Epidemiological study of bovine fasciolosis using coprological technique in district Mardan, Khyber Pakhtunkhwa, Pakistan

The basic aim of the present study was to investigate the prevalence and the associated risk factors responsible for occurrence of bovine fasciolosis causing huge economic losses in cattle in district Mardan. A total of (n=500) cattle of different age groups, breeds, sex and body condition score (BCS) were randomly selected for collection of feces and screened for presence of fasciola eggs using fecal sedimentation test. As a result, out of 500 cattle only 214 (214/500= 42.8%) were found positive for fasciola infection and overall prevalence was (42.8%).Non-probability statistics was applied on obtained data at 5% probability. The total population (n=500) was divided in to three age groups (1-3 years, 4-6 years and 7-9 years) where the highest prevalence was recorded at the age of 1-3 years(48.82%) followed by 4-6 years(39.74%) and the lowest at the age of 7-9 years(39.65%) and statistically highly significant association (p<0.001) was recorded. The breed wise study revealed the highest prevalence in cross Friesian cattle (46.78%) than local cattle (42.90%) and statistically non-significant (p>0.229) association was observed. The highest sex wise prevalence of bovine fasciolosis was recorded in female cattle (49.03%) than male (32.63%) and statistically significant association (p<0.003) was observed. Relationship between BCS and prevalence of bovine fasciolosis showed the highest rate of infection in those animals having poor body conditions (49.54%) followed by medium body conditions (45.61%) while the lowest prevalence was recorded in those animals having good body conditions (37.27%) and statistically

non-significant association (p>0.373) was recorded. As a result it was concluded that bovine fasciolosis was highly prevalent in the study area causing a huge economic losses and it should be mentioned in priority list for chemotherapy and preventive measures. Keywords: BCS; Fasciola; Lymnea; Snails; Trematodes Introduction In Pakistan, livestock is the backbone of economic sector and the integral part of agricultural sector which is the second largest sector; that almost adds 56.3% shares to agricultural added value. In livestock and agriculture more than 35 million populations are involved which donates approximately 11% to the total the national GDP. In Pakistan more than 62% of population are living in rural areas who are where directly or indirectly involved in Agro-livestock sector for their livelihood. According to our national herd survey the total number of cattle, buffalo, goat, sheep and camels are 29.6, 27.3, 53.8, 26.5 and 0.9 million while in district Mardan the total population of cattle are 2447445 [1]. Among all the parasitic diseases, fasciolosis is one of the major parasitic disease causing a huge economic losses, caused by trematodes belonging to genus Fasciola where migration occur in hepatic parenchyma and final development and establishment occur in bile duct [2]. The parasitic infestation is an emerging infection in livestock and human health directly or indirectly affecting economic sector globally [3]. Among all animal disease, parasitic infections are major constrain in economic development [4]. Fasciolosis is the major parasitic infection which enforces economic impact on production cattle and sheep [5]. The most common etiological species of Fasciola are Fasciola hepatica and Fasciola gigantica. Fasciolosis has been reported in all domestic animals, human beings and other wild species but severe economic losses observed in small ruminants. Its direct economic losses have been reported in the form of condemnation of liver, decrease in production of milk, lower weight calves at birth, poor weight gain and cost of treatment.

Study animals and sampling technique
In the present study, five different areas of district Mardan were selected where ten dairy farms were visited for collection of samples. For collection of data, a questionnaire was established indicating the useful information such as breed, age, sex, health status and history of deworming. Recorded all the basic information related to bovine fasciolosis during collection of the fecal samples. All the cattle were selected randomly in dairy farms. In the present study random sampling method was applied for collection of fecal samples and data was composed. Convenience sampling technique was used for the purpose of the ease of research and simplicity of sampling. The study was continued for a period of 12 months, started from January 2018 to December, 2018.

Ethics statements
Before visiting the dairy farms, prior permission was taken from the owners of the each dairy farm and provided free veterinary services such as vaccination; treatment and other necessary suggestions were given without any fee at a time of sample collection.

Sample size determination
Since this is the first study about the prevalence and identification of associated risk factors, the sample size was calculated by using the formula assumed by [9] where prevalence of 5% and 50% absolute precision was considered as 50%. According to the formula only 384 animals were to be sampled but here the sample size was increased up to 500 to increase the precise results.

Study design
A cross-sectional study was steered to determine the prevalence of bovine fasciolosis in five different selected zones of district Mardan and conducted from January, 2018 to December, 2018.

Coprological examination
All the fecal samples were directly collected from rectum of each animal using disposable gloves and mask. After collection of fecal samples, were placed in sterile open mouth bottles containing 10% formalin as a preservative. All the samples were properly sealed and labeled for necessary information such as age, sex, BCS and breed. All the samples were transported to the parasitological laboratory, Veterinary Research Institute (VRI) Peshawar. In laboratory sedimentation technique was applied to identify the fluke eggs according to the procedure as described by [10] Hansen and Perry (1994). Measured two gram of feces and placed in a cylinder containing 42ml of water. Then mixed properly with the help of glass rod and poured through a tea strainer for the removal of large particles. The solution was further passed through a sieve into flask and water was passed through sieve to make free of eggs. Waited for three minutes to filtrates to sediment and supernatants were removed. Then added two drops of methylene blue to filtrates and placed the entire sediment on the glass slide and placed the cover slip then viewed the slide under compound microscope (Labomed).On the basis of morphology and color characteristic, eggs of fasciola spp were identified. All the samples were stored in a refrigerator at 4°Cthat were not processed within twenty four hours [11]. Diagnosis of fascioliasis Tentative diagnosis was made on the basis of history, Physical examination and clinical examinations(Chronic diarrhea, dullness, rough body hair coat, bottle jaw, emaciation) while confirmatory diagnosis was made on the basis of coprological examination findings such as oval shape ova, presence of operculum and yellow brown in color.

Body condition scoring (BCS)
Scoring of the body conditions were determined by the formula as described by Mihrete et al. [12], which ranging from 0 to 5, where score (0-1) stands for poorest conditions of the body, score (2-3) stands for medium conditions of the body whereas score (4-5) stands for good health conditions of the body. All cattle under our study were divided into three classes on the basis of their body conditions such as poor (score 0-1), medium (score 2-3) and good (score [4][5].

Age detection
In the present study the age groups were classified on the basis of dental formula described by [13].

Management and statistical analysis
All the collected data was properly entered in to Microsoft Excel 2010 spread sheet and analyzed by applying SPSS version 20 statistical software. Descriptive statistics was applied to analyze and set as frequencies and percentages. Pearson Chi square (χ²) tests were applied to evaluate the statistical associations present between the prevalence of fasciolosis and different risk factors (age, sex, body conditions, breeds). A P<0.05 was considered significant in all statistical analysis Results

Overall prevalence of bovine fascioliasis
The result of the current study exposed 42.8% overall prevalence of bovine fasciolosis in district Mardan as described in (Table 1).

Bovine fasciolosis associated with age
In the present study, the total numbers of animals were divided in three age groups such as 1-3 years, 4-6 years and 7-9 years. As a result, the highest prevalence was recorded in the cattle at the age of 1-3 years (48.82%) followed by 4-6 years (39.74%) while the lowest at the age of 7-9 years (39.65%) and statistically significant variations (P<0.001) was observed among age group 1-3 and others two age groups 4-6 years and 7-9 years as presented in (Table  2).

Bovine fascioliasis associated with breeds
In the present study, breed wise prevalence of fasciolosis was also studied between local and cross friesian breeds where the highest prevalence was observed in cross friesian cattle (46.78%) followed by local cattle (42.90%). There was high resistance to fasciolosis in local breeds than cross friesians and statistically non-significant (P>0.229) association was observed (Table 3).

Bovine fascioliasis associated with sex
In the present study, the highest prevalence of fasciolosis was reported in female (49.03%) cattle while lower in male (32.63%) as presented in (Table 4) and statistically significant variations (P<0.003) were recorded.

Bovine fasciolosis associated with body condition score (BCS)
In the present study, the highest rate of infection was recorded in those animals having poor body conditions (49.54%) followed by medium body conditions (45.61%) while the lowest prevalence was recorded in those animals having good body conditions (37.27%) and statistically nonsignificant variations(p>0.373) was recorded ( Table 5).

Discussion
The results of our current study exposed 42

Conclusion and recommendations
It was concluded that bovine fasciolosis is highly prevalent in the study area with an overall prevalence was 42.8%. Bovine fasciolosis are responsible for heavy economic losses in form of low milk production and poor weight gain. Bovine fasciolosis is the primary concerned disease in the study area so it should be mentioned in priority list. This study explored that age is the key risk factor in bovine fasciolosis. Fasciolosis was one of the major constrain in development of livestock in district Mardan, Pakistan. The climate of our studying area is humid and hot, which is susceptible for growth and development of parasites. To control parasitic infection in bovines, epidemiological research is important for proper control. In Pakistan, few studies are available regarding bovine fasciolosis in the current study area.This study will help other researchers and field workers to overcome prevalence of fasciolosis in different parts of our country. As a result it was concluded from the present study that bovine fasciolosis is highly prevalent in the study area with an overall prevalence is 42.8%.Bovine fasciolosis are responsible for heavy economic losses in form of low milk production and poor weight gain. Bovine fasciolosis is the primary concerned disease in the study area so it should be mentioned in priority list. This study explored that age is the key risk factor in bovine fasciolosis. I recommend the following suggestions for future control such as regular use of different anthelmintic (flukicides) to reduce resistant to the drug proper control of intermediate hosts (snails) by spraying copper sulphate, avoid grazing of animals in heavily populated snail's area.