Introduction

Buffalo is one of the most important ani- mals as a source of milk and meat and it is also used for draught purposes in Bangla- desh. They are one of the major sources of animal proteins along with providing milk, meat, and good quality hides (Liu et al., 2009). Buffalo is the ‘Black gold’ of Ban- gladesh next to cattle. They are widely used for plowing and laddering, racking, thresh- ing, and crushing of sugarcane and oil seeds carting and transportation of goods in rural areas of Bangladesh, and due to its excel- lent draft and pulling capacity, buffaloes  are called the living tractor of the East (Ra- haman and Islam, 1992; Cockrill, 1968). The working life of buffalo is ranged up to 25 years of age which is higher than cattle (Cockrill, 1974). The livestock sub-sector contributes 1.54% to the GDP and in finan- cial year 2017-2018, the livestock popula- tion is 551.39 lakh (ruminant) whereas the buffalo is 14.85 lakh (Islam et al., 2017).

It is a  well-known  fact  that  parasitism is a far greater threat to the livestock than any other visible outbreaks of the diseas-  es. Gastrointestinal parasitism is a major cause of the reduced buffalo production and economic losses in Bangladesh and is of- ten ignored due to its chronic and deceptive nature (Jabber and Green, 1983; Sanyal, 1998; Chowdhury and Tada 1994). It causes significant losses in different parameters of health and production (Lebbie et al., 1994). Moreover, some parasites of buffaloes such as schistosomiasis and fascioliasis can transmit to humans where they cause signif- icant clinical diseases in several countries (Wang et al., 2006; Tum et al., 2007). Gas- trointestinal parasites in buffalo calves lead to reduced growth and constantly hinder the development of buffalo industry in Bangla-

desh. Epidemiological data regarding the parasites of buffalos are not abundant. No study was conducted regarding parasitic in- fection of the buffalo calves in the Sylhet district which is a suitable place for buffalo rearing in Bangladesh. Therefore, this study was conducted to identify the parasitic load found in the gastrointestinal tract in buffalo calves at the Sylhet district, Bangladesh.

Materials and methods

Study area and animals

The study was conducted in Sylhet Sadar, Jaintapur and Zakiganj Upazila of Sylhet district which is located in between 24°36' and 25°11' north latitudes and inbetween 91°38' and 92°30' east longitudes and where the majority of buffalo calves are reared as free – range (Figure 1). One hundred sev- enty buffalo calves ranging from 1 day old to 18 months of age of either sex were ran- domly selected from villages of the Sylhet district of Bangladesh for collection of fe- cal samples (Gunathilaka et al., 2018). The study was conducted from January to Sep- tember in 2018, considering March to June as summer and June to October as rainy season according to the seasonal clarifica- tion of Bangladesh.

Collection and preservation of fecal samples

Questionnaires were prepared and in- terviews with the farmers were conducted during the collection of the sample to ob- tain specific information regarding buffalo calves (age, sex, type of maintenance, the treatment that has been given, the other reared buffalo calves, type or material  of the enclosure, environmental condition, or livestock care). Samples were collected di- rectly from the rectum and brought to the

Figure 1. Map of Sylhet district illustrating the study location (Sylhet Sadar, Zakigonj and Jaintapur)

laboratory in mini zip-locked polythene bags. Samples were dispatched in a cool box to prevent the eggs from developing and hatching. Finally, samples were stored in the laboratory in the refrigerator (4 °C) until further processed.

Examination techniques

Fecal samples were examined by fecal floatation using the saturated salt solution, sedimentation techniques for the presence of eggs/oocysts of parasites, and finally, positive cases were counted through the McMaster technique (Storey, 2015; Mo- hammed et al., 2017).

Flotation technique

3 gr of faeces were placed into a contain- er and mixed with 50 ml flotation fluid. The faeces were mixed thoroughly with satu- rated salt (NaCl) solution using a tongue blade and the resulting faecal suspension was poured through a tea strainer into an- other container. Taking the faecal suspen- sion from the container the test tube with the suspension was topped off, leaving a convex meniscus at the top of the tube and a coverslip was carefully placed on top of

the test tube. After 20 min, the coverslip was carefully lifted off of the tube, togeth- er with the drop of fluid adhering to it, and the coverslip was immediately placed on a microscope slide and examined under the microscope (Kandasamy et al., 2013).

Sedimentation technique

Three gr of faeces was weighed or mea- sured in a container and mixed with 45 ml tap water with a tongue blade. The faecal suspension was filtered through a tea strain- er into another container and poured into another test tube. The mixture was allowed to sediment for 5 min and (pipette, decant) the supernatant was removed very careful- ly. The sediment was resuspended in 5 ml of water and allowed to sediment for 5 min. The supernatant (pipette, decant) was dis- carded and the sediment stained by adding one drop of methylene blue and transferring the sediment to a micro-slide covered with  a coverslip (Marco et al., 2019).

McMaster egg count

Two gr of faeces was weighed,and added to 60 ml of ZnSO4 flotation solution (Sp. Gr. 1.18-1.20*) in a flask or beaker. The

contents were swirled vigorously to break down and homogenize the faecal sample. The contents were poured through a sieve or a cheesecloth-lined funnel into a second container. A tongue depressor or spatula was used to squeeze as much fluid as possi- ble from the material left in the sieve or fun- nel. While swirling vigorously to keep the material in suspension, a sample of the mix- ture was aspirated with a pipette and care- fully transferred to one of the chambers of the McMaster slide. The procedure was re- peated to fill the adjacent chamber. for a pe- riod of 1-3 minutes was given to allow eggs to float to the top and debris to fall to the bottom of the chamber. Eggs that fell within the gridded area of both sides of the cham- ber were counted under the microscope. The total number of eggs in the 2 chambers were multiplied by 100; this is the eggs per gr of faeces (EPG) (Mohammed et al., 2017).

Statistical analysis

Data were analyzed by Statistical Package for Social Science (SPSS version 20, USA.)

using the F test. For comparative analysis, data were analyzed by using paired sample t-test (Mostafa, 1989). P≤0.05 was consid- ered as significant.

Results

During this study one hundred seventy

(170) buffalo calves were examined through fecal sample examination, of which 62 (36.47%), were found infected with gas- tro-intestinal (GI) parasites (Table 1). Two species of GI parasites were identified as 26.47% positive with Neoascaris vitulorum, 5.88% with Strongyloides sp. and 4.12% Strongyle type. EPG (Egg per gram of fe- ces) was also determined in the study and the range of EPG varied among the parasites. The highest EPG count was detected in N. vitulorum (50-1950) followed by Strongy- loides sp. (50-150) and Strongyle type (50- 150). Mean EPG count was also higher in N.vitulorum (252.22±60.86) followed by Strongyloides sp. (80±13.33) and Strongyle type (64.29±14.29).

Table 1. Overall prevalence of gastro-intestinal parasites in buffalo calves in Sylhet district

Name of parasite

No. of animal affected

Prevalence (%)

EPG (Egg per gram of Feces)

The prevalence of GI parasites in males and females was not significant (P≤0.05). Females (55.88%) were more vulnerable to parasitic GI infection than males (44.12%). In both the sexes, the prev- alence was the highest in the case of N.vi- tulorum. In the male highest prevalence

was observed in N.vitulorum (22.67%) followed by Strongyloides sp. (5.33%) and Trichostrongylus sp. (2.67%), whereas in the female the prevalence was 29.47% in N.vitulorum followed by 6.32% in Stron- gyloide sp. and 5.26% in Strongyle type (Table 2).

Table 2. Sex related prevalence of gastro-intestinal parasites in buffalo calves in Sylhet district

Categories                    Name of parasite (Sex)

No. of animal affected

EPG (Egg per gram of feces) Prevalence (%)                     

             
Level of significance                                                                                                                             0.078**

** P≤0.05, N = Total number of samples collected

In the present study, Table 3 showed that the seasonal effect of gastro intestinal parasitism in buffalo calves was not significant (P≤0.05). The prevalence of GI parasites was relatively higher in the summer season (58.82%) than in the rainy season (41.18%). Our study revealed that buffalo calves were more susceptible to GI parasitic infection in the summer season

than the rainy season. In the summer season, prevalence was somewhat higher in the case of N.vitulorum (27%) followed by Strongyloi- des sp. (4%) and strongyle type (2%) and in the rainy season, the prevalence was relative- ly higher in the case of N.vitulorum (25.71%), followed by Strongyloides sp.(8.57%) and strongyle type (7.14%).

             
Table 3. Seasonal prevalence of gastro-intestinal parasites in buffalo calves in Sylhet district

Categories

No. of ani-

EPG (Egg per gram of feces)

(Season)                    Name of parasite             mal affected           Prevalence (%)                                                                      

             
Level of significance                                                                                                                              0.056**

** P≤0.05, N = Total number of samples collected

Table 4 showed that the prevalence of GI parasites was significantly (P≤0.05) higher in calves aged > 1-day to 6 months than in calves of aged > 7-12 months. In calves (1-day to 6 months), the prevalence was relatively high- er in the case of N.vitulorum (36%) followed by Strongyloides sp. (2.67%) and strongyle type (2.67%). In calves of 7 to 12 months

age, N.vitulorum was the main gastrointesti- nal parasite recorded (23.64%) followed by Strongyloides sp. (5.45%) and strongyle type (3.64%). In calves of 13 to 18 months of age, the higher prevalence was also recorded  in the case of N.vitulorum (12.50%),  followed by Strongyloides sp.(12.50%) and strongyle type (7.50%).

             
Table 4. Age related prevalence of gastro-intestinal parasites in buffalo calves in Sylhet district

Categories

No. of ani-

EPG (Egg per gram of feces)

(Age)                      Name of parasite              mal affected           Prevalence (%)                                                                      

** P≤0.05, N = Total number of samples collected

Discussion

In the present study, 34.47% buffalo calves were found to be infected with GI parasites. Other studies reported that 47%, 75% and 73.58% buffalo calves were pos- itive for GI parasites in Sindh of Pakistan, Toba Tek Singh in Pakistan and  Punjab state of India respectively (Bachal et al., 2002; Bilal et al., 2011; Singh et al., 2012). Jyoti et al. (2011) reported that N. vitulo-

rumin buffaloes were 8.47% which is low- er than the present finding (26.47%),  but the present finding is lower than the other study where it was recorded that N. vitulo- rum in buffaloes was 33% (Bachal et al., 2002). Azam et al. (2002) observed that the overall prevalence of strongyle type was 21.19% in Khadagzai. This finding is also higher than the current findings (4.12%). Jyoti et al. (2012) observed that the prev-

alence of Strongyloides sp. was 28.45%, which was much higher than the present study (5.88%). In the present study, infec- tion of Strongyloides sp. (5.88%) is higher than strongyle type (4.12%). There was no significant (P≤0.05) difference in the rate of infection between the male and female buf- falo calves. It was observed that, the prev- alence of GI parasite was slightly lower in male (44.12%) than in female (55.88%) an- imals which agrees with the previous report of higher prevalence (48.30%) of helminths in female than in male (45.12%) in buffalo calves (Bachal et al., 2002). However, the findings of the present study disagree with other studies Jyoti et al. (2012) and Bilal et al. (2002) that recorded the prevalence of helminths in the male (74.49% and 88.38%) which was higher than the prevalence of the female (72.95% and 59.46%) respectively. There was no sex variation in GI parasitic infection reported by Azhar et al. (2002). He observed that buffaloes of either sex were equally affected. This difference be- tween the findings cannot be explained precisely. The alteration in the physiologi- cal condition of the female animals during pregnancy and lactation (production activi- ty) may be the cause of the higher percent- age of infection in the females. The individ- uals (female) are more susceptible to any infection due to the secretion of the higher levels of prolactin and progesterone hor- mones (Lioyd, 1983). Seasonal variation of the year had no significant (P≤0.05) effect on the prevalence of GI parasitic infection in buffalo calves. There was significant dif- ference in the prevalence of GI parasites in buffalo calves in different seasons of the year. In this study, relatively higher infec- tion with GI parasites was observed in the summer season (58.82%) than the rainy

season (41.18%) but Jyoti et al. (2012) re- ported that in the rainy season, the preva- lence was (74.67%) that was slightly high- er than the summer season (73.59%). The present findings showed that the highest prevalence N. vitulorum was 27% in summer and 25.71% in winter season respectively. The present findings are higher than Singh et al. (2012), who reported that the highest prevalence of N. vitulorum was (12.85%) in the rainy season. The contrast between the earlier and present findings may be due to the methods used in the study and also variation in the geographical locations of the studies. Moreover, in the present study, the study period was divided into two sea- sons for which different seasons had made some overlapping seasons of months that might have formed some contradictions. The age of the buffalo calves had no sig- nificant (P≤0.05) effect on GI parasitic in- fection in our study. The prevalence of GI parasitic infections was significantly higher (P≤0.05) in calves of 1-6 months (41.18%) than calves of 7-12 months (32.35%) and calves of 13-18 months of age (23.53%), re- spectively. The present findings were con- sistent with the earlier report of Bilal et al. (2011), who reported that the higher prev- alence of helminths infection was found in calves of 1-6 months of age (86.67%) com- pared to 7-12 months of age (60%). On the other hand, the present findings disagreed with Jyoti et al. (2012), who reported that calves of  > 3 months of age were more sus- ceptible to parasitic infection  than  calves of < 3 months of age. They recorded that parasitic infection in >3 months of age was 68.62%, and< 3months of age was 79.12%. The higher infection rate was recorded in older buffaloes than the young buffaloes (Azhar et al., 2002), which is not in agree-