Prevalence and Risk Factors of Bovine Fascioliasis in Kelantan, Malaysia: A Cross-Sectional Study

Bovine fascioliasis is an important zoonotic parasitic disease that causes significant economic losses to the livestock industry. The aim of this study was to determine the prevalence and risk factors of bovine fascioliasis in Kelantan. In this cross-sectional study, a total of 308 stool and blood samples of farmed cattle were collected from December 2017 to June 2018. The stool samples were examined microscopically for the presence of Fasciola spp. eggs following a formalin-ether sedimentation process. The blood samples were subjected to a commercial ELISA kit (Bio-X-Diagnostic, Rochefort, Belgium) for the detection of anti-Fasciola IgG antibody. The association between coprological findings and risk factors was determined using Pearson’s chi-square (χ2). The coproprevalence and seroprevalence of bovine fascioliasis was 14.6% and 37.3%, respectively. There were significant (P < 0.05) associations between the risk of infections and the sex, type of feedings, anthelmintic treatment and farm hygiene. Female cattle (OR: 3.104; 95% CI: 1.265, 7.615), feeding by grazing (OR: 4.458; 95% CI: 1.823, 10.90), untreated cattle (OR: 3.833; 95% CI: 1.620, 9.071), non-schedule anthelminthic treatment (OR: 3.927; 95% CI: 1.685, 9.152) and farm that have never been cleaned (OR: 2.829; 95% CI: 1.428, 5.608) showed higher odds of Fasciola spp. infection. These findings suggested bovine fascioliasis is a serious veterinary disease in Kelantan. Thus, appropriate control, prevention and monitoring strategies of this parasitic infection are urgently needed to reduce the burden of the disease.


INTRODUCTION
Fascioliasis is an important parasitic disease caused by the trematodes, Fasciola hepatica and Fasciola gigantica. The trematodes affect a large variety of animals including cattle, sheep, goats and buffaloes through ingestion of infective metacercariae (Hossain et al. 2011;Zainalabidin et al. 2015;Rahman et al. 2017;Mursyidah et al. 2017). Fasciola species have a complex life cycle which involves snail as an intermediate host where the miracidia develop into thousands of cercariae (Neges & Sahle 2018). Once released from the snails, the cercariae will encyst on aquatic vegetation as metacercariae and taken up by grazing animals. Each metacercaria release an immature fluke which will penetrate the small intestine and migrate through the hepatic parenchyma where it develops into adult and produces eggs.
The disease causes significant economic losses to the livestock industries due to the reduction of body weight, liver condemnation, decrease in milk yield production, increase cost of anthelmintic treatment and reduction of animals' fertility (Schweizer et al. 2005;Khoramian et al. 2014;El-Tahawy et al. 2017). Fascioliasis has been considered as a worldwide animal health problem as it was reported in both developed and developing countries (Kuerpick et al. 2012;Byrne et al. 2018;Zewde et al. 2019). In Africa, Asia, Oceania and Europe, the occurrence of fascioliasis was highest in cattle compared to other animals such as sheep, goats and buffaloes (Mehmood et al. 2017). Fascioliasis in cattle is estimated to cause global economic losses up to million dollars annually. In Europe and Australia, economic losses were from reduction of milk yield and reduced fertility of the livestock (Schweizer et al. 2005;Toet et al. 2014). In Asia, bovine fascioliasis is spreading widely in many regions including Iran, Iraq, Saudi Arabia, 3 Russia, Thailand, Turkey, China, Vietnam, Nepal, Japan, Korea, Philippines, Pakistan, Bangladesh and Cambodia with prevalence range from 0.71% to 69.2% in cattle and estimated global annual economic losses over 200 million US dollars (Mehmood et al. 2017). The respective losses were due to reduced meat production and condemnation of livers (El-Tahawy et al. 2017;Zewde et al. 2019).
In Malaysia, the prevalence of fascioliasis in cattle has been reported in several states including Johor, Pahang, Perak, Pulau Pinang, Selangor and Terengganu (Saleha 1991;Zainalabidin et al. 2015;Khadijah et al. 2017;Mursyidah et al. 2017;Rita et al. 2017;Diyana et al. 2019). The prevalence of cattle fascioliasis in Malaysia range from 1% to 95% based on six studies published from 1991 to 2019. The most endemic state in Malaysia is currently Terengganu with a prevalence of 95% (Khadijah et al. 2015). The prevalence was based on the surveillance of 40 cattle at two cattle farms located in Kuala Terengganu.
Although bovine fascioliasis has been reported in several states in Malaysia, the epidemiological picture of the disease in Kelantan is still unknown. Kelantan has a conducive tropical climate, with intermittent rain throughout the year and monsoon season from November to January which is a favourable weather for snail population and Fasciola spp. life cycle (Mas-Coma et al. 2018). Cattle farming are increasingly popular among farmers in Kelantan due to high demand of meat supply. Nevertheless, the possible threat of fascioliasis among farmed cattle in Kelantan is still unknown due to lack of research interest on this neglected tropical disease. Therefore, this study aimed to determine the prevalence and risk factors of Fasciola spp. infestation among farmed cattle in Kelantan.

Ethical Approval
The present study protocol was reviewed and approved by the Animal Ethics Committee of Universiti Sains Malaysia (AECUSM). Reference number: USM/ IACUC/2017(107)(852).

Study Area
This cross-sectional study was conducted in 10 districts of Kelantan, Malaysia

Study Population and Sample Collection
A total of 308 cattle were randomly selected from December 2017 to June 2018. The minimum sample size was based on the single proportion sample size calculation with estimated prevalence of 11.02%, desired margin of error of 3.67%, confidence level of 95% and dropout of 10% (Chakraborty & Prodhan 2015;Masrin et al. 2015;Zainalabidin et al. 2015). About 30 to 32 cattle of six months of age and above were selected in each district. The fresh stool samples were collected from the rectum of the cattle and transferred into labelled 60 mL stool containers prior to coprological examination. The blood samples were withdrawn from the jugular veins of the cattle into 10 mL plain blood tubes and allowed to clot at room temperature for 2 h followed by centrifugation for 10 min at 1000× g. About 2 mL of serum was aliquoted into a labelled 2 mL microcentrifuge tubes and kept at -20°C.

Collection of Demographic Data
A set of questionnaires was used to record data on animals' age, sex, breed, type of farm, source of animals' drinking water, type of food, frequency of anthelmintic administration and farms' hygiene. The age of the cattle was determined on the basis of farm's records. The sex of the cattle was determined by examining the presence of sexual organ. The breed of the cattle, source of animals' drinking water, type of food, frequency of anthelmintic administration and farms' hygiene were determined by the consultation with the farms' owners. The selected cattle farms were grouped into two groups: Dry farms and wet farms, based on the ability of the soil to sustain water.

Coprological Examination
The stool samples were subjected to formalin-ether sedimentation technique for the detection of Fasciola spp. eggs (El-Tahawy et al. 2017). The sediment was viewed at 100× and 400× magnification using a light microscope with eyepiece reticle (Zeiss, Germany). Identification of the Fasciola spp. egg was made based on the morphology as described in a previous study . Fasciola spp. eggs appeared as golden shiny colour, ellipsoidal, operculated and measured 130 µm-150 µm long by 60 µm-90 µm wide. Interpretation of a positive result for Fasciola spp. infestation was based on the presence of Fasciola spp. eggs in the stool samples. The microscopic positive slides were verified by two experienced veterinary parasitologists.

Serological Detection of Anti-Fasciola IgG Antibodies
Detection of IgG antibodies against Fasciola spp. in serum samples of the cattle was conducted by using a commercial ELISA kit purchased from Belgium, Mono Screen Ab ELISA (Bio-X Diagnostics). The assay was performed according to 6 manufacturer's instructions (Yildirim et al. 2007;Yasar & Burcak 2018). The positivity levels were categorised as no infestation (less than 15%), low infestation (15% to 45%), moderate infestation (45% to 75%) or heavy infestation (greater than 75%). The degree of positivity of each sample was determined by dividing the optical density value with the corresponding positive control and multiplies with 100 to express it as a percentage.

Statistical Analysis
Data analyses were performed using SPSS version 24.0 (IBM Corporation, Chicago, United States). The prevalence of Fasciola spp. infestation in cattle was calculated as percentage value. The associations between epidemiological data and Fasciola spp. infestation were determined using Pearson's chi-square (χ 2 ). A statistical association between variables was considered significant if the calculated P-value was less than 0.05 with 95% confidence level. A binary logistic regression analysis and 95% confidence intervals (CI) of odds ratio were calculated to quantify the association of the statistically significant variables. A probability level of 5% was used as statistical significance.

RESULTS
The coprological examination of the cattle stool samples showed 14.6% (45/308) positive Fasciola spp. eggs. Positive coproscopy results were found in eight out of the ten Kelantan districts, namely Gua Musang, Tumpat, Bachok, Pasir Puteh, Tanah Merah, Kuala Krai, Jeli and Machang. The highest coproprevalence of fascioliasis among cattle was recorded in Machang with prevalence of 32.3% (Fig. 2). In serological study, 37.3% (115/308) of the cattle serum samples were positive for anti-Fasciola IgG antibody. Seropositive cases were detected among cattle from all 10 districts in Kelantan. The highest numbers of seropositive cases were recorded in Pasir Puteh, Kuala Krai and Tumpat, with prevalence up to 50% in all three districts. Tanah Merah recorded the lowest prevalence of 20% (6/30). Of the 115 seropositive cases, 36% had a low antibody titre, 18% had a moderate antibody titre and 46% had a high antibody titre (Fig. 3). Of the 41 farms visited, 32 farms (78%) were positive for anti-Fasciola antibody. Out of 115 seropositive cattle, eggs were only found in 37 (32%) cattle with 18 (49%) cattle had a high titre of the antibody, 6 (16%) cattle had a moderate titre of antibody and 13 (35%) cattle had a low titre of antibody (Table 2).  The results of the association analysis of epidemiological data with the prevalence of Fasciola spp. infestation are presented in Table 3. There was a significant association between Fasciola spp. infestation and sex (P = 0.01). The female cattle have 3.104 times odds of Fasciola spp. infestation as compared 8 to the male cattle. Higher prevalence of Fasciola spp. infestation was observed among those cattle that were allowed for grazing. Cattle fed via grazing was at 4.458 times odds to have Fasciola spp. infestation (P = 0.001) as compared to those fed on silage. With regard to the anthelmintic practice, the prevalence of Fasciola spp. infestation was higher among those cattle that have never received anthelmintic and those cattle that were only given anthelmintic when suspected of being infected with worms (P = 0.004). Cattle that have never been administered with anthelmintic drugs are at 3.833 times odds of Fasciola spp. infestation, as compared to cattle given anthelmintic drugs every six months. Besides, cattle that were only given anthelmintic drugs when suspected of being infected with worms demonstrated a higher risk for Fasciola spp. infestation with 3.927 times odds as compared to cattle given anthelmintic drugs every six months. Regarding farm cleaning, cattle raised in farms that have never been cleaned demonstrated higher prevalence of Fasciola spp. infestation with 2.829 times odds than those raised in farms that are cleaned daily (P = 0.003).

DISCUSSION
Fascioliasis is a serious threat to the livestock industry due to significant economic losses that attribute to liver condemnation and growth retardation of infected cattle (Kozlowska-Loj & Loj-Maczulska 2013;El-Tahawy et al. 2017;Arbabi et al. 2018).
The present study showed the prevalence of Fasciola spp. infestation in cattle was 14.6% based on coprological examination and 37.3% based on serological detection of anti-Fasciola IgG antibody. The coproprevalence of Fasciola spp. infestation among cattle in the present study is lower than those in the previous studies in Terengganu (Khadijah et al. 2015;. The higher prevalence in the previous studies in attributed mainly to the smaller samples size which increase margin of error in the studies (Naing et al. 2006). The coproprevalence of Fasciola spp. infestation in this study was similar to studies found in endemic countries such as Egypt (28.6%) and Vietnam (23.4%) Nguyen et al. 2017). This indicates that cattle fascioliasis is a serious problem in Kelantan. Therefore, prevention and control measures need to be taken immediately to overcome the burden of the infestation. The prevalence detected by the serological approach was higher than that detected by the traditional coprological examination. The difference between the coproprevalence and seroprevalence of the infestation was similarly reported in a study in Turkey which showed higher prevalence based on serological approaches compared to the coprological method (Yildirim et al. 2007). The explanation for this difference is that anti-Fasciola spp. antibodies can be detected much earlier than the detection of Fasciola spp. egg which is only present after 12 weeks post infestation (Mohammed et al. 2018). Besides, antibodies also remain in infected cattle for up to six months post infestation (Castro et al. 2000). Therefore, cattle that were previously exposed and recovered from the infestation will also show presence of anti-Fasciola antibody.
An interesting result found in the present study was that 18% of the cattle positive for coprological examination were found negative by serological detection of anti-Fasciola spp. IgG antibody. These findings may reflect a low immune response to the antigenic stimulus from the migrating or mature flukes in the cattle which result in undetectable antibody (Afshan et al. 2013). Another explanation was a false positive coproscopic result due to ingestion of pasture contaminated with Fasciola spp. eggs (John et al. 2019). This study also found that antibody titre does not correlate with the presence of eggs in the stool. All ranges of antibody titres were observed among copropositive cattle. This observation was in agreement with the previous study in Turkey which demonstrated various titres of antibody among copropositive cattle (Yildirim et al. 2007).
The present study showed no significant association between Fasciola spp. infestation and cattle age. This finding is in agreement with the previous studies in Vietnam and Nigeria (Nguyen et al. 2017;Shinggu et al. 2019). However, several studies reported different results whereby higher prevalence was seen in cattle of above five years old compared to cattle aged below five years old . The plausible reason for the differences is the feeding techniques practiced by farmers. The cattle aged above five years old was more likely to be infested due to the cattle aged above five years old were let to graze freely around the farm making them more exposed to the causative agent of the infestation .
With regards to cattle sex, the present study showed female cattle have higher odds of association with Fasciola spp. infestation as compared to male cattle. This observation is in agreement with the previous studies in Terengganu which reported higher prevalence of Fasciola spp. infestation in females than males Mohammed et al. 2018). This might be due to most female cattle were kept for milking the young which can be considered as a stressful physiological factor that may affect their immunity against the infestation. In contrary, a study in Egypt found a significantly higher prevalence of Fasciola spp. infestation in males than females (El-Tahawy et al. 2017). The observation may probably be due to the practice of keeping females under healthier and nourishing conditions compared to males which are kept free to graze on the fields (Khan & Maqbool 2012).
In the present study, grazing was found to have a statistically significant association with Fasciola spp. infestation in which it showed more than four times the odds compared to silage. The reason for that is due to feeding silage helps in removing metacercaria attached to the grass before it is given to their livestock (John et al. 2019). This finding suggests that feeding silage is an effective strategy for the prevention and control of the infestation. The present study also showed that there was no significant association between the source of water and Fasciola spp. infestation. On the contrary, several studies reported that river as the main water source for the transmission of fascioliasis (Mas-Coma et al. 2018). This outcome revealed that the infestation sources vary according to geographical areas, depending on suitable environmental condition for the survival of intermediate hosts and infective stage metacercariae (Neges & Sahle 2018).
Undoubtedly, the animals which did not receive anthelmintic (albendazole, fenbendazole and macrocyclic lactone) treatment for fascioliasis are of higher risk of fascioliasis than the one with regular treatment. A higher prevalence of fascioliasis was observed among cattle without anthelmintic treatment (Nguyen et al. 2017). Fasciola spp. requires a host to complete its life cycle and grows its population in an area. Therefore, the regular administration of anthelmintic is important in ensuring effective control to prevent the outbreak of infestation. However, over-reliance on anthelmintic could lead to a serious problem which causes the development of anthelmintic resistance (Kelley et al. 2016). Understanding of confounding factors such as correct dosing, treatment duration and regular surveillance is now critical for effective management of the drug resistance.
This study also found a significant association between coprological findings and frequencies of farm cleaning. In this study, a farm cleaning refers to the disposal of animal waste from the farm areas. The prevalence of Fasciola spp. infestation among cattle in the daily-cleaned farms was lower than cattle in the farms that have never been cleaned. The practice of cleaning prevents the healthy cattle from being exposed to Fasciola eggs shed by the infested cattle, which might increase the risk of disease transmission especially in the grazing areas (Gupta 2014). Therefore, hygiene interventions on farms' area must be implemented to prevent a sustained transmission of Fasciola spp.

CONCLUSION
In conclusion, the present study unravelled the current status of Fasciola spp. infestation among farmed cattle in Kelantan. The epidemiological investigation of risk factors revealed a significant association between Fasciola spp. infestation with the sex, feeding techniques, frequency of anthelmintic treatment and farms' hygiene. Thus, the outcome of this study will contribute to helping cattle farmers and veterinary department in implementing effective strategies to control the risks of fascioliasis among farmed cattle in Kelantan.