Comparative study of neem leaf ( Azadirachta indica ) suspension and toltrazuril against coccidiosis in Sonali chicken

Background: Coccidiosis is recognized as the parasitic disease with the greatest economic impact on poultry industries worldwide. Sonali chicken are commonly affected by coccidiosis in Bangladesh. Recently, the concerns about possible drug resistance have aroused great caution in the usage of drug in the animal industry. As an alternative herbal extracts are used and Neem leaves (Azadirachta indica) have anti-coccidial property. This study was conducted to evaluate the comparative efficacy of Neem leaves suspension and toltrazuril on coccidial load, blood constituents, growth performance of Sonali chicken infected with Eimeria tenella . Methods: The study was conducted in the laboratory and experimental shed under the department of physiology and pharmacology, HSTU, Dinajpur during, 2 nd January to 3 rd February, 2019. Eighty (80) Sonali chicken of seven days old were randomly divided into four groups named T 0 , T 1 T 2 and T 3 and each group contained 20 birds. All groups were supplied E. tenella orally except T 0 group and after 3 days T 2 group was treated with Neem leaves suspension (5%) for 15 days and T 3 groups was treated with toltrazuril (topzuril ®) solution (1 ml/lit) for 2 days. Results: Results showed that protozoal load decreased significantly (P<0.01) in T 2 & T 3 groups whereas increased significantly (P<0.01) in T 0 and T 1 group. The total erythrocyte count and hemoglobin in different treatment groups were similar and the differences were non-significant (P>0.05) statistically except total leukocyte count (TLC). The body weight of T 0 , T 1 , T 2 and T 3 group at day 30 were 432.65, 469.81, 548.81, 489.22 gm respectively which is statistically significant (P<0.01) and highest body weight gain was recorded from Neem leaves suspension treated group. Conclusion: Neem leaves suspension have significant effects on protozoal load, body weight and no significant effect on haematological parameters except TLC.


Introduction
Bangladesh is an agriculture based tropical country where more than 80% of the country's 160 million people are living in rural areas and highly dependent on agriculture.Poultry farming is one of the major activities in rural areas as it provides immense employment opportunities to the local communities especially for youth &women that helps in poverty reduction, ensures food security and improves the nutritional status of the people.Poultry industry can produce very high quality proteins for human nutrition as well as a source of income for the community in many countries, therefore poultry production plays very important role in economic development of any country (Tarhyel et al., 2012).Poultry is a promising sector in Bangladesh which is increasing day by day.Poultry production is hindered by many problems among which various diseases namely bacterial, viral and parasitic infections are the most important (Ojok, 1993).
The northern part of Bangladesh is considered as a poultry hub especially of Sonali chicken, which is used as meat purpose.Sonali chickens are well adapted in the environmental conditions of Bangladesh, as it requires less care and attention as compared to other breeds.It is also easier for women to rear (Saleque and Saha, 2013).In spite of many positive reasons behind rearing of Sonali chickens, there are some causes that hamper in optimum production of Sonali chicken by smallholder households in Bangladesh (Biswas et al. 2006).Poultry diseases are one of the major constraints for developing poultry industry in Bangladesh (Islam and Samad, 2004).
Avian coccidiosis is a parasitic disease of intestinal tract caused by single cell protozoan parasite belonging to genus Eimeria.It causes massive destruction of the epithelial cells, which leads to bloody diarrhea, reduced weight gain and temporary reduction in egg production (Dalloul and Lillehoj, 2005;Razzaq et al., 2011).
Coccidiosis has been considered as a very harmful disease affecting growth and performance of poultry (Lin et al., 2006;Mujahid et al., 2007;Bachaya et al., 2012).Thus coccidiosis is probably the most expensive and wide spread infectious disease in commercial poultry systems.
In Bangladesh, a number of drugs are available for the treatment and prevention of chicken coccidiosis.Among these sulphaclozine sodium are used extensively throughout the country.In addition, ionophorous antibiotic i.e., maduramycin and lasalocid and some other drugs like amprolium, toltrazuril are also used in the field.It has been found that the efficacy of various anti-coccdial drugs varies considerably against coccidisis (Lee, 1978).
Toltrazuril is a symmetrical triazinetrione compound and 2.5% oral solution has been shown to be effective against all species of Eimeria infecting chickens (Mehlhorn et al., 1988).It is active against all intracellular developmental stages.
Chemoprophylaxis with Toltrazuril enhances immunity against poultry coccidiosis (Chapman, 1987).It has been proved that therapeutic medication with Toltrazuril protects the birds from clinical coccidiosis (Ramadan et al., 1997).
Drug resistance is very much alarming matter nowadays.New drug resistance mechanisms are emerging and spreading globally, threatening our ability to treat common infectious diseases, resulting in prolonged illness, disability, and death.So, we need to quickly replace of anticoccidial with other options.These new options should be inexpensive and available in everywhere as well as healthy for human society.It is thought that medicinal plants are a good alternative for this purpose.Since ancient times medicinal plants play important role in health management of traditional poultry production (Eevuri and Putturu, 2013).In recent years, the growing demand for herbal product has led to a quantum jump in volume of plant materials traded within and across the countries.
Secondary plant metabolites (Phytochemicals), previously with unknown pharmacological activities, have been extensively investigated as a source of medicinal agents (Krishnaraju et al., 2005).Thus it is anticipated that phytochemicals with adequate anticoccidial efficacy will be used for the treatment of the protozoal infections .Also, in contrast to anticoccidial, most active components of medicinal plants are readily absorbed in intestine and have short half life.
Neem elaborates a vast array of biologically active compounds that are chemically diverse and structurally complex.More than 140 compounds have been isolated from different parts of Neem.
The tender leaves along with piper nigrum Linn are found to be effective in intestinal helminthiasis.Since Neem leaf increases immunity against some common diseases which assists to reduce mortality , it is likely that the growth pattern of sonali chicks will proceed uninterrupted.
So, this study was undertaken to evaluate the comparative efficacy of Neem leaves suspension and toltrazuril on coccidial load, blood constituents, growth performance of sonali chicken infected with Eimeria tenella.

Materials and Method
The experiment was conducted at the experimental shed under the department of Physiology and Pharmacology, for a period of 65 days from 2 nd January to 3 rd February, 2018.

Preparation of house
First the room as well as the wire cages were washed by sweeping with tap water using hose pipe connected with the tap.The room was properly ventilated and disinfected with a phenolic disinfectant and allowed to dry leaving the room unused with the electric fan and the bulb switched on overnight.All the utensils required for the experiment such as feeder, water pot, beakers, pestle and mortar, syringe, needle etc. were set in the experimental shed.

Grouping of birds
Eighty (80) Sonali chicken were used to evaluate the dietary effect of Neem leaf suspensionon Eimeria tenella infection, growth performance.The Sonali chicken were assigned into four groups containing 20 birds in each groups.20 birds kept in each group were considered as an experimental unit.Sonali chicken were randomly distributed in every groups.
Group T0: The Sonalis were fed normal diet and given water ad-libitum and their body weight was recorded at every 15 days interval.Body weights, blood parameter and Eimeria tenella protozoal load were measured at the times when that of other groups were measured.This group was served as "Negative control" group.

Group T1:
The Sonali chicken were supplied with protozoa after acclimatization to induce Eimeria tenella infection in this group.No antiprotozoal treatment was given against Eimeria tenella in T1 group.Adequate feed and drinking water was given.This group served as "Positive control" group.

Group T2:
The Sonali chicken were supplied with Eimeria tenella protozoa after acclimatization to induce Eimeria tenella infection as like as T1 group.This group was left untreated for 24 hours to establish Eimeria tenella infection.After 24 hours, this group was treated with Neem leaves suspension as herbal antiprotozoal agent at a dose rate of 0.25 ml per 100 ml of drinking water.This group served as "Neem leaf suspension" group.

Group T3:
After acclimatization Sonali chicken of this group were supplied with Eimeria tenella protozoa as like as T1& T2 group.This group was left untreated for 24 hours to establish Eimeria tenella infection.After 24 hours, this group was treated with antiprotozoal drug (Topzuril liquid @ 1 ml per 2lit water).This group served as "Topzuril liquid" group.

Collection of feed
Polli mash commercial feed were collected from Griholokkhi Poultry Feed, Kalitola, Dinajpur from a reputed Sonali feed exporter.Mash and water were provided ad-libitum during the whole experimental period.
The ingredients that were used for the composition of mash feed are described in Table 1.

Acclimatization of Sonali chicken
Immediately after reaching the destination the sonali were shifted to wire cages.They were fed with Griholokkhi Poultry Feed, Kalitola, Dinajpur and drinking water ad-libitum.Glucose and vitamin C were supplied with drinking water for first three days to overcome the transportation stress.Sonalis were allowed to acclimatize in their new environment for 13 days before the commencement of the experiment.

Lighting
During the whole experimental period, all Sonali chicken were exposed to a 16 hours continuous photoperiod (natural light plus artificial light) in an open sided house.Electrical bulbs were used for additional light at night.

Collection and processing of neem leaves
Neem leaves were collected from the HSTU, Dinajpur.Young Neem leaves were collected and washed with fresh water.Before chopping it into small pieces, it was soaked with cotton for removing the adhesive water.Then the leaf was chopped into small pieces and was mashed with the help of pestle and mortar.Leaf juice was collected by squeezing mashed leaf.Then it had been produced 0.25% of suspension of grinded Neem leaf with distilled water.

Collection, preservation and administration of toltrazuril
Topzuril liquid was collected from Square pharmaceuticals Ltd.It was preserved in a dry place at room temperature and was administered at a dose rate of 1ml per 2 litre of drinking water for 3-7 days.

Recording of body weight
The effect of the Neem leaves extract and toltrazuril on body weight was recorded before and during administration of drugs.
Chickens under trial and control groups were weighed with Electric weighing machine.The weight of each chicken was taken before feeding in the morning, in noon and afternoon.The average of these three weights was calculated and recorded.

Collection and preparation of test organism
The test organism (Eimeria tenella) was collected from the Gopalgonj local farm area, Hajee Mohammad Danesh Science and Technology University (HSTU), Dinajpur.

Collection and examination of feces for parasitic oocyst investigation
Feces were examined by two different types of qualitative tests; namely direct smear and flotation techniques were used to examine the fecal samples to identify the morphological features of oocysts (Hendrin and Robinson, 2006;Soulsby, 1982).

Hematological parameters
Blood samples were collected from the wing vein of chicken of both control and treated groups at pre-feeding and during feeding (28

Statistical analysis
The data were analyzed statistically between control and treated groups of chicken by using SPSS version 22 and Microsoft Excel.Statistically significant differences between group means were determined by analysis of variance (ANOVA).

Total Leukocyte count (TLC)
The values of TLC are presented in Table 4.The values of TLC of T0 (Basal diet) and T3 (E.tenella plus topzuril liquid) group were similar.But in case of T1 (Basal diet plus E. tenella) and T2 (E.tenella plus Neem leaves suspension) groups the values differed.

Hemoglobin
Hemoglobin (Hb) is presented in Table 5.The values in all treated groups and control group were similar and the values were within normal range.

Table 2 .
The values of TEC in all treated groups and control group were similar and within the normal range at15th and 30th day of experimental period.

Table 4 .
TLC in different groups of Sonali chicken

Table 6
It can be used for the control of coccidiosis and as a good growth promoter, leukocyte enhancer in Sonali chicken.It may be stated that the neem leaf (Azadirachta indica) may provide a new therapeutic avenue against E. tenella because of its availability in our country and effectiveness.18.Gabriel, S. Mallet, M. Leconte, G. Fort, and M.Naciri.Effects of whole neem leaves feeding on the development of coccidial infection in Sonali chickens.Qazi AW, Gill, ZJ, Rizwan Q, Sajid MA.Evaluation of 2.5 percent Toltrazuril against coccidiosis in broiler chicks.Pakistan