14 days of age did also show the same pattern with the HI-conversion titres; more specifically,

Table1Experimentaldesignwithfivetreatments,threereplicates/treatmentand12birds/replicate TreatmentNDVvaccination*byeye dropatages(days)GrouplabelNDVchallenge†(age)

Agesattheessentialoil-treatmentbydifferentroutes(days) DrinkingwaterSpray 1NonvaccinatedNEOC28456716171825–393712131516192327313539 21,14VNEOC28NoneNone 31,14VEOC28456716171825–393712131516192327313539 4NonvaccinatedNNEOC28NoneNone 51,14VEOUUn-challenged456716171825–393712131516192327313539 *Izovacclone,IZOS.P.A.,ViaA.Bianchi,9,25124Brescia,Italy. †NDVchallengeintwobirds/replicateof12birds,eachbirdisinjectedintramuscularlywith104EID50ofvelogenicNDV;contactbirdsperreplicatearebetween9–10.

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the VNEOC birds had a nonsignificant decline from 166 to 20 (P> 005), while the negative challenge controls (VEOU) had a significant mean HI-titre decline from 112 to 03 (P< 005).

Discussion

The data presented in Table 2 showed the best survivals in the negative challenge controls (NDV vaccinated/

essential oil treated/unchallenged), which is clearly due to the nonexposure to the v-NDV; in addition, this high mean survival rate of 933% is indicative of the safety of NDV vaccination by the live strain and of the administered essential oil blend. It is documented in literature that the essential oil of eucalyptus and peppermint are devoid of any toxicity effects (Holley and Patel 2005;

Ayala-Zavala et al. 2009). In our study, we did not note any taints on the pectoral and thigh muscles of the birds

Table 2 Mean percent survivors of contact broilers up to market age of 42 days (2 weeks post the velogenic NDV challenge)

Treatment

Essential oil

treatment Vacc.* Challenged†

% survivors of contact birds in each replicate

Mean % survivors

1 2 3

1 Yes No Yes 00 00 00 00^A

2 No Yes Yes 200 111 200 170^A

3 Yes Yes Yes 800 777 800 792^B

4 No No Yes 00 00 00 00^A

5 Yes Yes No 1000 900 900 933^C

*Vaccination: IZOvac clone, Italy. Intraocular at d. 1 and 14.

†Challenge: two birds/replicate (contact/replicate=9–10 birds). The challenge is delivered intramuscularly in the pectoral muscle, containing 10⁴EID50 of vNDV/ml/bird.

A–CMean% survivors in the last column followed by different capital letters are significantly different (P<005).

Table 3 Mean live body weight of contact broilers at market age of 42 days (2 weeks post the velogenic NDV challenge)

Treatment

Essential oil

Average live body weight (g) in each of

the three replicates Mean live body

weight of the three replicates

1 2 3

1 Yes No Yes All dead All dead All dead –

2 No Yes Yes 1973 1977 1975 1975^A

3 Yes Yes Yes 2435 2448 2452 2445^B

4 No No Yes All dead All dead All dead –

5 Yes Yes No 2650 2645 2653 2649^C

†Challenge: two birds/replicate (contact/replicate=9–10 birds). The challenge is delivered intramuscularly in the pectoral muscle, containing 10⁴EID50of vNDV/ml/bird.

A–CMean live body weight of the three replicates in the last column followed by different capital letters are significantly different (P<005).

Table 4 Mean feed conversion of broilers deduced from the three replicates in each treatment

Treatment

Essential oil

Feed conversion in each of the 3 replicates

Mean Feed conversion

1 2 3

1 Yes No Yes All dead All dead All dead –

2 No Yes Yes 218 221 224 221^A

3 Yes Yes Yes 158 162 160 160^B

4 No No Yes All dead All dead All dead –

5 Yes Yes No 155 157 160 157^B

†Challenge: two birds/replicate (contact/replicate=9–10 birds). The challenge is delivered intramuscularly in the pectoral muscle, containing 10⁴EID50of vNDV/ml/bird.

A–BMean feed conversion in the last column followed by different capital letters are significantly different (P<005).

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administered the essential oils. Birds in the other treatment of VEOC were able to maintain around an 80%

survival by the 42 days of age compared to a significantly lower survival of 170% in birds belonging to VNEOC treatment (P<005). This result indicates the positive effect of the inclusion of essential oil treatment with the NDV vaccination in highering the survival of birds against a virulent challenge by the v-NDV; actually, the NDV vaccination alone did result only in a 170% survival rate against the v-NDV used in this experimental design. Published studies from India and China have indicated that natural plants are beneficial to human health and could have an immunomodulatory effect (Chao and Lin 2010; Shukla et al. 2012; Gopal et al.

2013). This is in agreement with previous documented literature on the inability of classical vaccines or vectored vaccines to provide protection against the v-NDV (Sa’idu and Abdu 2008; Chonget al. 2010; Hossein et al. 2010).

The deprivation of the birds from the NDV vaccination while including treatment with essential oil alone did not provide protection against the v-NDV challenge in the

NEOC treatment (00% survivals), a similar result to the survival of birds in the NNEOC treatment. This data indicate that the treatment with essential oil in the absence of NDV vaccination will fail in protection against v-NDV challenge. Most likely, the essential oil works as an immunopotentiator of NDV vaccination, helping in the improvement of protection.

The significant different mean live body weight in the contact birds of the three treatments that had the survivors at the market age of 42 days is shown in Table 3.

The mean of weight in decreasing order of performance was in the unchallenged birds (VEOU) followed by birds of the VEOC treatment and birds of the VNEOC treatment.

This weight performance data of Table 3 correlates positively with the survival rate data in Table 2, indicat- ing that the best survival and weight performance will be attained by preventing the birds from challenge and that the synergism between the NDV vaccination and the essential oil leads to better performance in the production of broiler chicken. Previous workers were able to get an

Table 5 Mean ELISA-conversion titres of the differently treated birds by age

Treatment no.

Essential oil

Mean ELISA-conversion titres‡at different ages in days

14 28 35

1 Yes No Yes 4533^A 330^B –

2 No Yes Yes 5143^A 1259^B 2103^A,B

3 Yes Yes Yes 4683^A 4753^A 17016^B

4 No No Yes 3606^A 1218^B –

5 Yes Yes No 4729^A 2274^A,B 850^B

†Challenge: two birds/replicate (contacts/replicate=9–10 birds). The challenge is delivered intramuscularly in the pectoral muscle, containing 10⁴EID50of velogenic NDV/ml/bird.

‡Mean maternal antibody titre at 1 day is 7662.

A,BMean conversion titres in a row followed by different capital letters are significantly different (P<005).

Table 6 Mean Haemagglutination-Inhibition (HI) seroconversion titres of the differently treated birds by age

Treatment no.

Essential oil

Mean HI-conversion titre‡at different ages in days

14 28 35

1 Yes No Yes 520^A 08^B –

2 No Yes Yes 166Â 30Â 20Â

3 Yes Yes Yes 00^A 30^A 537^B

4 No No Yes 128^A 20^B –

5 Yes Yes No 112^A 24^B 03^B

†Challenge: two birds/replicate (contact/replicate=9–10 birds). The challenge is delivered intramuscularly in the pectoral muscle, containing 10⁴EID50of vNDV ml ¹bird ¹.

‡Mean maternal haemagglutination-inhibition (HI) antibody titre at 1 day is equivalent to 1:448.

A,BMean conversion titres in a row followed by different capital letters are significantly different (P<005).

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improvement in broiler performance by essential oils used as a dietary supplement (Suket al.2003; Hernandez et al.2004; Crosset al.2007).

Other workers proved the presence of antiviral effects in the active ingredients of peppermint and eucalyptus essential oils (Siddiqui et al. 1996; Sivropoulou et al.

1997; Schuhmacher et al.2003; Ocaket al.2008; Barbour et al.2010). The potential antiviral effects in the essential oils used in this study could have alleviated the pathoge- nicity effect of the v-NDV challenge, helping the NDV vaccine to raise its impact on protection and production performance.

The insignificant statistical difference (P>005) between the feed conversion of the birds in the VEOU treatment and those of the VEOC treatments is essential for the economy of rearing broilers, as the feed is the major part in the cost of chicken production. The challenge by v-NDV did not affect negatively the conversion of feed to live weight in the birds that were NDV vaccinated and essential oil treated, while the same challenge in NDV vaccinated birds that were deprived of essential oil had a lower efficiency of feed conversion. These data again are in agreement with the survival and weight performance data, confirming the positive synergism between the NDV vaccination and the essential oil treatment. The improvement in feed conversions and other production performance by essential oil in challenged broilers is in agreement with previous documented literature (Suk et al. 2003; Hernandez et al.2004; Cross et al.

2007; Timbermontet al.2010; Barbour et al.2011a,b).

The success in obtaining a significant rise in ELISA- conversion titre of birds in VEOC-treated birds compared with its decline in birds of the VNEOC treatment might be responsible for the significant differences in the two treatments in relation to survival and production performance. Previous researchers showed the effect of eucalyptus essential oil in the stimulation of the immune response, by enhancing the phagocytic activity of monocytes (Serafino et al. 2008), and the induction of local immunity in the lower respiratory system by the sprayed micelles of <3lm (Cserep 2008). Moreover, the virucidal effects were proven to be present in both, the essential oil of eucalyptus and peppermint (Ocak et al. 2008; Barbour et al. 2010). The decline in ELISA seroconversion of birds in the VEOU treatment was expected, due to the absence of challenge by v-NDV, resulting in a decay of the titre produced by NDV-vaccination by time. It is worth noting that the ELISA-conversion titres presented in Table 5 reflect the antibody response against protective and nonprotective antigens of the NDV coated on the microtitre plates of the commercial kit (Folitse et al. 1997; Chen et al. 2003), while the Haemagglutination-Inhibition (HI) titres (Table 6)

are specific to the haemagglutinin protein of the velogenic NDV virus that is used in our protocol (Brown et al. 1990; Moro de Souza et al. 2000). The pattern of rise and decline seen by the ELISA-conversion titres was the same observed by the HI-conversion titres. The immunopotentiation in the birds of the VEOC treatment is confirmed by both the commercial ELISA and the in- house protocol of the HI test, using the antigen of the same velogenic-NDV strain that was used in the challenge. This agreement between the ELISA and the HI test specific for NDV antigens is in agreement with previous works (Marquardt et al. 1985; Kumar et al. 2011) correlating seroconversion titres with survivals and production performance.

In conclusion, the results supported the possibility of using the essential oils of eucalyptus and peppermint in broiler chickens to immunopotentiate the response to vaccination against velogenic NDV, which indirectly improve their survival and production performance. Further studies are needed to evaluate the efficacy of essential oils of eucalyptus and peppermint in the treatment of other economic diseases of poultry that threatens the livelihood of poultry farmers, poultry production, safety and security around the globe. Future work will focus at evaluating the effects of other essential oils against NewCastle disease (NDV) and other viruses of economic significance.

Conflict of interest

The authors have no conflict of interest to declare.

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