EFFECT OF AQUEOS EXTRACT OF Momordica balsamina AND Cucumis metuliferus FRUITS ON NEWCASTLE DISEASE IN PULLETS

The study was conducted to determine the effect of aqueous extract of Mormordica balsamina and Cucumis metuliferus fruits on Newcastle disease (NCD) in pullets. Ninety (90) pullets were used for the study. The pullets were divided into prophylactic and curative groups comprising 10 birds per group. For prophylactic groups aqueous extract of the fruits at concentration of 2.5g and 5.0g per 100litres of water were given to the pullets ad-libitum for two weeks and then challenged with Newcastle disease virus kudu 113 strain via intra ocular route while for curative groups the pullets were challenged with the virus and then treated with the aqueous extracts of the fruits at 2.5g and 5.0g per 100litres of water ad-libitum. Data collected were analyzed using one way analysis of variance (ANOVA) using Statistical Analysis Software version 9.1. The results showed that M. balsamina at 2.5g/100litres of water had very good curative abilities which recorded lower clinical signs, mortality, postmortem lesions, higher antibody titer and higher white blood cell activity with a statistically significant (p<0.05) association while the same fruit at 5.0g/100litres of water had the best results for prophylactic treatments recording least clinical signs and mortality rates and highest antibody titers, highest white blood cell count which was statistically significant (P<0.05). It is therefore recommended that Mormodica balsamina can be recommended at 2.5g/100litres and at 5g/100liters of water for the control and prevention of Newcastle disease respectively to farmers and that further studies should be carried out to establish optimum concentration levels for the aqueous and other (methanolic and ethanolic) extracts of the fruit for achievement of best prophylactic and curative effect.


INTRODUCTION
Poultry production is the fastest growing segments of the agricultural subsector in Nigeria due to the increased demand of animal protein with the ever growing population (Salami et al., 1989). However, one of the constraints to the development of the poultry industry is outbreak of diseases (Salami et al., 1989) one of which is Newcastle disease (ND). Newcastle Disease Virus (NDV) causes a highly infectious neurological, respiratory or enteric disease of poultry.
Cucumis metuliferus, locally called 'gautar kaji' in hausa language, belongs to the family Cucurbitaceae and is a monoecious, climbing, annual herb that can be grown practically anywhere, provided the season is warm (Benzioni et al., 1993). It is commonly known as African horned cucumber, melano, Jelly melon, and kiwano. The fruits occur in two forms: the bitter and non-bitter forms, which occur mostly in the wild state. The non-bitter form has been found to be less toxic and has also been widely cultivated (Enslin et al., 1954).
Momordica balsamina Linn commonly known as African pumpkin (or African cucumber), Balsam apple (or Balsam pear) and locally called "Garahuni" (Hausa Language), also belongs to the family Cucurbitaceae. A macerate of the whole plant to which salt has been added is used in Senegal as a galactogogue and to increase milk yield of cows (Burkill, 1985). The fruit mixed with olive or almond oil is used for the treatment of pile; for festers, inflammations, swellings, yaws, burns, intermittent fever, burning sensation of sole, night blindness, diabetes, asthma and cough (Burkill, 1985). The fruit has emetic and cathartics effects. Roodt (1998) reported various medicinal uses of Momordica balsamina. The fruit extract contains alkaloidal components which have antiviral properties reverted the negative effects of Newcastle Disease virus in chicks when injected at 600 mg/kg (Raza et al., 2015) Newcastle disease still remains a serious economic challenge to all segments of the poultry industry because of its high morbidity and mortality rates (Alexander, 2003). The only control method so far still remains vaccination, which does not confer 100% immunity in all vaccinated birds. Vaccine breaks and failures are also a major problem. It has also been reported that vaccination itself may cause disease and reduced growth in vaccinated birds (Alexander, 2003). Moreover outbreaks have been reported in vaccinated populations (Alexander, 2003).
Investigations show that local farmers in the study area claim to use Cucumis metuliferus and Momordica balsamina fruits to treat their birds for various diseases including Newcastle disease by immersing the whole fruits in drinking water of birds (Alhaji, 2017). The need to test the above theory and the need to find a cheaper and more accessible means to the local farmer for the treatment and prevention of Newcastle disease thus preventing huge economic loses informed this research.

Study area
The study was conducted at the Poultry Unit of the Department of Animal Science Teaching and Research farm, Federal University Dutsinma, Katsina State, Nigeria. Dutsinma lies within the coordinates of latitude 12.45N and longitude 7.49E and also on the altitude of 605m above the sea level (Isah, 2009). Generally, the climate varies considerably according to months and seasons of the year namely a cool dry (harmattan) season from December to February, a hot dry season from March to May, a warm wet season from June to September; and a less marked season after rains characterized by decreasing rainfall and gradual lowering of temperature during the months of October to November, (Ati et al., 2010).

Experimental design
The research adopted a Completely Randomized Design with 9 treatments, each treatment/group were made up of 10 birds. Day old pullets were sourced from reputable hatchery (Olam ® Hatcheries). They were brooded under strict biosecurity measures to prevent entry of pathogens. The birds were vaccinated against Infectious bursal disease at day seven of the arrival but not against Newcastle disease and they were fed with chick-mash till 8weeks when experiment started. Experimental groups consisted of Curative, Prophylactic and Control groups. Aqueous solutions of both M balsamina and C metuliferus were made at 2.5grams per liter of water and 5grams per liter of water. The curative group was made up of groups-CM2.5g/l, CM5.0g/l, CC2.5g/l and CC5.0g/l where CM indicates curative mormodica and CC curative cucumis. They were initially challenged with the Newcastle disease virus Kudu 113 strain at 8weeks and treated with aqueous extracts of Cucumis metuliferus and Momordica balsamina ad libitum in drinking water at above concentrations as soon as clinical signs were observed. The prophylactic groups, PM2.5g, PM5.0g, PC2.5g and PC5.0g, were treated with aqueous extracts of Cucumis metuliferus and Momordica balsamina ad libitum in water for 2weeks and then subsequently challenged with Newcastle disease virus Kudu 113 strain to observe if the treatments would prevent or reduce the intensity of infection. The control group was challenged with the Newcastle disease virus Kudu 113 strain but was not treated with any extracts at 8 weeks of age.

Study parameters
The clinical signs, mortality and post mortem lesions were observed and recorded daily according to the method of Agang, 2014.

Laboratory analysis
Blood was collected via wing vein of birds at week two of the experiment and at week four of the experiment. Whole blood was collected for Heamogram/blood picture and the serum for Heamagglutination inhibition test.

Data analysis
Data were subjected to oneway ANOVA (analysis of variance) using Statistical Analysis Software version 9.1. P values of <0.05 were considered significant.

RESULTS AND DISCUSSION Clinical signs
The clinical signs in pullets challenged with Newcastle disease for curative groups are presented in Tables 1. Signs observed included: Gasping, sneezing, watery greenish diarrhea, anorexia, torticollis, gasping, depression, swelling of the head, paralysis and emaciation. For curative groups CM2.5g showed the least clinical signs of anorexia, watery green diarrhea and depression while the control group were most severely affected recording all clinical signs (Table 1). Table 2 shows the clinical signs for the prophylactic groups.
For the prophylactic groups PM5g recorded the least clinical signs while PC5g showed most clinical signs (Table 2). Ibrahim and Tanya (2001) and Bukar-Koloet al., (2006) both reported Newcastle disease to be the major poultry diseases affecting pullets and its management and high cost of orthodox drugs lower pullet's production too.
Groups CM2.5g and PM5g both treated with aqueous extract of Mormodica balsamina at 2.5g/liter and 5g/liter recorded the least clinical signs. This is not unexpected as Hassan and Umar (2006) reported that M balsamina has high calcium, phosphorus and vitamins. These contents would naturally be expected to translate to healthy birds. Agang (2014) reported increase in weights of birds treated with M. balsamina when compared to untreated groups.  Control 10 9

Post Mortem Lesions (Curative Group)
The post mortem lesions of pullets challenged with Newcastle disease virus are indicated in Tables 4 and 5. Post mortem lesions observed included haemorrhage in the intestine, haemorrhage in trachea, haemorrhage on proventriculus, bile enlargement, cloudy air sack, haemorrhage on glandular surface of proventriculus and necrotic areas in caecal tonsils. Findings for post mortem lesions followed a similar pattern with group CM2.5g showing the least post mortem lesions (Heamorrhage on proventiculus, cloudy air sacs and necrotic areas on cecal tonsils) while CC5.0g showed most serious post mortem lesions for curative groups with all signs recorded observed in this group (Table 4). For prophylactic groups PC2.5g showed most serious signs when compared with other groups recording all signs except bile duct enlargement ( Table 5).
The post mortem lesions seen may be attributed to the route of challenge (Ocular) which may enhance the clinical signs and gross lesions observed in most cases as the Newcastle disease virus replicates in the Haderian gland before spreading to other sites. This is in agreement with the work of Agang (2014) with M. balsamina on pullets, who reported that severe clinical signs, mortalities and lesions observed showed low level of protection in untreated groups.  HII -Heamorrhage in intestine, HIT -Heamorrhage in trachea, HOP -Heamorrhage on Proventriculus, BE -Bile duct enlargement, ASC -Cloudy air sac, HGSP -Heamorrhage on glandular surface of proventriculus, NCT -Necrotic areas on cecal tonsils

Heamatology
The blood pictures of pullets challenged with Newcastle Disease Virus is presented in Table 6. Blood pictures showed highest total white blood cell counts and packed cell volumes for group CM2.5g in curative groups while CM5g showed the least TWBC.
The higher white blood cell count seen from blood pictures for group CM2.5g showed higher white blood cell activity (17.33*10 9 /l) and thus higher chances of the immune system fighting off the infection than other groups (CC2.5g, CC5g, CM2.5g and the control group). This can be seen to be translated in the lower clinical signs and mortality rates of the groups with higher TWBCs.  The blood pictures of pullets challenged with Newcastle Disease Virus is presented in Table 7. For prophylactic groups PM5g had the highest TWBC while group PC5g showed the least TWBC count. They attributed this to high amount of calcium, phosphorus and vitamin in ash (11.10%) of Mormodica balsamina. Hassan and Umar (2006) also mentioned that Mormodica balsamina has high calcium, phosphorus and vitamins. These minerals and vitamins could be the reason for the increased total white blood cell count, PCV, Heamoglobin and Total protein seen in CM2.5g group and PM5g groups for curative and prophylactic groups respectively.

Heamagglutination Inhibition
Heamagglutination inhibition titres of pullets challenged with Newcastle disease virus (Kudu 113) are presented in Tables 8  and 9. For curative groups CM2.5 showed highest antibody titre up to dilution rate of 1 in 128 (Table 8) while CC5g recorded lowest titre of 1 in 8. For prophylactic group PM5 recorded the highest antibody titer of I in 64 while PC2.5g had the lowest tire of 1 in 8 (Table 9).
Antibody titers for heamagglutination inhibition tests were indicated by highest titre levels at which agglutination became absent thus indicating no antigen antibody reaction at that titre. All groups showed an initial positive response. This could be attributed to maternal derived antibodies. Similar results of maternal antibodies conferring protection in all groups have been reported by Agang et al., 2019;Allan et al., 1978 andVerma et al., 1985. The results for HI were not surprising as the groups with highest antibody titres had lowest recorded the lowest clinical signs in curative and prophylactic groups respectively as well as mortality and postmortem lesions. Agang (2014) reported that mean antibody titres showed significant increased from day-old up to the 7th week in groups treated with M.balsamina. The initial rise in antibody titer is attributed to maternal derived antibodies and this is obvious as even pullets in the negative control group showed initial agglutinations.

CONCLUSION AND RECOMMENDATION
The study showed better curative and prophylactic effects at concentration of M2.5g while the same plant at concentration of M5g showed best prophylactic ability.
It is thus recommended that aqueous extract of Mormodica balsamina at 2.5g/100litres and 5g/100liters of water should be used for the control and prevention of Newcastle disease respectively by local poultry farmers. Further studies should be carried out to establish optimum concentration levels for the aqueous and other (methanolic and ethanolic) extracts of the fruit for achievement of best prophylactic and curative effect.