Prevalence and in Vitro Culture of Trichostrongylus Spp . in Goat at Trishal , Mymensingh , Bangladesh

Received: Revised: Accepted: 2014–08–1


INTRODUCTION
Bangladesh is a developing country where goat ranks second in meat, milk, and skin production, representing about 28%, 23% and 28% respectively of the total livestock in Bangladesh (Banglapedia /goat/ 2013).But the productions are hindered by various types of infection.Among them parasitic infection is most common.Geographical location and agro-ecological condition in Bangladesh are highly conducive to the growth and multiplication of a large number of parasite species.Trichosrtongylus spp. is one of the important nematode of alimentary canal that causes a great harm to the ruminants.Nematodes especially Trichosrtongylus spp. is prevalent worldwide (Dhar et al., 1982;Ha and Vu, 2013;Rehbein et al., 2012).Trichostrongylus spp. is one of the common, pathogenic and also prevalent nematodes recorded in Bangladesh (Haq and Shaikh, 1968).The development, distribution, or migratory behavior of the free living stages (i.e.eggs, developing and infective larvae) of gastro-intestinal nematodes on pasture is primarily weather related.Environmental factors influenced both the development and survival of the larvae on pasture and their distribution on the herbage.In the pasture, deposited eggs hatch and develop to the infective L3 stage.Under optimal conditions as moisture and temperature, larvae of Trichostrongylus spp.reach the infective stage in approximately 5-6 days.Developmental time depends on environmental condition.Eggs hatch and develop more slowly at lower temperatures.The rate of development increases to a maximum at higher temperatures after which development will be adversely affected and death of the larvae occurs (Soulsby, 1982).The primary factors that affect the hatching of eggs, development and survival larvae are temperature and moisture and vary according to the parasite.In vitro information about effects of temperature, pH, humidity, light and nutrients on the hatching of eggs and survival of larvae of Trichostrongylus spp.are essential to study the biology of the parasite.By considering these facts, the present study has been designed to determine the prevalence and to investigate the effects of temperature, pH,

Study Area
The study was carried out in different villages and slaughter houses of Trishal in Mymensingh, Bangladesh.Identification, hatching and observation of larvae were performed in the laboratory, Department of Parasitology, Bangladesh Agricultural University, Mymensingh.

Study Period
The study was conducted from January to May, 2014.

Selection of Goats
Goats (82) were selected randomly.The age of the goats were determined by examining the teeth (Samad, 2008).According to the age, goats were divided into two groups such as < 1.5 years (55) and >1.5 years (27) of age.The sex of the goats was also recorded before slaughter.

Collection of Fecal Sample
Eighty two (82) fecal samples were collected directly from the rectum of goats.After collection, samples were placed in a polythene bag and packed tightly by mixing with 10% formalin and brought to laboratory.

Collection of Visceral Sample
Sixty seven (67) visceral samples (abomasum, small and large intestine) were collected.After collection, sample was packed in a plastic bag and brought to laboratory.Collections of parasites from intestine were made following the standard procedure described by Rahman (1969).Identification of species with sex differentiation was based on the morphology as described by Soulsby (1982).

Recovery of Eggs
Matured eggs were recovered directly from the ovipositing female (Trichostrongulus spp.).For this, female parasites were crushed by using sterile mortar and pestle containing necessary amount of phosphate buffer solution (PBS).Debris was picked up with needles and the contents were transferred to a clear Petri dish through sieving.The suspension was gently stirred to make uniform solution.Eggs were counted by McMaster technique (Soulsby, 1982).

Effects of Temperature
To study the effects of temperature, the pre-counted numbers of eggs were suspended in a Petri dish containing culture media and incubated at various temperatures (37 °C, 27 °C, 18 °C, 8 0 C and 4 °C) up to 8 days.Identification of different stages of larvae was made on the basis of morphological descriptions described by Anon (1977), Soulsby (1982) and Rahman et al., (1996).

Effects of pH
To study the effects of pH, the different pH levels (2.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0) of the culture media were adjusted by adding glacial acetic acid or by adding Sodium hydroxide in PBS drop by drop with the help of dropper and stirred the media.The pH was detected by pH meter.Pre-counted eggs were mixed in the culture media having above mentioned pH levels and kept in room temperature.Observations were made by dissection microscope in every 24 hours up to 7 days.

Effects of Humidity
To determine the effects of relative humidity, the precounted number of eggs were mixed in the PBS and incubated at 70% -<80% and >80% -90% of relative humidity at constant 26 0 C temperature.Developmental stages were observed by dissection microscope for every 24 hours up to 7 days.

Effects of Light
To study the effect of light, pre-counted number of eggs counting culture media was covered with hard paper to make dark condition having facilities for air exchange keeping in room temperature.Control media was also kept in room temperature at light condition.Then observation was made in similar manner up to 8 days.

Effects of Different Media
To find out a suitable media for the cultivation of eggs, precounted eggs were cultured in phosphate buffer solution (PBS), normal saline (NS) and tap water (TW) and incubated at room temperature (26 0 C) and pH 6.5 in 80-95% Relative humidity and observed in similar procedure.

Effects of Different Nutrients
For the preparation of different nutrient media, 5%, 10%, 15% serum of goat was added separately to the PBS.A precounted number of eggs were suspended in each and every culture medium mentioned above and all were incubated at room temperature up to 8 days.Then the percentage of hatching eggs was calculated for every 24 hours.

Statistical Analysis
Student t-test was used to determine the significance (p < 0.05) among the different variables (Steel and Torrie, 1980).

Prevalence of Trichostongylus Spp. In Goats
In the present study, overall prevalence of Trichostongylus spp. in fecal sample and visceral sample were 20.73% (17/82) and 14.93% (10/67), respectively (Figure 1).More or less similar finding in fecal sample survey was also observed by Ha and Vu (2013).But the present study differs with Achi et al., (2003); Abebe and Esayas (2001) and Patel et al., (2001) who observed the prevalence rate were 46%, 64.3% and 9.2%, respectively.Females (23.08%) were 1.31 times more susceptible than males (18.61%) and it was statistically significant (p <0.001) (Figure 2).The exact cause of higher rate of infection of Trichostronglus spp.cannot be explained, but in general, higher level of prolactin and progesterone hormone suppress the immune system of the individual and make the individual more susceptible to any infection (Lloyd,1983).
During this study, it was also recorded that young (23.64%) were 1.78 times more prone to Trichostongylus spp.infection than adult (14.82%) and it was statistically significant (p < .001)(Figure 3).This may be due to low level of immunity in young animals.

In Vitro Culture of Trichostrongylus Spp. in Goats
Maximum Eggs were hatched at 18 0 C Temperature In the present study, developments of eggs were arrested at 4 0 C temperature and failed to develop even when returned to room temperature (15-20 °C) during the period of observations up to 15 days.Better results were observed at 37 0 C temperature but maximum (46.67%) hatching of eggs were at 18 0 C on day 5. Details were shown in Table 1.(1980) had similar observation.The exact mechanism of inactivation of eggs at lower temperature is not known but may be due to cold injury which squeezed the germinal mass and the eggs were devitalized.At 8 0 C, hatching of eggs started at day 3 which was relatively delayed than in other temperature.This finding was in concordance the findings of Soulsby (1982) who recorded that low temperature retarded the development of eggs and little development took place below 9 °C.In the present study, maximum hatching was observed at 18°C on day 5. Minato et al., (2008) found that 15°C temperature was shown to favor direct development, producing infective larvae.Islam and Ahmed (1987) observed that eggs hatched at 15-34 °C in 6 days which support the present study.At 27°C temperature, 4.76% eggs hatched on day 2 and maximum 38.09% of eggs hatched at day 5 and larvae attained infective stage at day 5.
Whereas Rahman et al., (1996) found that eggs hatched within 1-2 days at 26°C and reached at the infective stage within 3-4 days.Similar observations were made by Tripathi (1977) where he mentioned that 20°C -30°C was the most suitable temperature for hatching and development of Trichostrongylus spp.In this study, development and hatching of eggs was not observed in pH 2.0.Hatching of eggs was initiated from day 2 only in pH 6.0 and pH 7.0.Highest hatching was observed at pH 6.0 (32.30%) and lowest was at pH 4.0 (5.17%) at day 6 and day 7, respectively (Table 2).(1983) support this observation and interpret as it might be due to coagulation of germinal mass of the eggs due to high acidic pH level of the media.Ashad et al., (2011) and Stringfellow (1986) also found that maximum hatching of eggs and development of Trichostrongylus spp.larvae occurred at pH 6.0-7.0 which also support the present observation.Again developments were decreased at pH 9 (Table 2), which is possibly by the denaturation of egg mass in alkaline pH.Maximum (42.86%) eggs were hatched at >80-90 relative humidity at day 5 and minimum (14.28%) were observed at 70-<80 relative humidity on day 3. Eggs were arrested in their development on day 1 and 2 in both cases (Table 3).In this study, maximum (42.86%) eggs were hatched in case of relative humidity >80-90% which are less than the finding of Rahman et al., (1996), Rossanigo and Gruner (1994) and Berbigier et al., (1990) who found that optimum egg hatching and larval development and survival was 49.03%, 54% and 74%, respectively in the temperature range from 15°C to 20°C and at humidity from 79.5 to 95.5%.

Dark Condition is Favorable for Hatching of Eggs
Dark and light had no significant effect on development and hatching of eggs of Trichostrongylus spp.It was observed that no eggs were developed on first two days in both cases.But developments were better in dark than light in all the observed day (Table 3).Islam and Ahmed (1987) and Ashad et al., (2011) also found that dark condition was more effective than light on the hatchability of eggs of Trichostrongylus spp.

Maximum Hatchability of Eggs and Survivability of Larvae was Observed when Grown in PBS
Hatching of eggs was initiated on day 3 in all media such as Phosphate buffer saline (PBS), normal saline (NS) and tap water (TW).Initially, the rate of hatchability was relatively higher in PBS (16.67%) followed by TW (14.29%) and NS (7.10%).On three culture media, maximum hatchability was observed in PBS (38.89%) on day 5.It was found that 22.94% larvae survived in PBS whereas in NS and TW, 8.34% and 6.47% larvae survived, respectively on day 8 (Table 4).

Day
Effect of different humidity (%) Effect of light 70-<80 (N=21) >80-90(N=21) Dark (%)(N=16) Light (%)(N=16)  During this study, hatchability and survivability was maximum in PBS.These results were clearly similar to the results obtained by Islam et al., (2005) who achieved the highest L3 yield in PBS (23.82%).In this study, No development were seen in first two days.This observation differs with Veglia (1916) who found that L1 hatched from eggs within 14-17 hours.The variation in development and hatching of eggs may be due to other condition applied.

Nutrient is Essential on the Hatching of Eggs and the Survival of the Larvae of Trichostrongylus Spp.
In this study, hatching was initiated from day 2 and gradually increased in all the media but best result was found in PBS containing 15% serum which was initiated as 13.33% on day 2 and finally reached 46.67% on day 5. Death of larvae initiated on day 6 in all media; survival and longevity of larvae was maximum (19.08%) in PBS containing 15% serum (Table 5).PBS containing 15% serum is comparatively more enriched than other media.This enriched media ensured a balanced environment and provide protein and several vitamins.Paul (1965) supports the finding who recorded the increased survival of larvae in a uniform manner in a medium containing plentiful food source.
Values in the same row having different superscript are statistically significant (p<0.05);N= number of eggs and light on the hatching of eggs and survival of larvae of Trichostrongylus spp.

Figure 1 :
Figure 1: Over all prevalence of trichostrongylus spp. in goats

Table 1 :
Effects of Temperature on the hatching of eggs of Trichostrongylus spp.same column having different superscript are statistically significant (p<0.05);N= Number of eggs In the present study, No development were observed at low temperature (4 0 C).Ross and Small (1980) and Tripathi

Figure 2 :
Figure 2: Sex related prevalence of Trichostrongylus spp. in goats

Table 2 :
Effects of pH on the hatching of eggs of Trichostrongylus spp.

Table 4 :
Effects of media on the hatching of eggs and survival of larvae of Trichostrongylus spp.

Table 5 :
Effects of nutrients on the hatching of eggs and survival of larvae of Trichostrongylus spp.Values in the same row having different superscript are statistically significant (p<0.05);d= dead, l= live, D= day of experiment; N= number of eggs

Table 3 :
Effects of humidity, dark and light on the hatching of eggs of Trichostrongylus spp.