Research Article

Immunological Evaluation of Antiviral Activity of Methanolic Extract of Piper guineense against Newcastle Disease in Experimentally Infected Broiler Chickens  

Osho  I.B. , Adebayo I.A. , Ajayi O.I.
Department of Animal Production and Health, School of Agriculture and Agricultural Technology, Federal University of Technology, Akure, Nigeria
Author    Correspondence author
International Journal of Molecular Veterinary Research, 2016, Vol. 6, No. 2   doi: 10.5376/ijmvr.2016.06.0002
Received: 10 Oct., 2015    Accepted: 27 Mar., 2016    Published: 20 Jun., 2016
© 2016 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Osho I.B., Adebayo I.A., and Ajayi O.I., 2016, A Review of Immunological Evaluation of Antiviral Activity of Methanolic Extract of Piper guineense against Newcastle Disease in Exoerimentally Infected Broiler Chickens, International Journal of Molecular Veterinary Research, Vol.6, No.02 1-10

Abstract

Forty-eight day old broiler chickens kept in eight equal groups were given Piper guineense extract at dose levels of 50 and 100 mg /Kg of body weight administered in their drinking water for eight weeks. The birds were partitioned into five groups whereby the first group, the control group contained 8 birds that were vaccinated against Newcastle disease but were not given Piper guineense extract. The 2nd group contained birds that were vaccinated and given Piper guineense extract at the dosage level of 100 mg/Kg, while the 3rd group were also vaccinated and given 50 mg/Kg  Piper guineense extract. Birds in the 4th group were not vaccinated but were given plant extract at the dosage level of 100 mg/Kg. The 5th group were given Piper guineense extract at the dosage level of 50 mg/Kg body weight but weren’t vaccinated. The birds were observed daily for general performance. Both the control group and the treatment groups remained healthy. The feed intake of experimental birds were not significantly different from the control. This shows that the Piper guineense extract does not affect the feed consumption of the experimental birds when compared to the control group. On the fifth week, the birds were challenged with velogenic strain of Newcastle disease virus. After two weeks of the challenge, the birds that survived were bled to determine the level of antibody response by the chickens against Newcastle disease using ?-haemagglutination inhibition test (an immunological reaction). Haematological parameters of the experimental chickens were also determined in order to know the health status of the birds. The antibody titre level of the experimental chickens treated with 100 mg/Kg of Piper guineense extract (log 26) had the highest mean value titre (log 26). Therefore, 100 mg/Kg of Piper guineense fruits is recommended to prevent Newcastle disease outbreak. The haematological parameters of experimental birds were not significantly different from the control groups. Therefore, the plant extract at both 50 mg/Kg and 100 mg/Kg body weight were not toxic to the experimental broiler chickens.

Keywords
Piper guineense; Immunology; Newcastle disease; Broiler Chickens

1 Introduction
Newcastle disease is a highly contagious disease of birds affecting many domestic and wild avian species. The high susceptibility often causes mortality in poultry populations. Transmission occurs by exposure to faecal and other excretions from infected birds, or through contact with contaminated feed, water, equipment and plumage.
 
It is a highly infectious respiratory and nervous system disease of birds. Although found in many kinds of birds, Newcastle disease is primarily a disease of chickens. The disease is caused by a virus and is found all over the world. It is caused by virulent strains of avian paramyxovirus. Newcastle disease virus (NDV) which is the causative organism for Newcastle disease has been shown to be able to infect over 200 species of birds, but the severity of disease produced varies with both host and strain of the virus (Miller et al., 2010). Since its recognition in 1926 (OIE Terrestrial manual, 2012), Newcastle disease is regarded as being endemic in many countries. Vaccination is practised in all but a few of the countries that produce poultry on a commercial scale. Natural remedies in poultry production are primarily based on disease prevention through implementation of a biosecurity plan. While implementation of a cost-effective biosecurity plan is very important, it is also critical to have a plan in place to react to any health crisis that may occur. The wide spread of viral infections in domestic animals in Africa and the limited number of available drugs which are effective against them led to the investigations on antiviral potentials of plants easily grown in Africa which has immunomodulatory effect on birds affected with the virus when given at varying level (Anani et al., 2000; Gbeassor et al., 1996; Desouza et al., 1993). Rural poultry farmers are aware of the need to keep birds in good health and when they are sick to source for prescription and procure medicaments for treating rural poultry diseases. They often do so through various means and the use of traditional (indigenous) method of medicare seems to be the main method of treatment. The use of traditional medicine may be due to its low cost, availability and ease of application compared to modern veterinary medicine. The high cost of veterinary drugs in most pharmaceutical shops and local markets will have contributed to low patronage by farmers in sourcing drugs for treatment of sick chickens. Studies conducted in laboratories around the world revealed that traditional medicinal plants can provide a rich source of antiviral activities (Yip et al., 1991; Vietinck and Vanden – Berghe, 1991; Desouza et al., 1993, 1995; Gbeassor et al., 1996).
 
Piper guineense also known as black African pepper is a climbing forest biennial plant with gnarled branchelets spiralling on shrubs to about 10m. The leaves are elliptic in shape and highly aromatic when crushed. It is used as a flavouring and spice in food but it also has several medicinal uses (Iwu, 1993). One of the several uses of the plant is to treat Newcastle disease. Application of ethnoveterinary medicine in the control of poultry diseases is being embraced in many parts of the world for more profitable production. This study investigated the antiviral property of the Piper guineense extract from the African black pepper tree (Piper guineense) against Newcastle disease virus.
 
2 Materials and Method
2.1 Experimental site
The experiment was carried out at the poultry experimental unit of the teaching and research farm of the Federal University of Technology, Akure, Ondo State, Nigeria. The birds were acclimatized for 2 weeks and the experiment was conducted for 7 weeks. Also the laboratory analysis was carried out at the Department of Animal Production and Health in the same institution.
 
2.2 Experimental birds 
A total number of 250 day-old broiler chicks of mixed sexes were purchased from a reputable hatchery in Ibadan, Oyo state out of which 180 were used for the experiment. Brooding of the birds was carried out at the farm using deep litter system.
 
2.3 Preparation of plant extract 
Piper guineense fruits also known as African black pepper were purchased from Odopetu Market, Akure, Ondo State. The seeds were air-dried for one week and were ground into powdery form. The powdered form of the seeds were then soaked with methanol in the ratio 1:2 for 48 hours at room temperature to extract the active ingredients of the seed into the methanol. The materials used for the extraction includes: Pressure pump, buckner funnel, methanol, filter paper, sterile containers where the samples were put, blender, weighing balance, rotary evaporator, and sieve. This was carried out in such a way that the methanol covered the powdered plant completely.  After 48 hours, the stock solution was concentrated under pressure by using vacuum pump connected to a conical flask containing a buckner funnel on top. Inside the buckner funnel is the filter paper which separated the filtrate from the residue. The stock solution was poured inside the funnel and pressed with a spoon. The vacuum pump created an air tight condition inside the hose and the buckner funnel so that the filtrate can come out from the stock solution by pressure. The filtrate was poured inside a 2 litres keg while the plant residues were poured away. The filtrate was carried to the Biology laboratory in Obanla futa to separate the crude extract from the methanol using the rotary evaporator.
 
The methanol extracted was reused for the extraction of the same plant. The crude extract was put inside an oven at 40°C to reduce the moisture content for the solution to be in a pasty form. The crude extract of Piper guineense was stored inside a fridge until needed for use.
 
2.4 Experimental layout
Two hundred days old broiler chicks were purchased from a Commercial Hatchery at Ibadan, Oyo state. The breed of the broiler chicken purchased was Marshall and the birds were raised in the brooding pen at the back of Animal Production and Health Department, FUTA for five weeks from day old before the birds were separated into two. On the 13th of July, 2012, some of the birds were moved from each treatment from the brooding pen to the Teaching and Research Farm. Two birds were left in each treatment in the brooding pen and were challenged with velogenic strain of Newcastle disease virus obtained from National Veterinary Research Institute (NVRI), Vom near Jos, Plateau State. After being challenged, the birds were observed for various signs of Newcastle disease.
 
From day old the experimental birds were divided into 8 units with each unit (treatment) containing eight birds. This treatment was further divided into four groups and each group vary in the level of administration (dose) of Piper guineense extract and whether they are vaccinated or not. The groups vaccinated were given Newcastle disease vaccine made in National Veterinary Research institute (NVRI), Vom near Plateau state on the 10th day. The route of administration was ocular. The 2nd Vaccination was done on the 3rd week giving Newcastle disease vaccine (La Sota) to the broiler chickens.
 
Group one (T2R1 Vac): birds in these group were vaccinated and given Piper guineense extract according to the standard administration formula obtained from literature from past research which is 0.1 mg of plant extract to 1000 g of body weight into their drinking water.
 
Group two (T2R2 Vac): birds in these group were vaccinated and given Piper guineense extract according to the formula; 0.05 mg of plant extract to 1000 mg body weight into their drinking water.
 
Group three (T2R1 Unvac): birds in these group were not vaccinated but were given Piper guineense extract according to the formula; 0.1 g of plant extract to 1000 mg. The extract was reconstituted into their drinking water in small quantity in which the birds can consume quickly and later given pure drinking water.
 
Group four: (T2R2 Unvac): In this group, birds were not vaccinated but were given plant extract at the rate of 0.05 mg plant extract to 1 000 g body weight into their drinking water.
 
The plant extract (Piper guineense) was administered into their drinking water daily and placed under daily observation. 
 
The challenged birds left in the brooding pen behind Animal Production and Health Laboratories showed various signs of Newcastle disease such as: torticollis, paralyzed wings and legs, respiratory signs such as sneezing and coughing, moving in a cyclic motion and sudden death in prone or surpine position.
 
2.5 Data analysis
Data collected were subjected to 2x3 factorial experiment in completely randomized design using the SPSS package and the means were separated using Duncan multiple range test of the same software at 5% level of significance.
 
3 Results 
3.1 General observations
Starting from the first day of administration of the plant extracts the birds were observed well for their general performance. The experimental birds were healthy from day old till the fifth week. At the fifth week, they were challenged with Newcastle disease virus. Three of the birds did not show any sign of the infection with Newcastle disease virus while the remaining five did. Various signs of the viral infection shown include; torticollis, respiratory signs such as sneezing and coughing, wings and leg paralysis, moving in a cyclic motion and death in a prone or suprine position. Postmortem was carried out on birds that died. The experimental birds that survived were bled and the blood samples were taken to the laboratory for haematology and immunology.
 
3.2 Post mortem 
Various lesions were observed on different organs of the dead birds from each treatment:
T2R1 Vac: The two birds in this group died and the observations from different organs during post mortem are;
1st bird-Duodenum: catarrhal inflammation;
Proventriculus: haemorrhagic inflammation;
The liver was bigger than normal while the gall bladder was engorged;
Spleen: normal.
2nd bird- Proventiculus: flabby 
Liver-Streaking
Duodenum: hyperaemia
T2R2 Vac :
Liver: streaked and friable
Proventriculus: catarrhal inflammation
Spleen: normal
Duodenum: hyperaemia.
8 birds were put in this treatment with 2 birds per group. 5 out 8 birds survived while 3 died. The percentage of mortality was 37.5%.
 
3.3 Haematological indices of unchallenged broiler chickens
The results of the haematological values of the broilers used for the experiment on effects of Piper guineense are shown in Tables 1, Table 2 and Table 3 for the unchallenged birds. Table 1 indicates that there was no change in the mean values of ESR (2.00 mm/hr) of the vaccinated and unvaccinated birds. The highest mean value of PCV (36%), RBC ( 3.42x106 /mm3), Hb (12 g/ 100ml), and eosinophils (2%) were observed in both vaccinated and unvaccinated birds. The highest mean value of monocytes was observed 100 mg/Kg of vaccinated birds. Highest mean value of lymphocytes and basophils was observed in 50 mg /Kg.

 

Table 1 Haematological indices of unchallenged broiler chickens

 

As shown in Table 2, there were no significant changes (p<0.05) observed in the mean values of packed cell volume (PCV), haemoglobin concentration, and red blood cell (RBC), lymphocytes (lym), Heterophil (Het), monocytes (Mon), basophil (Bas), and eosinophil (Eos) of birds treated with 0 mg/Kg body weight (control group), 50 mg/Kg body weight and 100 mg/kg body weight for the unchallenged birds. The highest mean values of PCV (36%), RBC (3.42x106 /mm3), heterophil (26%), monocytes (13%), and eosinophils (2%) were observed in the birds treated with 0 mg/ Kg body weight (control group). There was significant change in the mean values of basophils (2.5%) , Hb (12 g/100 ml) and lymphocytes (60%) for 0, 50 and 100 mg/Kg body weight and no change the mean values ESR (2 mm/hr) in the graded level of 0 mg, 50 mg and 100 mg of plant extract per body weight. 

 

Table 2 Dosage effect of P. guineense on the blood indices of unchallenged broiler chickens

 

Table 3 indicates the effect of vaccinated and unvaccinated broiler chickens on the haematological indices of broiler chickens. There were significant changes (p<0.05) observed in the mean values of packed cell volume (PCV), haemoglobin concentration, and red blood cell (RBC), lymphocytes (lym), Heterophil (Het), monocytes (Mon), basophil (Bas), and eosinophil (Eos) of the vaccinated and unvaccinated broiler chickens. The highest mean values of the PCV, RBC, Hb, lymphocytes, monocytes, and eosinophils were observed in the vaccinated birds while the highest mean values of lymphocytes and basophils were observed in the unvaccinated birds.

 

Table 3 Effect of vaccination on the blood indices of  unchallenged broiler chickenss

 

3.4 Haematological indices of challenged broiler chickens
The results of Piper guineense effects on the haematological values of the broilers used for the experiment of are shown in Table 4, Table 5 and Table 6 for the challenged birds. Table 4 shows that there was no significant change in the vaccinated birds compared to the unvaccinated birds, the haematological parameters of the vaccinated birds has no difference from the unvaccinated birds at 0 mg Piper guineense extract/Kg body weight of broiler chickens, (control group). The birds treated with Piper guineense extract were not significantly different from the control group (0 mg/Kg).

 

Table 4 Haematological indices of challenged broiler chickens

 

As shown in Table 5, there were no significant changes (p<0.05) observed in the mean values of packed cell volume (PCV), haemoglobin concentration, and red blood cell (RBC), lymphocytes (lym), Heterophil (Het), monocytes (Mon), basophil (Bas), and eosinophil (Eos) of birds treated with 0 mg/Kg body weight (control group), 50 mg/Kg body weight and 100 mg/kg body weight for the challenged birds. The highest mean of PCV, RBC, Hb, neutrophils, monocytes, and eosinophil were observed in birds treated with 50 mg/Kg body weight of Piper guineense while the highest mean value of ESR, lymphocytes, and basophils were observed in birds treated with 100 mg/kg body weight of Piper guineense.

 

Table 5 Dosage effection of P. guineense on the blood indices of challenged birds

 

Table 6 shows that there is no significant difference in the haematological indices of broiler chickens that are vaccinated and the birds that are not. The highest mean values of PCV, Hb, neutrophils, monocytes and basophils were observed in the vaccinated birds while the highest mean values of ESR, Hb, lymphocytes, and eosinophils were observed in unvaccinated birds. 

 

Table 6 Effect of vaccination on the blood indices of challenged birds

 

3.5 Feed consumption of Broiler chickens given Piper guineense
The histogram in Figure 1 indicates that feed consumption of broiler chickens given the plant extract (Piper guineense) was normal compared with the control group that was not given the plant extract. The feed intake of the treatment groups and the control groups is not significantly different (p<0.05). From the histogram, it was also observed that the feed intake increased drastically starting from 4th day to the 10th day. This is due to the fact that broiler chickens consume more feed as they grow up. Control groups took the highest feed intake on the 5th day, more than the control and treatment group. On the 1st day, T2R1 Unvac consumed the lowest feed (1.15 Kg) and there is no difference in the feed consumed by birds in T2R1 Vac and T2R2 Vac. Feed intake of the treatments with control is not significantly different. On the 2nd day, the highest feed intake is in control while T2R2 Vac is the lowest. There is no significant difference between the treatment group and the control. On the 3rd day, the highest feed intake is in T2R2 Unvac and there is no significant difference between the treatment and control group. On 4th day, there was no significant in the feed intake of birds in the treatment group and the control. Starting from the 5th day, there is increase in the feed intake of the chickens. On the 10th day, there was no significant difference in the feed intake of the treatment and control group.

 

Figure 1 Feed intake of broiler chickens given Piper guineense extract

 

3.6 Haemagglutination inhibition test
The result of the haemagglutination inhibition test of broiler chickens challenged with velogenic strain of Newcastle disease virus is shown in Figure 2 below. The level of antibody synthesized by the birds from different groups varies from one another. Birds in treatment 1 and 3 were given 100 mg of the Piper guineense extract per Kg body weight before they were challenged and birds in treatment 2 and 4 were given 50 mg Piper guineense extract. The group that has the highest level of antibody were the broiler chickens in group 1 which are not vaccinated and broiler chickens in treatment 3 which are vaccinated against Newcastle disease with antibody titre of log 26. This indicates that there is no significant difference in the immune response between these groups against Newcastle disease and compared to control group with the antibody titre of log 25, the plant extract has helped to boost the immunity of these groups. The treatment group with lowest antibody titre level are birds in group 2 which are not vaccinated against Newcastle disease virus is log 25. There is slight significant difference between group 2 and group 4. With the dose of 50 mg plant extract/Kg of body weight given to unvaccinated birds, there is no significant difference with the control group which are birds left only to natural immunity.

 

Figure 2 Antibody titre level of broiler chickens fed with varying level of Piper guineense

 

4 Discussions
The experimental birds which were given piper guineense extract were healthy and consumed feed normally as the control group did until they were challenged with Newcastle disease virus which led to various signs of Newcastle disease outbreak from the infected birds.
 
At the dose of 100 mg plant extract/Kg body weight, administration of Piper guineense plant into the drinking water of chickens proved to be a good preventive measure against Newcastle disease with both the vaccinated and the unvaccinated birds. This is of great advantage to people in the rural area where Newcastle disease vaccines are scarce and costly. The use of this plant extract to prevent the disease will also help them to avoid the danger of reversal to virulence of live vaccines which occurs because of poor electricity to maintain the cold chain of the vaccines. 
 
From the results gotten from the haematological parameters, the mean values of RBC, Haemoglobin concentration, PCV, basophils, and eosinophils obtained from all the experimental birds are perfectly within the range of  normal haematological values of chicken reported by Ross et al. (1978), but in great contrast with the lymphocytes, and basophils (%) reported by Ross et al. (1978).
 
The high mean values observed in the lymphocytes of the challenged birds are as a result of viral infection. This is in line with the findings of Dieterian-Lievre (1988).
 
The difference observed in the percentage lymphocytes in the challenged broiler chickens shows that the highest mean value of lymphocytes was observed in the birds treated with 100 mg of Piper guineense extract/Kg body weight of broiler chickens. Increase in the number lymphocytes is as a result of the viral infection and the plant extract at 100mg /Kg had helped to increase the number of lymphocytes produced by the infected birds to combat the foreign agent (virus). This as a result of boost in immunity of the treated birds at this dosage. Therefore, it is best to use the mehanolic Piper guineense extract at 100 mg/Kg to boost the immunity of the chickens exposure to viral infection.
 
Heterophils and monocytes protect the body against pathogenic micro organisms and eat up small particles of foreign matter. The analysis of the neutrophils and monocytes of the treated birds were not significantly different from those of the control groups which are within the normal range reported by Ross et al. (1978). This indicates that the plant extract at both 50 and 100 mg/Kg did not affect their immune status.
 
The antibody titre level of the experimental birds indicates that the immunity of chickens at the dose of 100 mg/Kg (log26) was the highest compared to the ones given 50 mg plant extract/Kg body weight and the control group. Therefore, piper guineense extract at 100 mg/Kg body weight can be given to chickens to prevent Newcastle disease outbreak.
 
The challenged birds that showed the signs of the disease started by sneezing and coughing on the second day after the infection followed by wings and leg paralysis, movement in a cyclic mortion and death in a prone or suprine position. Some of the infected birds did not show signs of Newcastle disease before their sudden death. Post mortem was carried out dead birds and some of the lesions seen were haemorrhagic proventriculus, streaked liver, inflammed duodenum, engorged gall bladder and so on (Figure 3).

 

Figure 3 Gross pathology of birds’ organs infected with Newcastle disease

 

Since the major constraint to profitable broiler production is disease outbreaks it has become imperative to seek means of achieving effective biosecurity and control measures which ethnoveterinary preparations could offer (Gueye, 1999; Musa et al., 2008). This project wok had shown that Piper guineense has antiviral properties in line with the findings of Negbenebor et al. (1999).
 
5 Conclusion and Recommendations 
5.1 Conclusion
The study showed that the administration of methanolic extract of Piper guineense into the drinking water of broiler chickens at both 50 mg/Kg and 100 mg/Kg body weight were not toxic to the experimental birds.  Results of antibody titres level of experimental birds also showed that the dosage level of 100 mg/Kg body weight has the highest immune response against Newcastle disease. This is in line with many literature reports that showed that Piper guineense helps to prevent Newcastle disease by increasing the immunological response of the birds to this disease.
 
5.2 Recommendations 
From the foregoing, it is recommended that Piper guineense extracts at the dose of 100 mg/Kg body weight should be administered to the drinking water of the chickens as a preventive measure against Newcastle disease as this will help to boost the immunity of the chickens.
 
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