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Continental J. Veterinary Sciences 2: 1 - 11, 2008 ©Wilolud Online Journals, 2008. IN-VIVO EVALUATIONS OF THE STEM BARK OF COMBRETUM MOLLE “R.BR/G. DON” (KEAY,

1989) FOR ANTHELMINTIC PROPERTIES.

¹Simon M.K., ²Ajanusi J.O., ²George B.D., ³Abubakar M.S., ¹Meduna J.A ¹Federal College of Wildlife Management, New-Bussa, Niger State

²Department of Veterinary Parasitology and Entomology, Faculty of Veterinary Medicine, A.B.U, Zaria ³Department of Pharmacognocy, Faculty of Pharmaceutical Sciences, A.B.U, Zaria

ABSTRACT

The anthelmintic activity of partitioned portions of the crude methanolic extract of Combretum molle was evaluated in-vivo in rats’ model experimentally infected with Nippostrongylus. The crude methanolic extract of the plant was obtained after extraction with absolute methanol; and further partitioned between three solvents (i.e. petroleum ether, chloroform and N-butanol). The extract portions (with the exception of petroleum ether) were tested for anthelmintic activity against Nippostrongylus in rats. Phytochemical screening conducted on the extracts revealed constituents that have anthelmintic effect such as; alkaloids, steroids, saponins, tannins, flavonoids and glycosides. The anthelmintic activity was assessed by comparing the number of worms recovered from rats treated with the portions to those from non-treated infected controls. This study considered deparasitization rate of 70 % or greater as significant. The aqueous methanol, chloroform and N-butanol portions produced significant (p<0.05) deparasitization rate of 86.98 %, 79.20 % and 72.72 % respectively when a maximum tolerated dose of 1000 mg/kg‾¹ was administered. Thus the extracts are recommended for further studies in ruminant to validate their efficacy in them.

KEYWORDS: in-vivo, anthelmintic, Combretum molle, Nippostrongylus braziliensis

INTRODUCTION Parasitic nematodes are among the most common and economically important infectious disease organisms of grazing livestock especially ruminants around the world (Perry et al., 2002; Alawa et al., 2001). In Nigeria, helminthosis can result in mortality rates exceeding 50 % with an estimated loss of $144 million annually in animal products and services (Jegede et al., 2006; Perry and Randolph, 1999). Conventional anthelmintics are good remedies for the control of livestock parasitosis, but constraints such as high cost, adulteration, resistant strain development, residual effect on man and the declining funding of veterinary services especially in developing countries are limiting the use of such drugs (Ademola et al., 2004; Uza et al., 1996). However, efforts are now directed towards screening herbal medicinal plants claimed by traditionalists and pastoralists as remedies for helminthosis in both in vitro and in vivo studies (Githiori et al., 2003b, Alawa et al., 2003; Ademola et al., 2004). Plants produce valuable organic substances that have potential value in the treatment of diseases. It is necessary therefore to investigate such plants and to isolate and identify their active principles (Abdul Ghani, 1990)

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Simon M.K et al: Continental J. Veterinary Sciences 2: 1 - 11, 2008 Moreover; the rational use of drugs requires the standardization of their crude forms, dosage formulation as well as the determination of their mode of action and any toxic manifestations which they may produce (Vercruysse et al., 2001; Ibrahim et al., 1984). Combretum molle ‘R.Br/G.Don’ (Keay, 1989) is a tree distinguished by its rough bark and dense crown. It is commonly referred to as wuyan damo (Hausa), damoruhi (Fulani) and aragba (Yoruba); the common English name is obscured. There was no reported medicinal use of this plant apart from the purported claims of the nomads and the pastoralists that its stem bark possessed anthelmintic activity in animals when given as concoction (Alawa et al., 2000; Atawodi et al., 2000). In view of public concern over perceivable drug residues in animal products, the increasing prevalence of anthelmintic-resistant strains of nematodes, the rising cost of such organic substances, there is an urgent need for development of sustainable alternatives to conventional anthelmintics in ruminant production system. This can be achieved through research into newer anthelmintics from natural resources. Thus this work is aimed at assessing the in vivo anthelmintic effect of the crude methanolic extracts (CME) and the partitioned portions of the bark of Combretum molle against adult Nippostrongylus braziliensis in experimentally infected rats with the view of extrapolating the findings in ruminant production system. Also to identify the most active portion of the partitioned extracts, with the view of providing scientific basis for their use in ethno-veterinary practices. MATERIALS AND METHOD The study was conducted in April, 2007 at the Department of Parasitology and Entomology, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, Nigeria. Plant collection, identification and preparation Five kilogram’s of the stem barks of Combretum molle were collected from Zaria, Nigeria and properly identified by a botanist, and a voucher number (V/No 297) was given. Samples were sun dried, powdered using mortar and pestle into fine powder and sieved as described by Onyeyili et al (2001). Five hundred grams of the powdered sample was extracted exhaustively with 2.5litres of absolute methanol in a soxhlet apparatus (Youn et al, 2003; Onyeyili et al, 2001).The extract was concentrated to dryness in a vacuum using a rotary evaporator coupled to a thermo-regulator (Hordegen et al, 2003). Twenty grams of the dried crude methanolic extract was further partitioned in a step-wise separation process in petroleum ether, chloroform, and N-butanol using a separating funnel (Assis et al, 2003). The solvents were evaporated leaving the dried portions which were then tested in-vivo for anthelmintic activity against N. braziliensis in a rat model (Ibrahim et al., 1984; Suleiman et al, 2005) PPhhyyttoocchheemmiissttrryy Phytochemical screening of the crude methanolic extract and the partitioned portions was conducted using standard techniques described by Trease and Evans (1983) and Brain and Turner (1975). Experimental animals (Rats) Sixty albino Wister rats (6 to 7 weeks old) of both sexes and weighing between 100 to 160 g were used in the study. The rats were acclimatized to laboratory conditions for two weeks and fed on commercially prepared feed; water was given ad lib. Forty two rats used for the anthelmintic study were dewormed using albendazole at 200 mgkg‾¹ two weeks before experimental infection (Suleiman et al., 2005).

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Simon M.K et al: Continental J. Veterinary Sciences 2: 1 - 11, 2008 Evaluation of maximum tolerated dose (MTD) of the crude methanol extracts Due to lack of information on the precise dosage of the plants preparation used by traditional herdsmen and the pastoralists, a maximum tolerated dose (MTD) experiment described by Lorke (1983) was carried out to determine the experimental dose of the plant using the crude methanolic extract in eighteen rats. Post-mortem findings Rats were randomly selected one from each of the dose levels of the preliminary maximum tolerated dose trial, one from each of the partitioned portions after the experimental treatment and salvaged for gross and histological changes on the visceral organs resulting from the administration of the plants extracts (Suleiman et al., 2005). Experimental infection/design Forty two worm-free rats were infected by injecting subcutaneously in the cervical region with 200 viable third stage larvae (L3) of N. braziliensis in .2 ml of water using an 18-gauge needle attached to an insulin syringe (Suleiman et al., 2005). Five days post infection, rats were screened for evidence of infection through faecal screening using simple floatation technique (Soulsby, 1982). Rats not shedding ova of N. braziliensis were excluded from the experiment. The infected rats were randomly allocated to three (3) treatment groups (A-C). Group ‘A’ had six rats and treated with albendazole at 200 mgkg‾¹ body weights as a positive control group (Suleiman et al., 2005). Group ‘B’ (having 24 rats), were sub-divided into four groups of six rats each and treated with the crude methanolic extract, chloroform, N-butanol and aqueous methanol dried portions at 1000 mgkg‾¹ (Vercruysse et al., 2001). Group ‘C’ having 12 rats and were subdivided into two groups of six rats each and given water and propylene glycol at 5 mlkg‾¹ as negative controls (Ibrahim et al., 1984). Worm counts On Day 2-post treatment, all rats were fasted for 24 hours, salvaged for adult worm count, using the WAAVP guides (Powers et al., 1982). The first 15 cm of the small intestine was removed, cut longitudinally and placed between two clean 20 cm glass slides. The section was examined at x40 magnification of a dissecting microscope. Visible worms were counted and recorded (Suleiman et al., 2005). The portion that showed the highest reduction in worm count and did not produce any behavioral changes in the rats was considered to be the most active portion (Githiori et al., 2003). Percentage efficacy Percentage efficacy (deparasitization) of the crude methanol extract and the various portions of each plant was calculated according to the method used by Cavier (1973). The percentage efficacy was calculated using the formula:

N-n % Efficacy = ----- X 100%

N

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Simon M.K et al: Continental J. Veterinary Sciences 2: 1 - 11, 2008 N= number of worms counted in the placebo-treated rats n= number of worms counted in the plant extracts or albendazole-treated rats. Statistical analysis Means of data obtained were analyzed statistically using the software package for Graph-Pad prism (version 4.0/2003). Statistical significance for the anthelmintic effect of the crude methanol extract, chloroform, N-butanol portion and aqueous portion was assessed by ANOVA. Subsequently Borferroni’s multiple comparison test was carried out to determine the most active portion. P value < 0.05 was considered significant. RREESSUULLTTSS Methanolic Extraction of plant material and solvent partitioning of crude methanolic extracts One hundred and fifty grams (30 %) was obtained after subjecting of 500 g powdered plant material to extraction with absolute methanol. Using petroleum ether, chloroform and N-butanol as separating solvents, the respective yields following partitioning of 20 g crude methanolic extract were 0 g (0 %), 5 g (25 %), 7 g (35 %) and 8 g (40 %) as the aqueous methanol portion PPhhyyttoocchheemmiiccaall ssccrreeeenniinngg The phytochemical screening revealed that the crude methanolic extract of the plant had alkaloids, steroids, saponins, carbohydrate, flavonoids, tannins and cardiac glycosides as constituents; the petroleum ether portion of the plant revealed the presence of only alkaloids and steroids; while the chloroform portion, when screened, showed the presence of alkaloids, steroids, flavonoids, tannins and cardiac glycosides. The screening of the N-butanol portion revealed the presence of carbohydrate, in addition to the constituent seen in the chloroform portion. The screening of the aqueous methanol portion revealed the presence of steroids, flavonoids, tannins, carbohydrate and cardiac glycosides. Table 1: Phytochemical screening for the crude methanol extract and various portions of A. africana. ____________________________________________________________________ Portions Constituents crude methanol extract pet ether chloroform N-butanol aq methanol

Alkaloids + + + + - Cardiac glycoside + - + + + Carbohydrate + - - + + Flavonoids + - + + + Saponins + - - + + Steroids + + + + + Tannins + - + + + Key Present (+) Absent (-) Determination of maximum tolerated dose. The crude methanolic extract at a dose range of 10 to 1000 mgkg‾¹ did not cause any visible toxic effect in the rats- the rats were active 6 to 12 h after recovering from the stress of the administration procedure. On the other hand, at a dose range of 1600 to 5000 mgkg‾¹, there were indications of toxicity – weakness after 24 hrs of administration, a case of mortalities at 24 h in the group given 2900 mgkg‾¹, and two mortality at 12 h in the group given 5000 mgkg‾¹ (Table 2), thus 1000 mgkg‾¹ was chosen as experimental dose.

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Simon M.K et al: Continental J. Veterinary Sciences 2: 1 - 11, 2008 Table 2: Maximum tolarated dose/toxicity of crude methanol extract of C. molle ___________________________________________________________________ • Dose(mg/kg‾¹) 10 100 1000 1600 2900 5000 ___________________________________________________________________ Initial number of rat 3 3 3 3 3 3 Mortality 0 0 0 0 1 2 Observation a a a b c d Inference - - - + ++ +++ ___________________________________________________________________ Key a = rat active 6-24 hrs and beyond, b = rats showed weakness for more than 24 hrs, c = rats showed weakness for more than 48 hrs, d = rats showed weakness for more than 7 days, - = no sign of toxicity, + = slightly toxic, ++ = less toxic, +++ = toxic. PPoosstt mmoorrtteemm ff iinnddiinnggss There were no gross pathological and histological lesions on the visceral organs resulting from the administration of the tested doses of the crude methanolic extract and various portions. AAnntthheellmmiinnttiicc eeff ffeecctt ooff eexxttrraaccttss oonn NN.. bbrraazzii ll iieennssiiss Rats that had oral infection of 200 L3 followed by treatment with crude methanolic extract at 1000 mg/kg‾¹ had a mean worm count of 6.17; while those treated with chloroform, N-butanol and aqueous methanol portions at 1000 mg/kg‾¹ each and albendazole at 200 mg/kg‾¹ had mean worm count of 3.0, 3.5, 1.67 and 0 respectively; compared to the mean worm count of 12.83 and 12.0 from the negative controls treated rats. The extracts produced percentage deparasitization of 48.61 %, 75.0 %, and 72. 72% and 86.98 % for crude methanolic extract chloroform, N-butanol and aqueous methanol respectively. Table 3: Worm count and percentage deparasitization 7 days after treatment with crude methanol extract and fractions of C. molle ---------------------------------------------------------------------------------------------------------------------------------

Worm cout after treatment with • CME Chloroform N-butanol Aqueous Albendazole Placebo1

Placebo2 • Rat (1000mgkg‾¹) (1000mgkg‾¹) (1000 mgkg‾¹) (1000 mgkg‾¹) (200 mgkg‾¹) (5 ml-water) (5 ml-

p. glycol) --------------------------------------------------------------------------------------------------------------------------------- • 1 7 2 6 2 0 4 6 • 2 2 3 0 0 0 17 16 • 3 7 3 5 0 0 18 15 • 4 8 4 3 4 0 12 10 • 5 7 3 3 2 0 14 11 • 6 6 3 4 2 0 12 14 Mean ± SD 6.17 ± 2.11b 3.0 ± 0.63a* 3.5 ± 2.09a* 1.67 ± 3.37a* 0.0 ± 0.0a* 12.83± 5.0b 12.0

± 3.74b % DPZ 4 8.61 75.0 72.72 86.98 100 0 0 --------------------------------------------------------------------------------------------------------------------------------- Mean with * within the column are significantly different at p<0.001, while those with the letter ª and ъ

show no significant difference between their means at p>0.05 as determined by Borferroni’s multiple comparison test. %DPZ= percentage deparasitization

Compared to the negative controls, the crude methanolic extract and the various portions albendazole gave 100 % deparasitization

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Simon M.K et al: Continental J. Veterinary Sciences 2: 1 - 11, 2008 The deparasitization produced by the chloroform, N-butanol and aqueous methanol portions were significant (p<0.05) when compared to that produced by the negative control rats, while the deparasitization produced by the crude methanolic extract was non-significant (p>0.05) (Table 3).

Alben Aqu Chlo N-but CME water p.gly0

5

1 0

1 5

t r e a t m e n t s

worm

count

Fig 1: Mean ± SD of worm count after treatment with the various fractions of C. molle extracts, albendazole and the placebos DDIISSCCUUSSSSIIOONN Recent harmonizations on anthelmintic efficacy guidelines in ruminants have indicated that for a drug to be considered efficacious, a 90 % reduction in total worm count should be achieved (Vercruysse et al., 2001). However, the in-vivo anthelmintic effect of the plant extracts is unknown. Thus, it was considered ‘a priori’ that the efficacy of the extracts would be biologically significant if a reduction in total worm count above 70 % occurred (Githiori et al., 2003). Rats treated with chloroform, N-butanol, and aqueous methanol portions showed anthelmintic activity. The aqueous methanol portion showed the highest reduction in total worm count 86.98 %. This was followed by chloroform and N-butanol portions with reduction in total worm count of 75.0 % and 72.20 % respectively. Treatment with the crude methanolic extract did not produced the required significant biological reduction in worm count in comparison with the untreated control groups. The results of this study also demonstrated that the parasites N. braziliensis was highly sensitive to albendazole with complete deparasitzation at a dose of 200 mg/kg‾¹ (Suleiman et al., 2005). The outcome of the phytochemical screening revealed that plant extracts have constituents including tannins, alkaloids, flavonoids, cardiac glycosides and steroids which may have anthelminthic activities (Athanasiadou et al., 2001, 2005; Gamenara et al., 2001; Lahlou., 2002; Onyeyili et al., 2001; Lateef et al., 2003; Prasharth et al., 2001) Kahiya et al. (2003) in in-vitro studies reported that condensed tannins from the leaves extract of Acarcia nitotica inhibited the development of H. contortus larvae from goats. Tannins polyphenols from bryophytes were shown to have anthelmintic activity against N braziliensis (Gamenara et al., 2001). Athanasiadou et al. (2001) in in-vitro and in-vivo studies reported the anthelmintic activity of condensed tannins extracted from Quebracho on the larvae of H. contortus, Teladorsagia circumcinta and Trichostrongylus vitrinus. Anthelmintic activity of tannins is attributed to their capacity to binds to free proteins available in the gastrointestinal tract of the host reducing nutrients available to the parasites resulting into starvation and death (Athanasiadou et al., 2001). Also tannins are capable of binding with the glycoproteins on the cuticle and leading to death of the parasite (Thompson and Geary, 1995). Also tannins have vasoconstriction effect and could be advantageous in preventing worm implantation onto the mucosa of the gastrointestinal tract thus making their expulsion from the GIT easier (Aguwa and Nwako, 1988; Min and Hart, 2003).

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Simon M.K et al: Continental J. Veterinary Sciences 2: 1 - 11, 2008 Tannins-containing plants increase the supply of digestible protein by animals thus improving the immunity against gastrointestinal parasites (Coop and Kyriazakis, 1999; Coop and Holmes, 1996). Tannins contain mixtures of phenols which combine with plasma proteins rendering them resistant to proteolytic enzymes secreted by the worms (Mitcell et al., 1983; Kahn and Diaz-Hernandez, 2000). It is therefore reasonable to assume that the herdsmen/pastoralists claims may be right since the plants used in this study contain tannins and could have had anthelmintic effect similar to the ones earlier described. Hashizume et al. (1978) reported that flavonoids offer some protection against ulcer by increasing capillary resistance and through improved microcirculation which renders the cell less injurious to predisposing factors such as worms. Flavonoids are also believed to stimulate intestinal motility similar to that produced by acetylcholine (Akendenque, 1992), there by causing rapid worm expulsion from the gastrointestinal tract. Lahlou (2002) reported that flavonoids are phytochemicals that have anthelmintic effect. Having identified flavonoids in almost all the portions used in this study, it is possible that they had a significant anthelmintic effect on the N. braziliensis resulting in the observed deparasitization. In in-vitro and in-vivo studies, Al-Qarawi et al. (2001) reported that alkaloids extracted from both the latex and leaves of Calotropis procera, were effective in inhibiting the exsheathment of L3 of H. contortus to L4 in sheep. Lateef et al. (2003) also reported that alkaloids and their glycosides extracted from the roots of Adhatoda vestica were effective against mixed gastrointestinal infections in sheep. Also, Onyeyili et al. (2001) reported that tannins and alkaloids, the active principles of Nauclea latifolia bark were effective against mixed infections in sheep. The present study has shown that alkaloids are present in all the portions except the CME. It is possible that the presence of alkaloids had a role in the significant deparasitization observed. Conversely, the absence of alkaloids in the CME may have accounted for the very poor and insignificant deparasitizatioin observed with this portion. Glycosides extracted from the root of Adhatoda vestica were found to be effective against mixed gastrointestinal nematode infection in sheep (Lateef et al., 2003). Also this compound has been shown to induce tonic contraction that resulted in the expulsion of the worms from rat’s gastrointestinal tract (Kim et al., 1992). Cardiac glycosides were identified in the plant extracts used in this study and could have had the same effect. Steroids were identified as active principle in Butea monosferma seed and reported to have in-vitro anthelmintic activity against adult Caenorhabditis elegans, a free-living nematode (Prasharth et al., 2001). However, it is unknown whether the steroids identified in all the portions of both plants could have had the same effect in-vivo. The maximum tolerated dose trials or otherwise referred to as dose determination studies (Vercruysse et al., 2001) were carried out on the premise that the plant extracts under investigation had no alternative data to support any intended dosage. In this work, the injurious dose and the maximum tolerated dose were determined. The plant extracts produced lethal effect at a dose of 1600 to 5000 mgkg‾¹. However there was no sign of toxicity at a dose of 10-1000 mgkg‾¹. Therefore 1000 mgkg‾¹ was selected as the maximum tolerated dose as well as the experimental treatment dose. CCOONNCCLLUUSSIIOONN Results from this study demonstrated that the aqueous methanol, chloroform and N-butanol portions of the plant extracts were effective against experimental N. braziliensis infection in rats at a non-toxic dose of 1000 mgkg‾¹. The chemicals believed to constitute the active principles in the plant have significant anthelmintic efficacy whereas albendazole was found to be highly efficacious. The investigation of the effects of chemical compounds on helminths from natural products is fundamentally important for the

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Simon M.K et al: Continental J. Veterinary Sciences 2: 1 - 11, 2008 development of new anthelmintics drugs, especially in view of the vast worldwide flora. Thus a quality controlled extraction of C. molle and the isolation of their bioactive compounds could be a promising alternative to conventional anthelmintics for the treatment of gastrointestinal helminths of ruminants in the future. Unknown factors may also have an influence on the anthelmintic activity of these plants. For instance, the rats may have generated a strong T. cell-depedent immune response that brings about expulsion of the worms from the intestine (Mitcell et al., 1983) More detailed studies are needed to isolate, characterize and evaluate the active components and the mechanism of action of the identified active principles. Also preliminary studies on the toxicity, evaluation of the effect in-vivo against economically important gastrointestinal nematode species and the establishment of adequate doses for sheep, goats and cattle are needed. REFERENCE Abdul Ghani. (1990). Introduction to pharmacology. 1st edition ABU Zaria Press, Zaria. Nigeria. 2-8. Ademola, I.O., B.O. Faagbemi and S.O. Idowu, 2004. Evalaution of the anthelmintic activities of Khaya Senegalensis extracts against gastrointestinal nematodes of sheep. In vitro and In vivo studies. Vet. Parasitol. 112. 151-164. Aguwa, C.N and S.O.Nwako, 1988. Prelimnary stuidies on the root bark extracts of Nauclea latifolia Smith for anti-ulcer properties. Nig. J. Pharm. Sci. 4/1. 16-23. Akendenque, B. (1992). Medicinal plants used by the Fang traditional healers in Equatorial Guinea. J. Ethnopharmacol.37 (21), 167-143 Al-Qarawi, A.A., O.M.Mahmoud, E.M.Haroun, and S.E.Adam, 2001. A preliminary study on the anthelmintic activity of Calotropis procera latex against Haemonchus contortus infection in Najdi sheep. Vet. Res. Com. 25, 61-70. Alawa, C.B.I., J.O. Gefu, N.P. Chiezey, P.A. Abdu, S.O. Magaji, L.O. Eduvie and I.A. Adeyinka, 2000. Screening of Vernonia amygdalina for antelmintic properties. In: Ethnoveterinary Practices, Research and Development. Proc. Int. Workshop on Ethnoveterinary Practices (Ed: Gefu, J.O., P.A Abdu and C.B.I. Alawa) held in Kaduna, Nigeria. August 14th-18th, 2000 NAPRI/ABU, Zaria Alawa, C.B.I., A.K. Mohammed, O.O. Oni, I.A. Adeyinka, O.S. Lamidi and A.M. Adamu, 2001. Prevalence and seasonality of common health problems in Sokoto Gudali cattle at a research station in the Sudan ecological zone of Nigeria. Nig, J, Anml. Prod. 28(2) 224-228. Alawa C.B.I., A.M.Adamu, J.O. Gefu, O.J. Ajanusi, P.A. Abdu, N.P. Chiezeny, J.N. Alawa and D.D. Bowma, 2003. In vitro screening of two Nigeria medicinal plants (vernonia amygdalina and Annona Senegalensis) for anthelmintic activity. Vet. Parasitol. 113. 73-81. Assis, L.M., C.M.L. Bevilaqua, S.M. Morais, L.S.Vieira, C.T.C.Costa and J.A.L Souza, 2003. Ovicidal and larvicidal activity in vitro of spigelia anthelmia Linn. extracts on Haemonchus contortus. Vet. Parasitol. 117. 43-49. Atawodi, S.E., M. Usman, S.T. Bulus, J.C. Atawodi, L. Wakawa and D.A. Ameh, 2000. Herba treatment of some peotozoan and parasitic diseases of poultry in the middle of Nigeria. Proc.Int. Workshop on EthnoVet erinary Practice. Kaduna Nig. 2000. pp79-84

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Simon M.K et al: Continental J. Veterinary Sciences 2: 1 - 11, 2008 Athanasiadou, S., I. Kyriazakisi, F. Jackson and R.L. Coop, 2001. Direct anthelmintic effects of condensed tannins towards different gastrointestinal nematodes of sheep. In vitro and in vivo studies. Vet. Parasitol. 99. 205-219. Athanasiadou, S., O. Tzamalouka, I. Kyriazakis, F. Jackson and R.L. Coop, 2005. Testing for direct anthelmintic effects of bioactive foreages against Trichostrongylus columbriformis in grazing sheep Vet. Parasitol. 127. 233-243. Brain K.R and Turner T.D. (1975). The practical evaluation of phytopharmaceuticals and therapeutics. Wright-Scientechnica, Bristol, pp 10-30 Cavier, R., 1973. Chemotherapy of internal nematode. In: Chemotherapy of Helmithiasis. Ed. by Hawking, F. International Encyclopedia of Pharmacology and Therapeutics. First edition, Pergamon Press Ltd Headington Hill Hall. Oxford. Vol 1.pp 437-500. Coop R.L and I. Kyriazakis, 1999. Nutrition-parasite interaction. Vet. Parasitol. 84, 187-204. Coop, R.L and P.H. Holmes, 1996. Nutrition and parasite interaction. Int.J. Parasitol. 26, 951-962. Gamenara, D., E. Pandofli, J. Saldana, L. Dominguez, M.M. Martinez and G. Seoane, 2001. Nematocidal activity of natural polyphenols from bryophytes and their derivatives. Arzncimittelforschung 51, 506-510 Githiori, J.B., Jahan Hoglund., J.W. Peter and Leyden Baker. 2003. Evaluation of Anthelmintic properties of extracts from some plants used as livestock dewormers by pastoralist and smallholder farmers in Kenya against Heligrnosomoides polygyrus infection in mice. Vet. Parasitol. 118. 215-226. Where is Githiori 2003a? if none delete letter ‘b’ from 2003b and do same in the text Harbone, J.B., 1973. Phytochemical methods. First edition, Chapman and Hall, London, 14pp. Hashizume, T., A. Hirokawe, S. Aibara, H. Ogawa and A. Kashara, 1978. Pharmacological and historical studies of gastric mucosa lesions induced by serotonin in rats. Arch. de Internationale et Pharmacodyne, 236. 96-108 Hordegen, P., H. Hertzberg, J. Heilmann, W. Langhams and V. Maurer, 2003. The anthelmintic efficacy of five plants products against gastrointestinal trichostrongylids in artificially infected lambs. Vet. Parasitol. 117. 51-60. Ibrahim, M.A., N. Nwude, R.A. Ogunsusi and Y.O. Aliu, 1984. Screening of West African plants for anthelmintic activity. ILCA Bull. 17, 19-22 Addis Ababa Ethiopia Jegede, O.C., O.J. Ajanusi, A.O. Adaudi and R.I.S. Agbede, 2006. Anthelmintic efficacy of extracts of Spigelia anthelmia Linn on experimental Nippostrongylus braziliensis in rats. J. Vet. Sci. 7 (3), 229-232 Kahiya C., S. Mukaratirwa and S.M. Thamsborg, 2003 Effects of Acarcia nilotica and Acacia karoo diets on Haemonchus contortus infection in goats. Vet. parasitol. 115, 265-274. Kahn, L.P and A. Diaz-Hernandez, 2000. Tannins with anthelmintic properties, In: Tannins in Livestock and Human Nutrition: Proc. Int. Conf. (Ed: J.D. Brooker) Australian Centre for International Research, Adelaide, Australia, May 31-June 2 1999, pp. 130-139. Keay,R.W.Y.(1989). Tress of Nigeria. 3rd Edition. Clavendon press oxford. Pp146-216.

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Simon M.K et al: Continental J. Veterinary Sciences 2: 1 - 11, 2008 Kim, Y.K., H.H. Valdivia, E.D.B. Maryon, P. Anderson and R. Coranado, 1992. High molecular weight proteins in nematode C. elegans bind (H) ryanodine and form a large conductance channel. Biophy. J. 63, 1379-1384. Lateef, M., Z. Iqbal, M.N. Khan, M.S. Aktar and A. Jabbar, 2003. Anthelmintic activity of Adhatoda vestica roots. Int. J. Agric .Biol. 5, 86-90 Lahlou, M. (2002). Potential of Origanum Compactum as a cercaricide in Morocco. Ann. Trop. Med. Parasitol. 96, 89-90. Lorke D. 1983. A new approach to practical acute toxicity testing. Arch. Toxicol. .54: 275-287. Min, B.R and S.P. Hart, 2003. Tannins for suppression of internal parasites. J. Anml Sci. 81, E102-E109 Mitcell, L.A., Wescott, R.B., Perryman, L.E. 1983. Kinetics of expulsion of the nematode, Nipposatrongylu braziliensis in mast cell deficient W/W mice. Parasite Immunology 4. 1-12. Onyeyili P.A., J.D. Amin, H.I. Gambo, C.O.Nwosu and G.I. Jibike, 2001. Toxicity and anthelmintic efficacy of ethanolic stem bark extract of Nauchlea latifolia. Nig. Vet. J. 22(1) 74-79. Perry B.D and T.F. Randolph, 1999. Improving the assessment of the economic impact of parasitic diseases and of their control in production animals. Vet. Parasitol. 84: 145-168. Perry. B.D., T.I. Randolph, J.J. McDermoh, K.R. Stones and P.K. Thornton, 2002. Investing in animal health research to alleviate Poverty. International livestock research institute (ILRI). Nairobi. Kenya pp 148. Powers, K.G., I.B. Wood, J. Eckert, T. Gibson and H.J. Smith, 1982. World Association for the Advancement of Veterinary Parasitology (WAAVP). Guidelines for evaluating the efficacy of anthelmintics in ruminants (bovine and ovine). Vet. Parasitol. 10. 265-284. Prasharth, D., M.K. Asha, A. Amit and R. Padmaja, 2001. Anthelmintic activity of Butea monosperma. Fitoterap 72. 421-422. Soulsby E.Y. 1982. Helminths, Arthropods and protozoa of domestic animals 7th ed. FLBS Barrierve Tindal London. 1982. Suleiman, M.M., M. Mamman, Y.O. Aliu and J.O. Ajanusi, 2005. anthelmintic activity of the crude methanolic exract of Xylopia aethiopica against Nipposstrongylus braziliensis in rats Veterinarski arhiv. 75(6), 487-495. Thompson, D.P and T.G. Geary, 1995. The structure and function of helminth surfaces. In: Biochemistry and Molecular Biology of Parasites (J.J. Marr. Ed.), 1st ed,. Academic Press. New York, pp.203-232. Trease, G.E and W.C. Evans, 1983. Pharmacognosy. First edition. Bailliere Tindall, London, pp. 370. Uza, D.V., N.N. Umunna and E.O. Oyedipe, 1996. The productivity of muturu cattle (Bos brachycerus) under the traditional management system. Herd health. Bull. Anim Hlth Prod. Africa 444 151-51. Vencruysse, J., P. Holdsworths, T. Letonja, D. Barth, G. Conder, K. Hamamoto and K. Okano, 2001. International harmonization of anthelmintic efficacy guidelines. Vet. Parasitol. 99. 171-193.

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Simon M.K et al: Continental J. Veterinary Sciences 2: 1 - 11, 2008 Youn, H.J., J. Lakritz D.Y. Kim, G.E. Rothinghars and E.A. Marsh, 2003. Antiprotozol efficacy of medicinal herbs extracts against Toxoplasma gondii and Neospora cannun. Vet. Parasitol. 116. 7-14. Received for Publication: 27/03/2008 Accepted for Publication: 12/06/2008 Corresponding Author: SSiimmoonn MM..KK.. FFeeddeerraall CCooll lleeggee ooff WWiillddll ii ffee MMaannaaggeemmeenntt,, NNeeww--BBuussssaa,, NNiiggeerr SSttaattee

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Continental J. Veterinary Sciences 2: 12 - 21, 2008 ©Wilolud Online Journals, 2008.

AVIAN INFLUENZA: AN OBSTACLE TO POULTRY DEVELOPMENT.

Otuma, M. O and Uchewa, E. N.

Department of Animal science, Ebonyi State University, P. M. B. 053. Abakaliki Nigeria.

ABSTRACT Viral infections replicate in millions so as to out number the antibodies of the host and cause great harm to eventually result to death. More in general, the bird flu outbreaks can be considered as part of the process of global change. Traffic and trade dynamics create conditions for viruses, bacteria and parasites to hitch hike around the world affecting people, animals and ecosystems. Consequently, the best approach to ameliorate the present situation is to maintain good hygienic practices (bio-security), which are the first line of the defense and attack against epidemics of bird flu. All persons working with poultry should avoid bringing in virus (bio-exclusion) and prevent virus existing (bio-containment) if it has already entered a flock, region or village. Avian influenza virus like moist and dirty conditions (where it can get attenuated fast and explode). However, the use of detergents is of paramount importance because the virus is simpler to destroy than many viruses since it is very sensitive to detergents, which destroy the fat containing outer layer of the virus. Key words: Avian influenza, Virus, Epidemics, Hygiene, Poultry and Breeds.

INTRODUCTION Nigeria has the biggest national poultry population in Africa. It is estimated to be 104 million (Federal Livestock Department, 1992) of which only a 10th is of exotic breeds kept in commercial farms, mostly around cities in the Southern parts, and as smaller flocks throughout the country. The rest constitutes village chickens of local breeds kept as free roaming and in backyards. Others are scattered populations and typically are non-descript in type because of indiscriminate inter-breeding (Sonaiya et al.,1999), although they are hardy and well adapted to their local environment. To date, the best estimate of the poultry population in Nigeria is the one obtained during the National Livestock Census undertaken in 1992 (Federal Department of Livestock, 1992). Table 1 summarizes the results of the survey. However, the emergence of the avian flu can be a great obstacle to this poultry population as stated above. The Avian Influenza (AI) is an infectious viral respiratory disease of birds caused by type “A” subtype of the influenza virus (Menn et al. 2005). It was originally termed “fowl plague” and recognized as an infectious disease of birds in chickens in Italy 1878 by Perroncito. Due to a former hot spot in the Italian Upper Po Valley, it was also referred to as “Lombardian disease”. Although Centanni and Savonuzzi, in 1901 identified a filterable agent responsible for causing the disease, it was not before 1955 that Schafer characterized these agents as influenza A viruses (Brahmbhatt, 2005). The aim of this review therefore is to increase farmers knowledge of the epidemic, mode of transmission and its preventive measures. EPIDEMIOLOGY The flu virus appears naturally among birds. Wild migratory aquatic birds such as ducks, geese, gulls and shorebirds are natural carriers of the virus, but are resistant to severe infection from the virus. They are carriers of the full variety of influenza virus A subtypes and thus most probably constitute the natural reservoirs of all influenza A virus (Alice and Edler 2006). While all bird species are thought to be susceptible, some domestic poultry species-chickens, turkey, guinea fowl, quail and pheasants are known to be especially vulnerable to the sequence of infection and can cause very sever consequences.

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Otuma, M. O and Uchewa, E. N: Continental J. Veterinary Sciences 2: 12 - 21, 2008 Avian influenza A viruses generally do not cause disease in their natural hosts. Instead, the viruses remain in an evolutionary stasis i.e. the host and virus seems to exist in a state of a meticulously balanced mutual tolerance, clinically demonstrated by absence of disease but in efficient viral replications (FAO, 2006). AETIOLOGY Avian Influenza Virus (AIV) is a member of the Orthomyxoviridae family. Genus Influenza Virus type A. They are classified as types A, B, and C. The influenza A virus genome comprises eight negative-sense RNA segments. These segments encode ten proteins, two of which are glycoprotein- hemagglutin (HA) and neuraminidase (NA). H5NI is a subtype of the Influenza A virus, with the H5 and N1 subtypes. Subtypes of the influenza A virus are designated as HXNY where H stands for hemagltuinin and N stands for neuraminidase. X is a number that can be one of the 16 types of hemagglutinin and Y is a number that can be 1-9. The most pathogenic subtypes of avian influenza in birds are H5NY of H7NY although both low pathogenic avian influenza (LPAI) and high pathogenic avian influenza (HPAI) exist for the same subtypes (C. Li. et al. 2005). CLINICAL DIAGNOSIS AI is diagnosed in human by isolating the virus from nasal secretions by testing methods. VIRUS THAT CAN CAUSE HPAIV Only virus of the H5 and H7 subtypes are known to cause the highly pathogenic form of disease. However, not all viruses of the H5 and H7 subtypes are highly pathogenic and not all will cause severe disease in poultry. Presently, H5 and H7 are introduced to poultry flocks in their low pathogenic form. When allowed to circulate in poultry populations, the viruses can mutate, usually within a few months, into a highly pathogenic form. This is why the presence of an H5 and H7 viruses in poultry is always a cause for concern, even when the initial signs of infections are mild. SYMPTOMS OF LPAIV IN BIRDS INCLUDE; � Ruffled feathers. � Transient reductions in egg production. � Weight loss combined with a slight respiratory disease. SYMPTOMS OF HPAIV IN BIRDS INCLUDE; � In laying flocks, a cessation of egg production is apparent. � Oedema, visible at feather-free parts of the head. � Cyanosis of comb, wattles and legs. � Greenish diarrhea. � Emaciation. � Sneezing. � Laboured breathing. � Coughing. � Decreased feed intake. Symptoms of highly pathogenic H5NI avian influenza in humans include; � Fever, � Cough, � Sore throat, � Muscle aches, � Eye infections (Conjunctivitis),

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Otuma, M. O and Uchewa, E. N: Continental J. Veterinary Sciences 2: 12 - 21, 2008

� Pneumonia, � Severe respiratory diseases such as acute respiratory distress. The severity of the infection will depend to a large part on the state of the infected person’s immune system and if the victim has been exposed to the strain before. Affected systems of Tracts in humans are: � Respiratory � Urogenital � Nervous � Digestive Incubation period: for birds: 3-7 days; humans: 2-17 days Diffusion rate - Rapid Effects on production � High mortality. � Egg drop. � Retarded growth. � Chronic Respiratory Disease (CRD). MODE OF TRANSMISSION Avian influenza is transmitted by contact with infected birds or surface contaminated with nasal secretions or excretions from infected birds.

• To poultry – exposure of poultry to infected water fowl. • Within a flock- bird to bird by direct contact. • Farm to farm- movement of infected poultry, equipment and people.

Infected birds transmit H5NI through their saliva, nasal secretions, feaces and blood. Other species of animals may become infected with the virus through direct contact with these bodily fluids or through contact with surfaces contaminated with them. H5NI remains infectious after over 30 days at 00C (32.00F) i.e. over one month at freezing temperature or 6 days at 370C (98.60F) which is one week at human body temperature so at ordinary temperatures it will remain in the environment for weeks. Since migratory birds are among the carriers of the highly pathogenic H5N1 virus, it spreads to all parts of the world. H5N1 is different from all previously known highly pathogenic avian flu viruses because of its ability to be spread by animals other than poultry birds. According to Webster and Walker, 2003, transmission in humans is by direct contact with infected poultry, or surfaces and objects contaminated by their feaces. This is presently considered the main route of human infection. To date, most human cases have occurred in rural or periurban areas where many households keep small poultry flocks, which often roam freely, sometimes entering homes or sharing outdoor areas where children use for recreations. As infected birds shed large quantities of virus in their feaces, opportunities for exposure to infected droppings or to the environments contaminated by the virus are abundant under such conditions (FAO, 2004). Moreover, because many households in Asia depend on poultry for income and food, many families sell or slaughter and consume birds when signs of illness appear in a flock, and this practice has proved difficult to change. Exposure is considered most likely during slaughter, defeathering, butchering and preparation of poultry for cooking. However, transmission in humans requires very close contact with an ill person. The strains without asterisk are in the low pathogenic form. In most cases, it causes minor sickness or no noticeable signs of diseases in birds. It is not known to affect humans at all. The only concern about it is that, it is possible for it to be transmitted to poultry and in poultry may become lethal and mutate into a highly pathogenic form.

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Otuma, M. O and Uchewa, E. N: Continental J. Veterinary Sciences 2: 12 - 21, 2008 ENVIRONMENTAL SURVIVAL OF AVIAN FLU Avian flu virus can last indefinitely at a temperature dozens of degrees below freezing point, as is found in the northern most areas of Asia where migratory birds frequent (Jennifer, et. al. 2004). Influenza A viruses can survive:

• On clothes, paper and tissues for 8-12 hours. • On hard non-porous surface such as plastic or stainless steel for 24-48 hours. • Decades in permanently frozen lakes. • 6 days at 370C (98.60F) i.e. one week at human body temperature. • Over 30 days at 00C (32.00F) i.e. over one month at freezing temperature.

Inactivation of the virus occurs under the following conditions:

• Exposure to disinfectants e.g. formalin, iodine compounds. • Acidic conditions pH less than 0.05. • Presence of oxidizing agents such as sodium dodecyl sulfate, lipid solvents, etc. • Ordinary levels of chlorine in tap water kill H5N1 in public water systems. • Heat inactivates H5N1 i.e. the virus therefore, while cooking poultry to 700C (1580F) kills the

H5N1 virus, it is recommended to cook meat to 1650F to kill all food borne pathogens. Generally, to kill avian flu virus, the “World Health organization recommends that environmental surfaces by cleaned by the following; � Bleaching powder 7g/Litre with 70% available chlorine for toilets and bathrooms. � 70% alcohol for smooth surfaces, Table tops, and other surfaces where bleach cannot be used. � Disinfectants such as sodium hypochloride, 1% in use dilution, 5% Solution to be diluted 1:5 in clean water, for materials contaminated with blood and body fluids. PRESENT SITUATION IN NIGERIA In December, 2005, before the emergence of HPAI into Nigeria in February, 2006, the Federal Livestock Department of Nigeria (FDL) constituted a Technical Committee of experts on the prevention and control of HPAI in Nigeria. The committee was mandated to map out strategies for prevention, disease surveillance, networking and contingency plan for an HPAI emergency in Nigeria (Federal Department of Livestock 2005). The Committee also suggested the risk factors that could facilitate the emergence of the disease into the country to include;

a) Through migratory birds, b) Presence of HPAI in Southeast Asia and South Africa and increased trade as well as human

traffic with Nigeria, c) Nigeria’s long and porous borders and informal livestock moment/trading across the borders,

especially at border markets. d) Smuggling/illegal movement of poultry and poultry products into Nigeria from infected

countries and e) Inadequate veterinary quarantine facilities and manpower.

Unfortunately, before the full implementation of the committee’s plan, the disease was reported for the first time in Nigeria. The first outbreak occurred in a commercial poultry layer farm in Jaji, Kaduna State, where ostriches and geese were also kept. About 50,000 birds were affected. It is still unclear if the outbreak has been triggered by migratory birds or by the trade and movement of poultry or poultry products. Subsequent outbreaks happened in farms in Plateau, Katsina, Kano States and the Federal Capital Territory and so many other unconfirmed farms.

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Otuma, M. O and Uchewa, E. N: Continental J. Veterinary Sciences 2: 12 - 21, 2008 Daily Trust Newspaper reported on 21st February (2008), that poultry farmers in Kebbi state have been warned on dangers of absence of bio-security facilities in their farms which could cause outbreak if avian influenza. A survey conducted by an avian influenza control project team in the state showed that most of the poultry farms visited were either lacking proper bio-security or it is completely absent. However, bio-security entails proper sheltered, periodic checks by qualified veterinary doctors and separation of new birds from old ones. In maintaining a healthy poultry farm, these modern facilities must be provided, otherwise the birds could be prone to diseases (Ito, 2001). Risk factors abound in Nigeria that could facilitate the spread and continued presence of HPAI in the country. Some of the identified factors are;

(a) Presence of wet lands where free flying wild birds and domestic ducks visit and rest, which could be the source of the recent emergence of the disease into the country.

(b) Sale and consumption of sick and dead birds. (c) Improper disposal facilities for poultry carcasses. (d) Lack of funding for compensation of livestock/flock owners in the event of slaughter of their

animals for purposes of disease control. (e) Difficulties in the clinical differentiation of HPAI from other epidemic poultry diseases like

Newcastle disease, fowl cholera, Mycoplasmosis, etc. (f) Inadequate earl warning and early reaction capabilities including inadequate experience of

most animal health workers in the recognition and diagnosis of HPAI. (g) The rearing together of poultry flocks of different species and different ages. (h) Lack of registration and licensing of poultry farms/hatcheries and the related establishments

as provided by the law. (i) Lack of organized poultry marketing and existence of open live markets characterized by

interspecies mixing and poor sanitary conditions. (j) Uncontrolled livestock and poultry movement within the country as a result of lack of

enforcement of animal disease control laws and regulations in the country.. (k) Structure of poultry industry in Nigeria consisting predominantly of family poultry with little

or no bio-security, and peri-urban commercial poultry production with minimum to moderate bio-security and constant introduction of new birds from relatively unknown and unverifiable sources.

CONTROL OR PREVENTIVE MEASURES Avian Influenza is a viral disease and no cure has been made except the death of the host. You have to kill the host, burn and bury it. However, some control measures have been carried out generally to prevent lateral spread to other farms or areas. Such measures include;

(1) Quarantining of infected and contact farms. (2) It is pivotal that movement of live poultry and also possibly, poultry products, both within and

between countries are restricted during outbreaks. (3) Rapid culling of all infected or exposed birds. (4) Proper disposal of carcasses. (5) Testing and culling of acutely infected holdings with H5 and H7 subtypes of LPAI in poultry

so as to reduce the risk of a de novo development of HPAIV. (6) For sanitary reasons, wash your hands after handling any poultry.

Specific problems of this eradication or control measures may arises areas;

(i) With a high density of poultry populations (ii) Where small backyard holdings of free roaming poultry prevail.

Due to the close proximity of poultry holding and intertwining structures of the industry, spread of the disease is faster than eradication or control measures (FAO 2003).

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Otuma, M. O and Uchewa, E. N: Continental J. Veterinary Sciences 2: 12 - 21, 2008 ROLE OF VACCINES Two drugs (in the neuraminidase inhibitor class), oseltamivir (commercially known as Tami flu) and Zanamivir (commercially known as Relenza) can reduce the severity and duration of illness caused by seasonal influenza (Timm, et al; 2006). In other words, can sometimes inhibit the influenza virus from spreading inside the user’s body. As regards to the drugs, WHO says that Tami flu’s real effectiveness remains unsure. As for a vaccine, work cannot start on it until the emergence of a new virus, and will take six to nine months to develop it. Therefore for the moment, they cannot by any means count on a potential vaccine to prevent the spread of a contagious influenza virus whose various precedents in the past 90 years have been highly pathogenic. Then Nigeria in 2006, the government under President Olusegun Obasanjo had handled the outbreak very well by banning the importation of poultry products into the country. If not, Nigeria would have been a dumping ground for avian flu infected products. Other measures undertaken include; stamping out, quarantine, disinfection of infected premises/establishments and movement control inside the country. ECONOMIC CONSEQUENCES Outbreaks of highly pathogenic avian influenza can be catastrophic for single farmers and for the poultry industry of an affected region as a whole. Economical losses are usually only partly due to the direct deaths of poultry from HPAI infection (Brahmbhatt, 2005). Measures put to prevent further spread of the disease leaves a heavy toll. Nutritional consequences have been devastating in developing countries where poultry is an important sources of animal and human protein. Once outbreaks have become widespread, control is difficult to achieve and may take several years (WHO 2005, WHO 2006). REFERENCES Alice, A and Edler, M.O (2006). Avian flu (H5NI): Its epidemiology, prevention and implications for anesthesiology. Journal of Clinical Anesthesia 18(1): 1-4. Brahmbhatt, M. (2005). Avian and Human Pandemic Influenza- Economic and Social Impacts. World Bank, November 2005,www.who.int/mediacentre/events/2005/World-Bank-milan-Brahmbhattv2.pdf. C. Li., K.Yu, G. TiaG, D Yu, L. Liu, B Jing, J. Ping, H. Chen (2005). “Evolution of H9N2 influenza viruses from domestic poultry in mainland China”. Virology 340. Daily Trust Newspaper. (2008). Report on Avian Influenza. 21st February 2008 FAO (2003); Prevention and Control of the Avian flu in the small poultry farms, Hanoi; 2003. FAO (2004); Special Issue: Avian Influenza: EMPRES, NO 25, 2004. FAO (2006); Update on the Avian Influenza situation as of 23/01/2006. FAOAIDE News, Issue No. 39. Federal Department of Livestock (1992): Nigerian Livestock Resources Survey. Resource Inventory and Management Limited, Jersey, UK. Federal Livestock Department, (1992). National Livestock Census. Federal Livestock Department, Nigeria,. Federal Department of Livestock (2005): Highly pathogenic avian influenza (HPAI) in Nigeria: Strategies for prevention of introduction, disease surveillance networking and contingency plan for disease

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Otuma, M. O and Uchewa, E. N: Continental J. Veterinary Sciences 2: 12 - 21, 2008 emergency. Report of the Technical Committee of Experts on the Prevention and Control of HPAI in Nigeria. Federal Department of Livestock and Pest Control Services, Abuja Nigeria. Food and Agricultural Organization of the United Nations (2005). Wild Birds and Avian Influenza. Retrieved on 2006-04-18. Ito, T. (2001). Generation of a Highly Pathogenic Avian Influenza A Virus from an A-virulent field isolate by passaging on Chickens. Journal of Virology, May 2001, 75(9):4439-4443. Jennifer, C., Hess, D.V.M., Jean, A. and Pare, D.M.V (2004). Viruses of Waterfowl. Seminar on Avian and Exotic pet medicine volume 13. Issues 4:176-183. Menn, D.J and Tran, T.H. (2005): Avian Influenza A (H5NI) Review: Journal of Clinical Virology in Press. 2005. Sonaiya, E.B., Brankaert, R.D.S. and Gueye, E.F. (1999): Research and Development Options for family Poultry. Introductory paper to the first INFPD/FAO Electronic Conference on family poultry “The Scope and Effect of family poultry Research and Development (7 December 1998-5 March 1999)”. Swayne, D.E. and King, D.J. (2003): Zoonosis update: Avian influenza and Newcastle disease, Vet. Med. Today. JAVMA 222 (11) 1534-1540. The Prevention and treatment of viral respiratory disorders. Retrieved on 2007-09. Thomas, M.E. et al (2005). Risk factors for the introduction of high pathogenicity Avian Influenza virus into poultry farms during the epidemic in the Netherlands in 2003. Timm, C., Harder and Werner, O. (2006). Avian Influenza. In: Influenza Report, eds. B.S. Kamps et al; Flying Publisher, Paris 2006. Webster, R.G., and Walker, E.J. (2003). “The world is teetering on the edge of a pandemic that could kill a large fraction of the human population” American Scientist 91(2): 122, doi: 10. 1511/2003.2.122. World Health Organization (2005); Avian Influenza A (H5NI). Weekly Epidemiology Rev 2004; 79:65-70. World Health Organization (2006) Avian Influenza assessing the pandemic threat. http:www.who.int/csr/disease/influenza/WHO-CDS-2005_29/2n, accessed 06 January 2006. Table 1: National Poultry Population Estimates, Nigeria Species Rural area Urban Total Chickens 68,244,195 4,156,161 72,400,856 Ducks 11,220,461 573,507 11,793,968 Guinea fowls 4,621,670 58,237 4,679,907 Pigeons 13,566,775 1,593,091 15,159,866 Turkeys 207,219 16,144 223,363 All Poultry 97,860,320 6,397,640 104,257,960 Source: Federal Livestock Department, Nigeria, (1992).

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Otuma, M. O and Uchewa, E. N: Continental J. Veterinary Sciences 2: 12 - 21, 2008 Table 2: Documental Human Infections with A.I. Viruses Date Country/Area Strain Cases Symptoms Source (Deaths) 1959 USA H7H7** 1 Respiratory Overseas travel 1995 UK H7H7 1 Conjunctivitis Pet ducks (shared lake with migratory birds) 1997 Hong Kong H5N1** 18(6) Respiratory/pneumonia Poultry 1998 China (Guangdong) H9N2 5 Unknown Unknown 1999 Hong Kong H9N2 2 Respiratory Poultry, Unknown Feb Hong Kong H5N1** 2(1) respiratory Unknown 2003 Mar. Netherlands H7N7** 89(1) Conjunctivitis (Pneumonia, Poultry 2003 respiratory in-sufficiency in fatal case) Dec.2003 Hong Kong H9N2 1 Respiratory Unknown 2003 New York H7N2 1 Respiratory Unknown 2003 Vietnam H5N1** 3(3) Respiratory Poultry 2004 Vietnam H5N1** 29(20) Respiratory Poultry 2004 Vietnam H5N1** 17(12) Respiratory Poultry 2004 Canada H7N3** 2 Conjunctivitis Poultry 2005 Vietnam H5N1** 61(19) Respiratory Poultry 2005 Thailand H5N1** 5(2) Respiratory Poultry 2005 China H5N1** 7(3) Respiratory Poultry 2005 Cambodia H5N1** 4(4) Respiratory Poultry 2005 Indonesia H5N1** 16(11) Respiratory Poultry 2006 Turkey H5N1** 3(3) Respiratory Poultry Source: WHO, (2006). ** Highly pathogenic for poultry. The strains without asterisk are in low pathogenic form. In most cases, it causes minor sickness or no noticeable signs of diseases in birds. It is known to affect humans at all. The only concern about it is that, it is possible for it to be transmitted to poultry may become lethal and mutate into a high pathogenic form.

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Otuma, M. O and Uchewa, E. N: Continental J. Veterinary Sciences 2: 12 - 21, 2008

Table 3: Confirmed human cases and mortality rate of avian influenza (H5N1) As of February 28, 2008.

Report dates Country 2003 2004 2005 2006 2007 2008 Total Azerbaijan 8 5 63% 8 5 63%

Cambodia 4 4 100% 2 2 100% 1 1 100 7 7 100%

PR China 1 1 100% 8 5 63% 13 8 62% 5 3 60% 3 3 100% 30 20 67%

Djibouti 1 0 0% 1 0 0%

Egypt 18 10 56% 25 9 36% 1 0 0% 44 19 43%

Indonesia 20 13 65% 55 45 82% 42 37 88% 12 10 83% 129 105 81%

Iraq 3 2 67% 3 2 67%

Laos 2 2 100%

Myanmar 1 0 0%

Nigeria 1 1 100%

Pakistan 1 1 100%

Thailand 17 12 71% 5 2 40% 3 3 100% 25 17 68%

Turkey 12 4 33% 12 4 33%

Vietnam 3 3 100% 29 20 69% 61 19 31% 8 5 63% 4 4 100% 105 51 49%

Source: World Health Organization Communicable Disease Surveillance and Response (CSR) (http://www.who.int/csr/disease/avianinfluenza/country/en).

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Otuma, M. O and Uchewa, E. N: Continental J. Veterinary Sciences 2: 12 - 21, 2008 Table 4

Key Form and final concentration Contact time and notes 1. Soaps and detergents Leave in contact. 10 minutes 2. Oxidizing agents 2a. Sodium hypochlorite Liquid, dilute to final 2-3% Not good for organic materials. 10-30 Available chlorine (20g/litre minutes contact Powder, 30g/L solid 3. Alkalis 3a. Sodium hydroxide (caustic soda) 2% (-20g/Litre) 10 minutes. Do not use in presence of NaOH) do not use with aluminum Aluminum And like alloys. 3b. Sodium carbonate anhydrous 4% (=40g/Litre) from powder 10 minutes. Recommended for use in (Washing soda) NaCO3 10H2O 100g/L from crystals presence of organic materials as above 30 minutes 4. Acids 4a. Hydrochloric 2% (20ml/litre) Corrosive, use only when better not available 4b. Citric 0.2% (2g/litre) 30 minutes, safe for clothes and body decontamination. 5. Formaldehyde gas Special generation required 15-24hrs. Toxic, only if others cannot be used. Source: AUSVETPLAN Operational Procedures Manual, Decontamination-http://www.aahc.com.au/ausvetplan/decfnl2.pdf) Received for Publication: 03/07/2008 Accepted for Publication: 25/08/2008 Corresponding Author: Uchewa, E. N. Department of Animal science, Ebonyi State University, P. M. B. 053. Abakaliki Nigeria. Email: euchewa@yahoo.co.uk

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Continental J. Veterinary Sciences 2: 22 - 26, 2008 ©Wilolud Online Journals, 2008.

EFFECT OF ENZYME SUPPLEMENTATION ON FEED INTAKE, GROWTH RATE AND EFFICIENCY OF FEED CONVERSION IN BROILER BIRDS FED SINGLE PHASE AD-LIBITUM

A. AREMU1, M.N. HARUNA2, M.E. GAWU3 and M. Z. SHABA4

1School of Agriculture and Agricultural Technology, Department of Animal Production, Federal University of Technology, Minna.

2, 3 and 4Livestock Department, Ministry of Agriculture, Minna, Niger State.

ABSTRACT An eight-week trial was conducted to study the effect of enzyme supplementation on feed intake, growth rate and efficiency of feed conversion in diets of broiler birds fed single phase ad-libitum. One hundred and forty four day old broiler chicks were randomly allotted to four different treatment groups with three replicates per treatment. Four (4) rations were formulated at different enzyme inclusion rate of the recommended levels of 0%, 50%, (1.2g), 75% (1.88g) and 100% (2.5g) and were tagged T1, T2, T3 and T4 respectively. The chicks were fed the diets for a period of eight weeks. Data on body weight gain, feed intake and efficiency of feed conversion were taken. Data collected were analysed using analysis of variance (ANOVA) while means were separated using Duncan multiple range test. The result showed that birds on enzyme supplemented diets had better weight gain than those on the control diet (T1). This difference was significant (P<0.05). Birds on 50 percent, 75 percent and 100 percent enzyme supplementation had 5 percent, 5.10 percent and 8.77 percent weight gain more than those on the control diet respectively. Feed intake was highest for birds on 50 percent enzyme supplementation with 13.54 percent over birds on the control diet and this was significant (P<0.05). Data on efficiency of feed conversion showed that birds on 50 percent enzyme supplementation recorded the best value of 1.62 compared to birds on the control diet with a value of 1.50 and these were also significantly different (P<0.05). Corn based diets supplemented with enzymes can be used in poultry to improve their performance. KEY WORDS Enzyme supplementation, feed, growth parameters, broiler birds, single phase ad-libitum.

INTRODUCTION Advances in the field of enzyme technology have brought benefits to the pig and poultry industries for almost 10 years. By adding enzymes to feed, the majority of problems traditionally associated with cereal-based rations can be alleviated. Use of enzymes to target feeds such as corn or sorghum and containing soy bean meal is a more recent concept. In corn and sorghum, as with the grain-based diets, xylase enzymes are effective in degrading the fibrous cell walls of feed grains, releasing nutrients previously inaccessible to the animal. Furthermore, recent studies have revealed that in a normal bird, starch digestion may not be completed (Acamovic, 2001 and Chesson, 2001). Just like xylanase, proteases also contribute to the digestive process. The subtilism protease is highly active and can degrade soy protein, namely the storage proteins, and the soybean anti- nutritional factor such as trypsin inhibitors, lectins and antigenic proteins. Although the application of feed enzymes to improve the nutritive value of sorghum and corn soy bean meal diets is still in the developmental stage, such enzymes that can improve the performance of diets

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A. AREMU et al: Continental J. Veterinary Sciences 2: 22 - 26, 2008 based on corn and soy bean meal can offer broiler producers cheaper alternative, without compromising on bird performance. Cost saving can be made by practical application of enzymes to feed formulations. The two different methods of enzyme supplementation include:- (1) the simple over the top addition to an existing formulation to improve the broiler performance cost efficiency. (2) the second option is to change the feed formulation to reduce the cost of feed. Although this would normally lead to a reduction in performance, the addition of enzymes improves the value of the feed, resulting in a similar performance to the normal, more expensive, feed formulation. Depending on normal feedstuff prices, this can give a net saving of approximately N360 - N600 ($3 – 5) per ton of feed. Developments in feed enzyme technology may be the key to maintaining future profitability. It is against this background that this work was designed to evaluate the effect of enzyme supplementation on feed intake, growth rate, and efficiency of feed conversion in broiler birds fed single phase ad-libitum. MATERIALS AND METHODS Location of experiment and source of enzyme The experiment was carried out at the Teaching and Research farm of the School of Agriculture and Agricultural technology, Federal University of Technology, Minna, Niger State, between the months of June and October 2006. The enzyme (Nutraser xyla) was obtained from the Department of Animal Science of the University of Ilorin, Ilorin.

Experimental Diet. Prior to the arrival of the birds, four experimental diets which were isonitrogenous and iso-caloric (20% CP and 2800 Kcal/kg ME) and designated T1 to T4 were compounded. They contained enzyme at inclusion levels of 0% (o.g), 50% (1.2g), 75% (1.88g) and 100% (2.5g) of the recommended value respectively. Feed compounding was carried out in Feed Mill unit of the University Teaching and Research farm. Feeds compounded were fed single phase ad-libitum to the birds. Experimental birds and their Management At the arrival of the birds they were randomly distributed into four treatment groups each comprising of three replicates. The replicates contained 12 birds each which were weighed on arrival and subsequently weekly weights were recorded. Drinking water and feed were provided ad-libitum. Also the temperature of the brooding house was maintained at optimum level. Adequate medication was provided for the birds while feed intake, body weight were determined weekly accordingly. The efficiency of feed conversion was also calculated. Samples of the diets were collected from each treatment group for proximate analysis. The experiment was subjected to completely randomized design while data collected were subjected to analysis of variance (ANOVA). Means were separated using Duncan multiple range test (Duncan 1955).

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A. AREMU et al: Continental J. Veterinary Sciences 2: 22 - 26, 2008 Table 1 Composition of experimental diets supplemented with varying levels of enzyme (%) fed single phase to broiler birds. _____________________________________________________________________ Treatment diets (%) _________________________________________________________________ T1 T2 T3 T4 Ingredients _________________________________________________________________ Maize 55.43 55.43 55.43 55.43 Groundnut cake 21.57 21.57 21.57 21.57 Fish meal 7.00 7.00 7.00 7.00 Rice husk 9.00 9.00 9.00 9.00 Lime stone 4.00 4.00 4.00 4.00 Bone meal 2.00 2.00 2.00 2.00 Methionine 0.25 0.25 0.25 0.25 Lysine 0.25 0.25 0.25 0.25 Vitamin/min Premix 0.25 0.25 0.25 0.25 Salt 0.25 0.25 0.25 0.25 Total 100.00 100.00 100.00 100.00

T1 = control diet with 0% enzyme inclusion level T2 = diet with 5% (1.20g) enzyme inclusion level T3 = diet with 75% (1.88g) enzyme inclusion level T4 = diet with 100% (2.50g) enzyme inclusion level Calculated T1 – T4

Energy level = 2798.76 Kcal/kg ME Protein level = 20.15% Formulated T1 – T4

Energy level = 2800 Kcal/kg Protein level = 20.00%.

Table 2 proximate composition of experimental diets supplemented with varying levels of enzyme (%) fed single phase to broiler birds _____________________________________________________________________ Treatment diets (%)

T1 T2 T3 T4

_____________________________________________________________________ Nutrients (%)

Dry matter 94.97 95.27 95.23 95.42 Crude protein 20.40 20.02 20.24 20.32 Ash 4.51 4.63 4.57 4.70 Crude fibre 6.50 5.66 5.72 5.54 Ether extract 4.98 4.88 4.87 4.79 Nitrogen free extract 58.58 59.90 59.83 60.07 Metabolizable energy Kcal/kg ME 2830 2840 2840 2840

T1 – T4 = same diets as in Table.

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A. AREMU et al: Continental J. Veterinary Sciences 2: 22 - 26, 2008 Table 3 Growth performance of broiler chicks fed single phase on diets supplemented with varying levels of enzyme (%). _____________________________________________________________________ Treatment diets (%)

T1 T2 T3 T4 SEM

Parameters__________________________________________________________________ Initial body weight/ bird (g) 49.68d 47.99c 46.83a 46.97b 0.29 Final body weight/ bird (g) 1130.33a 1182.73b 1215.00c 1222.40d 9.37 Final body weight over and above T1 0% 4.64% 7.49% 8.15% Weight gain per bird (g) 1080.65a 1134.74b 1168.17c 1175.43d 9.66 Weight gain over and above T1 0% 5.00% 8.10% 8.77% Feed intake per bird (g) 1618.77a 1837.92d 1794.43b 1803.28c 21.99 Feed intake over and above T1 0% 13.54% 10.85% 11.40% Efficiency of feed conversion 1.50a 1.62d 1.54c 1.53b 0.01 Efficiency of feed Conversion over and above T1 0% 8.00% 2.67% 2.00%

T1 – T4 = same as diets as in Table 1 abcd = means not having the same super script along the row are statistically significant (P<0.05).

RESULTS AND DISCUSSION Table 1 shows the composition of the experiment diets supplemented with varying levels of enzyme. The diets were isonitrogenous and isocaloric. Table 2 on the other hand shows the proximate analysis of the four experimental diets. The analysis shows that the diets were similar in nutrient values in all respects. Table 3 shows the growth performance of the broiler chicks fed the experimental diets. Birds on enzyme supplemented diets had better weight gain than those on the control diet (T1). This difference was significant (P<0.05). Birds on 50 percent, 75 percent and 100 percent enzyme supplementation had 5 percent, 8.10 percent and 8.77 percent weight gain more than those on the control diet respectively. Feed intake was highest for birds on 50 percent enzyme supplementation with 13.54 percent over birds on the control diet this was followed by birds on 100 percent enzyme supplementation with 11.40 percent over the birds on the control diet. The least value of feed intake by birds was recorded by birds with 75 percent enzyme supplementation. This differences were also significant (P<0.05). The efficiency of feed conversion shows that birds on 50 percent enzyme supplementation recorded the best value of 1.62 compared to birds on the control diet with a value of 1.50. This was followed closely by birds on 75 percent enzyme supplementation with a value of 1.54 while birds on 100 percent enzyme supplementation recorded a value of 1.53. These values were also significant (P<0.05). The result for weight gain is in agreement with the works of Marquard et al (1994), who reported that enzyme supplementation of cereal based diets significantly improved chicks performance by increasing the rate of gain. The result is also in agreement with that of Al-Bustany (1996) who reported that enzyme supplementation of mash diets also tended to have more pronounced effect on

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A. AREMU et al: Continental J. Veterinary Sciences 2: 22 - 26, 2008 weight gain than supplementation of pelleted diets. Al-Bustany (1996) further stated that enzyme supplementation significantly increased live weight of chicks by 7.8 percent. This could be observed in 75 percent and 100 percent enzyme supplemented diets with values of 7.49 percent and 8.15 percent over birds on the control diet respectively. The result of feed intake is in consonance with that of the National Research Council (1994) that studied the effects of a local sunflower meal diet containing 19.75 percent crude fibre supplemented with enzyme, on growth performance and feed intake of broilers, raised to market age (42 days) on floor pen. The observation was that high fibre content can be successfully included in the diet of broilers fed in mash form up to market age to give increase in growth rate and feed intake. This study revealed that birds on enzyme supplemented diets had above 10 percent feed intake over those on the control diet. The result of efficiency of feed conversion in this experiment agrees with the findings of Marquard (1994), who reported that enzyme supplementation improved the feed conversion ratio and feed to gain ratio of birds. Patrick and Schaible (1980) also reported that the beneficial effects of enzyme treatment include, improvement in growth, feed utilization and litter conditions of chicks. CONCLUSION This experiment shows that corn based diets supplemented with enzyme improved feed intake, body weight gain and efficiency of feed conversion over and above those chicks whose diets were not supplemented. This implies that enzyme can be used in poultry diets at various recommended inclusion level to improve the performance of the birds. REFERENCES Acamovic T, (2001). Commercial application of enzyme technology for poultry production. World’s Poultry Science Journal 57 (3): 225 – 242. Al- Bustany .Z. (1996). The effect of pelleting on enzyme supplemented barley based broiler diet. Animal Feed Science and Technology. 58(3&4), 283 – 284. Chesson . A, (2001). Non – Starch Polysaccharide degrading enzymes in poultry diets: Influence of ingredients on the selection of activities*. World’s Poultry Science Journal 57 (3): 251 – 263. Duncan, D.B. (1955). Multiple range and multiple R. tests. Biometrics 11: 1-42. Marquard, R. R., Boros .D., Guenter, W. and Crow, G. (1994). The nutritive value of barley, rye, wheat and corn for chicks as affected by the use of atrichoderma reesei enzyme preparation. Animal Feed Science Technology 45 : 363 – 378. National Research Council (1994). Nutrition requirements of poultry 9th revised edition. National Academy of Science, National Academy Press. Washington D.C. Patrick, K. H. and Schaible, P. J. (1980). Poultry feeds and nutrition 2nd edition. Avi. Publishing company Inc. Westport, Connecticut. Pp 23 – 73.

Received for Publication: 03/09/2008 Accepted for Publication: 25/10/2008 Corresponding Author: A. AREMU School of Agriculture and Agricultural Technology, Department of Animal Production, Federal University of Technology, Minna.

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Continental J. Veterinary Sciences 2: 27 - 32, 2008 ©Wilolud Online Journals, 2008.

NUTRIENT DIGESTIBILITY, HAEMATOLOGY AND CARCASS EVALUATION OF INDIGENOUS WILD GUINEA FOWL (NUMIDA MELEAGRIS GALEATA PALLAS) FED GRADED LEVELS OF PROTEIN UNDER

INTENSIVE MANAGEMENT.

Kudu, Y.S., Egena, S.S.A., Ayanwale, B.A. and Alabi, J.O. Department of Animal Production, Federal University of Technology, P.M.B. 65, Minna, Niger State, Nigeria.

ABSTRACT Nutrient digestibility, haematology and the carcass of indigenous wild guinea fowl reared under intensive management were evaluated for 20 weeks. The guinea fowl keets were randomly allotted to 4 treatments groups (designated as T1, T2, T3 and T4) of 2 replicates each. They were fed on a common starter diet containing 24%CP during the brooding period which lasted 8 weeks. After 8 weeks, the birds were fed diets containing 18%, 22%, 24% and 26%CP representing the various treatments. The keets fed the 24%CP diet were used as the control after the brooding period. Results show that the treatment significantly affected (p<0.05) dry matter (DM), crude protein (CP) and ether extract (EE) digestibility while crude fibre (CF) and Nitrogen free extract (NFE) digestibility were not affected (p>0.05) at the end of the experiment. The haematological indices measured were not affected (p>0.05) by the treatments. Evaluation of the cut-up parts of the carcass showed that the neck, drumstick, thigh, leg and head were not significantly affected (p>0.05) while the live weight, slaughtered weight, dressed weight, back, wing and breast were affected (p<0.05) by the treatment. It was concluded that feeding indigenous wild guinea fowls kept under intensive management graded levels of protein led to marked differences in their ability to utilize nutrients, their blood constitution as well as their carcass quality. KEYWORDS: Nutrient digestibility, haematology, carcass, guinea fowl, graded level of protein, intensive management.

INTRODUCTION Guinea fowl usually obtained from the wild are cherished and widely eaten by Nigerians mainly because of the distinctive flavour of both its meat and eggs (Ayeni and Ayanda, 1982; Okaeme, 1982). It is indigenous to West Africa mostly found north of the equatorial forest where they occupy the guinea savanna region. With an estimated population of 43 million in captivity in Nigeria (Ayeni, 1980) it represents a great potential as a source of meat and egg especially as the nation strive towards a speedy bridging of the protein deficiency inherent in its population. The bird is socially accepted and there is no religious taboo against its consumption. Its meat has a higher protein content (about 28%) compared to the 20% of the domestic fowl (Ayeni, 1980). Guinea fowls are mostly kept under semi-intensive management. Ayeni (1980) opined that keets managed this way grow best on 20-24%CP diet and that it could be further reduced to 18%CP from the 8th week of age. Rearing guinea fowl commercially under semi-intensive management however exposes the keets to a lot of hazards. The intensive production of guinea fowls in Nigeria which is just beginning is likely to be accelerated as the birds’ potential as an easy and quick grown source of meat and egg becomes more fully realized. Research into its biology and performance will provide opportunities for an increase in the commercial production of the bird (Ayorinde and Ayeni, 1983; Ayorinde and Okaeme, 1984). Most of the works carried out using guinea fowls are in the southern part of the country. There is a dearth of information therefore on how the birds will react to confinement in other parts of the country. This study was undertaken therefore to investigate the effect of feeding graded levels of protein on nutrient digestibility, haematology and carcass quality of indigenous wild guinea fowls reared under intensive management.

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Kudu, Y.S et al: Continental J. Veterinary Sciences 2: 27 - 32, 2008 MATERIALS AND METHODS The experiment which lasted 20 weeks was conducted in the poultry unit of the Department of Animal Production, School of Agriculture and Agricultural Technology of the Federal university of Technology Minna, Niger State, Nigeria. Minna lies within the southern guinea savanna area of Nigeria with an average annual rainfall of 1200mm. Table 1: Dietary composition of experimental feed (%).

Starter diet Finisher diet

T1 T2 T3 T4

Ingredients 24 18 22 24 26

Maize 47.71 69.93 53.11 47.71 42.33

GNC 34.54 18.32 29.14 34.54 39.92

Rice bran 5.00 5.00 5.00 5.00 5.00

Fish meal 1.00 1.00 1.00 1.00 1.00

Blood meal 5.00 5.00 5.00 5.00 5.00

Oyster shell 2.50 2.50 2.50 2.50 2.50

Bone meal 3.50 3.50 3.50 3.50 3.50

Salt 0.50 0.50 0.50 0.50 0.50

Premix 0.25 0.25 0.25 0.25 0.25

Total 100.00 100.00 100.00 100.00 100.00 CP% 24.00 18.00 2.00 24.00 26.00 Energy (Kcal/kg) 3,100 3,100 3,100 3,100 3,100 Table 2: Average feed intake, body weight, body weight gain and feed Conversion efficiency of indigenous wild guinea fowl keets during brooding on starter diet. Average feed Average body Average body Feed conversion Weeks intake (g) weight (g) weight gain (g) (%) 1 21.74 44.20 11.40 1.91 2 39.51 56.10 11.90 3.32 3 57.63 78.60 22.50 2.56 4 79.60 116.00 37.40 2.13 5 88.40 166.60 50.60 1.74 6 81.94 195.83 29.60 2.77 7 119.72 230.99 35.16 3.41 8 165.67 268.87 37.67 4.40 Source of feed and experimental diet All the materials used for the experiment were sourced locally. These include: maize, groundnut cake (GNC), fish meal, bone meal, oyster shell; premixes were obtained from Pfizer feed. Four different isocaloric diets were formulated using these ingredients. These are a starter diet (with 24%CP) fed to all the keets from day old to 8 weeks and finisher diets made up of 18%, 22%, 24% and 26%CP respectively. The graded protein levels represent the different treatments. The compositions of the experimental diets are as shown in Table 1.

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Kudu, Y.S et al: Continental J. Veterinary Sciences 2: 27 - 32, 2008 Housing The birds were raised on deep litter. At 8 weeks, the birds were divided into 4 treatments with 2 replicates each in a completely randomized design. Prior to the arrival of the birds, the pens were cleaned, washed, disinfected and the floor covered with wood shavings. 60 watts bulbs were used to provide heat during brooding and subsequently as a source of light through out the remaining part of the trial. Chick feeders and drinkers were used for the first 4 weeks and thereafter changed to bigger ones. The partitioning of the pens was raised up to roof level using wire mesh because of the flighty nature of the wild guinea fowl. Feed and water were supplied ad libitum and appropriate medications given as of when due. Table 3: Apparent nutrient digestibility by indigenous wild guinea fowls fed graded levels of protein. Dietary protein levels (%)

T1 T2 T3 T4

Parameters 18 22 24 26 SD

Dry matter 54.33c 59.24ab 62.68b 71.25a 6.76*

Crude protein 30.69c 38.09c 47.16b 61.06a 2.32*

Crude fibre 48.75 50.55 57.76 52.43 4.74ns

Ether extract 92.33b 94.42a 93.81ab 95.08a 1.17*

Nitrogen free extract 97.99 94.33 98.46 96.82 2.92ns

Means denoted by different alphabets along the same row are significantly different (p<0.05), ns: not significant (p>0.05), SD: Standard deviation. Table 4: Haematology values of indigenous wild guinea fowls fed graded levels of protein. Dietary protein levels (%)

T1 T2 T3 T4

Parameters 18 22 24 26 SD

Total protein (g/100ml) 5.44 4.69 4.58 4.78 0.39ns

Albumin (g/100ml) 2.69 2.36 2.63 2.63 0.15ns

Globulin (g/100ml) 2.75 2.33 1.95 2.15 0.34ns

Glucose (g/100ml) 330.47 317.13 301.28 329.38 31.60ns

ns: not significant (p>0.05), SD: Standard deviation. Digestibility trial Two birds were selected from each treatment and used to carry out digestibility trial. Samples of faeces were collected from the birds in the metabolic cages after a 5 days adjustment period and stored in the refrigerator. These were later dried at 650C until a constant weight was achieved and used for laboratory analysis to ascertain the level of nutrient utilization by the birds. Blood analysis Blood sample (5 ml ) was collected via the wing veins and used for haematological study. Glucose content was analyzed using the method of Jain (1986) while blood analyzer (model 6300 Ames Company USA) was used for the determination of plasma protein and albumin.

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Kudu, Y.S et al: Continental J. Veterinary Sciences 2: 27 - 32, 2008 Carcass analysis Two birds from each treatment were selected, slaughtered by severing the jugular vein, bled and used for carcass analysis. Statistical analysis Data collected during the study were statistically analyzed by the method of Steel and Torrie (1980) and means separated where significant differences exist by the method of Duncan (1955). Table 5: Effect of feeding graded levels of protein on the cut-up parts of indigenous wild guinea. Dietary protein levels (%)

T1 T2 T3 T4

Parameters 18 22 24 26 SD

Live weight (g) 566.0c 672.5a 695.0b 781.0a 88.45*

Percentages of live weight

Slaughtered weight 97.69a 97.32a 96.69b 97.79a 0.56*

Dressed weight 73.32b 76.25a 77.33a 75.15a 1.71*

Back 13.84c 14.43b 16.72a 15.54a 1.27*

Drumstick 9.12 9.48 9.60 11.31 0.95ns

Neck 4.77 4.93 5.13 4.25 0.38ns

Wing 11.75a 11.80a 10.93b 11.92a 0.45*

Breast 19.06c 22.65b 24.52a 22.44b 2.25*

Thigh 11.08 11.64 11.75 12.06 0.41ns

Legs 3.75 3.33 3.16 3.03 0.31ns

Head 4.28 3.53 3.40 3.44 s0.42ns

Means denoted by different alphabets along the same row are significantly different (p<0.05). ns: not significant (p>0.05), SD: Standard deviation.

RESULTS AND DISCUSSION Table 2 shows the general performance of the guinea fowl keets from day old to 8 weeks of brooding. It shows that feed intake, body weight and body weight gain of the keets increased with age. The result of the feed conversion efficiency did not follow any specific trend. Table 3 shows the apparent nutrient digestibility of the diets fed the birds. Dry matter, crude protein and ether extract digestibility were significantly elevated (p<0.05) as a result of feeding graded levels of protein to the wild guinea fowls. In all the parameters measured except Nitrogen free extract, the guinea fowls fed 18%CP diet had lower values compared to those fed the 22%, 24% and 26%CP diets. The lower value observed (the 18%CP diet) especially for dry matter digestibility is at variance with the findings of Obioha and Okonkwo (1983) who reported that dry matter digestibility is increased when lower levels of protein are fed to guinea fowls. This work shows that a direct relationship possibly exist between protein level and nutrient digestibility. All the birds were able to utilize the carbohydrate component of the diets to the same degree hence the non significant (p>0.05) nature of NFE digestibility. This agrees with Vogt and Stute (1994) who in their comparative study of the guinea fowl, found out that the birds utilize Nitrogen free extract and lignin components of feed better than the domestic fowl. The result obtained for ether extract and crude protein digestibility however agrees with the findings of Agwunubi (1984).

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Kudu, Y.S et al: Continental J. Veterinary Sciences 2: 27 - 32, 2008 The result of the blood analysis is presented in Table 4. No significant effect (p>0.05) was noticed as a result of the feeding of graded levels of protein to the guinea fowls. The low serum total protein observed for birds fed 22%, 24% and 26%CP is indicative of the fact that dietary protein is better utilized at higher levels by guinea fowls. The range of value obtained in this study is not too different to that reported by Olowokurum et al. (1983) although in their own work, no feeding regime was used. The almost similar values observed for glucose reflects the similarity in the energy portion of the diets. Table 5 shows the cut-up parts expressed as a percentage of live weight. The increase in the proportion of the cut-up parts obtained from birds fed 22%, 24% and 26%CP diets could be attributed to the tendency of the body parts to grow in proportion to body weights of the birds. The results shows that as the protein level increased in the diets, there was an increase in the ability of the guinea fowls to retain more of it in the form of muscle. This might be the reason why better weights were observed for the cut-up parts of guinea fowls fed 26%CP diet compared to those in the other treatment groups. The guinea fowls on slaughter in this trial had a dressing percentage of between 73.32-77.33% of edible parts which quite agrees with the range of 50-80% posited by Ayeni (1980). CONCLUSION The result of the study showed that indigenous wild guinea fowls respond to different levels of protein and the respond is positively related to the level of protein fed. The guinea fowls fed the 26%CP diet performed better in most of the parameters measured. REFERENCES Agwunubi, L.N., 1984. Comparison of protein and energy requirements of broiler guinea fowl and chicken. Post graduate seminar, Department of Animal Science, University of Ibadan, Nigeria. Ayeni, J.S.O., 1980. The biology and utilization of the helmet guinea fowl (N.m.galeata pallas) in Nigeria. PhD thesis, University of Ibadan, Nigeria. Ayeni, J.S.O. and J.O. Ayanda, 1982. Studies of the husbandry practices and social acceptance of guinea fowl in Nigeria. Bull. Anim. Health and Prod. Afr. 30(2): 139-148. Ayorinde, K.L. and J.S.O. Ayeni, 1983. Comparison of the performance of different varieties of indigenous guinea fowl (N. m galeata) and imported stock (N. meleagris) in Nigeria. KLRI Annual Report. Pp: 170-182. Ayorinde, K.L. and A.N. Okaeme, 1984. All year guinea fowl-how feasible? African Farming and Food Processing. March/April. Pp: 21-22. Duncan, D.B., 1955. Multiple range and multiple F-test. Biometrics 11: 1-42. Jain, N.C., 1986. Schalm’s veterinary haematology. 4th edition, Lea and Febiger, Philadelphia. Obioha, F.C. and I.U. Okonkwo, 1983. Energy and protein requirements of guinea fowls. In: Ayeni, J.S.O., Aire, T.A. and Olomu, J.M. (Eds.), The helmet guinea fowl (N. m. galeata pallas) in Nigeria. Pp: 129-136. Okaeme, A.N., 1982. Guinea fowl production in Nigeria. World Poultry Science Journal (38): 36-39. Olowokurum, M.O., Makinde, M., Aire, T.A. and J.S.O. Ayeni, 1983. Guinea fowl compared with the Nigerian local fowl. In: Ayeni, J.S.O., Aire, T.A. and Olomu, J.M. (Eds.), The helmet guinea fowl (N. m. galeata pallas) in Nigeria. Pp: 85-91. Steel, R.G.D. and J.H. Torrie, 1980. Principles and procedures of statistics. McGraw-Hill, New York.

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Kudu, Y.S et al: Continental J. Veterinary Sciences 2: 27 - 32, 2008 Vogt, H. and F. Stute, 1994. Digestibility of some carbohydrate fractions in hens and guinea fowls. Archi. Fur. Gefi. U geikunde 38: 117-118.

Received for Publication: 03/06/2008 Accepted for Publication: 25/08/2008 Corresponding Author: Alabi, J.O. Department of Animal Production, Federal University of Technology, P.M.B. 65, Minna, Niger State, Nigeria.

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Continental J. Veterinary Sciences 2: 33 - 37, 2008 ©Wilolud Online Journals, 2008.

INCIDENCE OF FOOTROT INFECTION IN SHEEP AND GOATS IN MINNA

J.Y. Adama and Y.S. Kudu

Department of Animal Production, Federal University of Technology, P.M.B. 65, Minna, Niger State, Nigeria

ABSTRACT The incidence of footrot infection in sheep and goats was studied in the University Research Farm, Federal University of Technology, Minna, Nigeria for a period of one year. The percentage number of animals affected in the herd was 32.6% and 32.4% for both sheep and goats respectively. Infection was found to be more prevalent during the wet season of the year. The source of infection was linked to different market areas within Niger state where the animals were purchased and brought to the farm at different times. All the animals used in the experiment were managed through semi-intensive system. The experiment tends to reveal that through supplementary feeding coupled with the treatment, the weight of the animals appreciated. It was discovered that treatment methods using antibiotics, like penicillin-streptomycin at a higher dose combined with feet trimming gave the best result of 62.5% and 66.6% cure respectively, while the other method using same antibiotic at a lower dose combined with foot bath gave a lower result of 28.5% and 50% cure for both sheep and goats respectively. KEYWORDS: Footrot, penicillin-streptomycin, feet trimming, foot bath, supplementary feeding, 10% copper sulphate, Gram stain.

INTRODUCTION Footrot is an infectious disease of ruminants particularly sheep, cattle and goats which causes severe lameness and economic loss from decreased flock production. It is caused by an interaction of two anaerobic gram negative (G-ve) bacteria, Bacteroides nodosus (formerly Fusiformis nodosus) and Fusobacterium necrophorum (formerly Spaherophorus necrophorus). Fusobacterium necrophorum is a normal inhabitant of the ruminant digestive tract and in wet weather may interact with another organism, corynebacterium pyogenes, to produce foot scald, an infection of the skin between the toes. This infection sets up the foot for invasion by bacteroides nodosus, which working in conjunction with the fusobacterium, produces the condition referred to as footrot. Footrot is a costly disease in ruminant livestock population particularly during the wet season. Treatment, costs of labour, drugs and equipment, decreased flock productivity, losses from sales of breeding stock etc make this disease of economic importance for producers (Dee, 1996). Introducing an infected animal into a non-contaminated herd can create herd contamination. The causative agents can also be carried to the soil on visitors’ boots. The disease causes stress to the animals and can affect weight gain, reproductive rates and wool production. Therefore, such conditions as this which tend to limit livestock production ought to be given adequate attention in terms of control and preventive measures such that livestock production particularly in Niger State will be able to meet up the protein needs of the society (FDLPC, 1992). This study was therefore carried out to determine the best treatment approach to footrot and equally to observe the response of the animals to supplementary feed, as indicated by Said and Tolera (1993) that good quality roughage and legume tends to increase intake, and digestibility (Roger et al., 1999). MATERIALS AND METHODS This study was carried out in the University Research Farm, Federal University Technology, Minna, Nigeria from May, 2007 to April, 2008. The animals were purchased from Beji, Mariga and Tunga Mallam livestock markets in

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J.Y. Adama and Y.S. Kudu: Continental J. Veterinary Sciences 2: 33 - 37, 2008 Niger state. The animals were managed through semi intensive system. They were usually released for grazing from 10am to 3.30pm and returned to their pens daily and supplementary feed was usually supplied ad-libitum along with salt licks. The pen was well ventilated through side windows and illumination was enhanced by use of electricity. The animals were routinely dewormed using albendazole. A total of 46 sheep (Yankasa breed) and 37 goats (Sokoto brown breed) were kept in the farm. Each specie of animals were kept separately in different pens. The floors of the pens were not (cemented as such they were muddy particularly during the raining season. In all these animals, particular attention was paid to the following clinical signs, limping, holding of limbs above the ground, reluctance to walk, presence of pus and foul smell from the interdigital spaces and possible loss of appetite. Samples of pus obtained from the interdigital spaces were taken to the state veterinary centre Bosso, Minna for bacteriological examination using Gram stain method. The affected animals were culled from the various pens and divided into two treatment groups. Each of the treatment group was further divided into two replicates. 15 sheep were divided into two replicates of 8 and 7 sheep while 12 goats were divided into two replicates of 6 goats each. Two treatment methods were used in the management of the affected animals using penicillin-streptomycin (4ml/10kg body weight) combined with feet trimming and at (3ml/10kg body weight) of the same drug combined with foot bath using 10% CuSO4 respectively for 2 weeks. Responses to treatment were monitored for about 4 weeks in order to assess the level of response of the animals to each of the treatment methods. At the end of the experiment, the results obtained were subjected to descriptive statistical analysis using percentage to determine the extent of cure of each of the treatment methods. Table 1 Composition of Supplementary Feed Ingredients % Level of Inclusion Groundnut hay 25 Maize bran 35 G/N cake 10 Beans haulms 30 Total 100kg Table 2 Average Feed Intake of Sheep and Goats (kg)

GRP A (Sheep) GRP B (Goats) Period R1 R2 −

X FCE R1 R2 −

X FCE

1st Week May-July

4.1 3.9 4.0 0.30 3.1 2.8 2.95 0.32

2nd Week Aug. – Sept.

5.2 5.4 5.3 0.38 4.2 4.2 4.2 0.44

3rd Week Nov. – Jan.

6.5 6.6 6.6 0.48 4.3 4.4 4.35 0.43

4th Week Feb. – April

8.3 8.4 8.35 0.59 5.6 5.7 5.65 0.54

Total 24.1 24.3 24.2 0.31 17.2 17.1 17.15 0.30 Key

R1 = Replicate 1, R2 = Replicate 2, GRPA = Sheep, GRPB = Goats, −X = Average

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J.Y. Adama and Y.S. Kudu: Continental J. Veterinary Sciences 2: 33 - 37, 2008 Table 3 Average Weekly Weight Gain (kg) GRP A (Sheep) GRP B (Goats) Period R1 R2 −

X R1 R2 −

X 1st Week May-July

13.1 13.20 13.15 9.01 9.10 9.10

2nd Week Aug. – Sept.

13.80 13.70 13.75 9.40 9.50 9.45

3rd Week Nov. – Jan.

14.4 14.1 14.25 10.01 10.03 10.02

4th Week Feb. – April

14.7 14.5 14.6 10.30 10.40 10.35

RESULTS AND DISCUSSION The response of the animals in terms of feed intake and feed conversion efficiency is shown in Table 2. It revealed that as the treatment progresses feed intake progressively increased. This agrees with the findings of Ayoade et al (1999) that goat’s performance is enhanced when fed solely or partially on legume feed or forage allowance. Table 3 revealed that as the feed conversion efficiency amongst the animals tends to increase in this experiment, it thus translates to weight increase relatively, such that, for the sheep it increase from the initial weight of 13.15kg to 14.6kg, while for the goats it increase from 9.10kg to 10.25kg, this positive response might be similar to the findings of Galyean and Goestsch, (1993), that legume digestibility might be attributed to histological make up of legumes in that the majority of cell wall matrix of legumes are easily degraded and penetrated by microbial enzymes than that of the grasses which constitute majority of the feed picked up by the animals when released. The result of the bacteriological examination obtained in Table 4 is similar to the earlier findings reported (Gyang et al, 1986), in which fusobacterium spp was observed from the pus that was cultured using Gram stain method. The results of the two treatment methods used in this study for sheep and goats affected with footrot were expressed in simple percentages (Table 5). The use of penicillin-streptomycin at 4ml/10kg body weight combined with feet trimming gave the best result of 62.5 and 66.6% cure for sheep and goats while the use of the same antibiotic combined with foot bath using 10% CuSO4 gave a lower result of 28.5% and 50% cure for sheep and goats respectively. The above findings agrees with earlier reports (Casey, 1988; Leite-Browning, 2007), that keeping feet trimmed of overgrown tissues will reduce mud and manure packing and decrease the chances of the survival of microorganisms since anaerobic environment will develop. Therefore, combining feet trimming with high dose of antibiotic at 4ml/10kg body weight has proven to give the best result. However, Helen (1990) reported that approximately 75% of the affected feet of sheep were completely healed when given antibiotic treatment without feet trimming with in 4 weeks. Since the animals used in the study were purchased form different sources and introduced into the farm at different times, the infection might have been introduced through infected animals from where such animals may have been in the last 2 weeks before purchase (Egerton et al, 2002). Environmental factors of rainfall, topography and soil type are known to influence the outbreak of footrot. The situation is such that the research farm is located in a muddy soil environment and the incidence of the disease was found to be high during the raining season which is in agreement with earlier findings (Dee, 1996), that footrot outbreaks occur often during persistent raining weather along with high temperature, when animals walk across wet pastures and muddy soil which are favourable for microbial growth and possible transmission.

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J.Y. Adama and Y.S. Kudu: Continental J. Veterinary Sciences 2: 33 - 37, 2008 Table 4 Morphological and Biochemical characteristics of Bacteria Isolates Found in Pus

Sugar Morphology Colour Gram

rxn Catalase Coagulase Lactose Sucrose Glucose Fructose Maltose organisms

Cocci in shape Grayish + - - A A A A A Streptococus spp Long rod in chain and single

Whitish and pink

+ + - + - + - + Lactobacillus spp

Cocci in cluster Yellowish + + + + A A A A Staphylococcus spp Rod shaped Reddish - + - A A A A A Fusobacterium spp Short rod Grayish

white - + - AG A AG AG A e. COLI

Key: + = Positive Reaction, - = Negative Reaction, A = Acid Production, AG = Acid and Gas Production Table 5 The Response of Sheep and Goats to Two Treatment Methods Animal sp Total No Herds Total affected Penstrep + hoof

trimming Grp A Penstrep + Foot bath. Grp B

No cured Grp A

No cured Grp B % cured Grp A % cured Grp B

Sheep 46 15 8 7 5 2 62.5 28.5 Goats 37 12 6 6 4 3 66.6 50

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J.Y. Adama and Y.S. Kudu: Continental J. Veterinary Sciences 2: 33 - 37, 2008 In conclusion, with the influx of livestock population, particularly small ruminants into Niger state from semi arid regions of the country as a result of desertification, more research efforts are needed in order to curb the rate of spread of diseases of economic importance such as footrot within the state thereby creating an enabling environment for optimal performance in livestock production. REFERENCES Ayoade, J. A., Ogebe, P. O. Okuvori, A.L. and Ogbede, T. O. (1999) Proceedings of Nigerian Society of Animal Production Conference (NSAP). Abeokuta Nigeria pg 55-56. Casey, R. H. (1988). Effect of foot paring of sheep affected with footrot on response to zinc sulphatedium laory sulphate foot bathing treatment. Australian Vet. Journal. 65: 258 - 259 Dee, W. (1996). Control, treatment and elimination of footrot form sheep. Journal of Virginia Cooperative Extension. 12: 410 – 428. Egerton, J. R. , Ghimire, S. C., Dhungyel, O. P Shrestha, H. K., Joshi, B. R. (2002). Eradication of virulent footrot from sheep and goats in an endemic area of Nepal and an evaluation of specific vaccination, The Veterinary Record, 151 (10) 290-295. FDLPC, (1992). Nigerian Livestock Resources, Jersy, U. K. pg 19-63. Galyean, M.L. and Goestch, O. L. (1993) Processing of forage cell wall structure and digestibility. A. SSA USA pg 33-62. Gyang, E. O. Umoh, J. U, Ezeokoli, G. D. Momodu, J. O. and Abdulkadir, I (1986). Epidemiology of lameness in small ruminants in Zaria. The livestock farmer 6(1): 21-23. Helen, A. S (1990). Footrot control in sheep. In Lincoln University at Jefferson City of Missouri and the U. S Department of Agriculture, distributed in furtherance of Food and Agricultural Act, 1977. pg 95-113. Leite-Browning, M. L. (2007). Footrot and foot-scald goats and sheep. Extension Animal Scientist, Alabama A and M University. Pg 1-4. Said, A. N. and A. Tolera, 1993. Supplementation of Poor Quality Roughage with Forage Legume. Livestock Production Science, 33: 229-237. Roger C. M., R.P., Kevin, C. B. Joseph and S.F Dwight, 1999. Anim. Feed Sci. Technol., 82:107-120. ACKNOWLEDGEMENT Special acknowledgement goes to my research partner as well as Mal. Danjuma, the Head of Livestock Research Farm for the co-operation received throughout the period of this study. Received for Publication: 03/11/2008 Accepted for Publication: 15/12/2008 Corresponding Author: J.Y. Adama Department of Animal Production, Federal University of Technology, P.M.B. 65, Minna, Niger State, Nigeria E-mail: adama_live@yahoo.com

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