Haematological and biochemical changes in Fasciola gigantica infected buffaloes fed on diet containing deoiled mahua (Bassia latifolia) seed cake

Abstract

A study was conducted to evaluate the effect of condensed tannins and saponins from deoiled mahua seed cake (DMSC) on the development of fasciolosis. Fifteen male buffaloes were randomly divided into three groups of five each and were orally infected with Fasciola gigantica metacercaria at 800 per animal. DMSC was included in the concentrate mixture of group 2 (M5) and group 3 animals (M10) at 5 and 10%, respectively and group 1 remained as the non-supplemented control (C) group. Hypo-albuminemia, hyper-globulinemia and decrease in Albumin:Globulin (A:G) ratio were the significant features in C and M5 group. The group M10 was characterised by higher haemoglobin and packed cell volume and lower plasma total protein concentration, antibody response and faecal egg count than C and M5 group. This combined with higher plasma albumin, creatinine and lower globulin concentration in group 3 animals were conclusive of lower intensity infection as compared to other groups. It is concluded that condensed tannin and saponins of DMSC (10% of DMSC in concentrate mixture) possess remarkable anti-fasciolic activity.

Keywords:

• fasciolosis
• deoiled mahua seed cake
• condensed tannins
• blood parameters

Introduction

Fasciola gigantica is one of the most important parasites of cattle and buffaloes in the tropical areas of the world. Even though a wide range of control strategies are available, none is completely effective and free from side effects. Therefore, novel and inexpensive control procedures are essential for control of Fasciola. Recent trend is towards the phytotherapy where the plant secondary compounds are effectively used for helminth control. Forages containing condensed tannins (CT) and saponins have the potential to control anthelminthic resistant gastrointestinal parasites. They have been shown to decrease faecal egg counts in sheep and goats and may decrease hatch rate and larval development in faeces (Nguyen et al. 2005). The CT at high concentrations from L. leucocephala was most effective in reducing migration of exsheathed larvae (Kahn and Diaz-Hernandez 2000). The present study was carried out to assess the effect of CT and saponins of deoiled mahua seed cake (DMSC) (Bassia latifolia) on haematological and biochemical parameters in buffaloes experimentally infected with F. gigantica.

Materials and methods

Fifteen male Murrah buffaloes (2 years, 284±5.41 kg) were dewormed and divided into three groups of five each. All of them were orally administered with 800 freshly collected metacercaria of F. gigantica. Animals of all the groups were fed standard ration to meet out their nutrient requirement. Group 1 (control, C) animals were fed on concentrate mixture (CM) containing deoiled soyabean meal. DMSC was included in the ration of group 2 animals at 5% level (M5) and group 3 at 10% level (M10) partially replacing deoiled soyabean meal. The DMSC contained 5% CT and 5.1% saponins. The experimental diets were isonitrogenous and isocalorific. They were adapted to the dietary regimen for one month prior to the infection. Clean and fresh drinking water was provided twice daily.

Faecal samples were collected from the rectum weekly. The number of F. gigantica eggs was determined by Stoll's dilution method (Soulsby 1982) which was modified suitably. Blood samples were collected from all the animals at 0, 8, 16 and 24 weeks post infection (WPI) to assess haematological, biochemical and humoral antibody response. The blood was processed for the assessment of total leukocyte counts (TLC), haemoglobin (Hb) and PCV according to standard procedures. Plasma was harvested and stored (–20°C) until further analysis. Total protein, albumin, globulin, glucose, creatinine, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were estimated in serum using kits of Span diagnostic limited. Antifasciola antibody in serum was determined by ELISA (Kumar et al. 2008).

The data were subjected to analysis of variance for completely randomised design using SPSS software (10.0). Post hoc mean comparisons were carried out on all significant treatment means using Duncan's test.

Results and discussion

The most noticeable feature in haematology was that Hb and PCV were significantly (P<0.01) higher due to supplementation of 10% DMSC. A temporal decrease in Hb concentration was noted at 16 and 24 WPI (Table 1). Following the trend in Hb, an elevated PCV was observed in group M10. Blood loss and subsequent anaemia were less in group M10 compared to other groups. In a study by Ganga et al. (2007), it was observed that haemoglobin and PCV started dropping from 4 WPI to 10 WPI in infected buffaloes. It could be sequel to traumatic lesion caused and hepato-toxic effect of metabolites released by the juvenile flukes in the liver and impaired erythropoiesis. The TLC remained unaffected among the groups.

Table 1. Haematological and blood biochemical parameters in buffalo calves experimentally infected with F. gigantica metacercaria.

	Periods (weeks)
Periods	0			8			16			24
Treatments	C	M5	M10	C	M5	M10	C	M5	M10	C	M5	M10	SEM	T	P	T×P
Hb (%)	12.24	13.70	14.26	13.68	14.34	15.20	11.38	12.98	14.54	12.80	11.10	13.40	0.861	0.013	0.048	0.491
PCV (%)	37.40^ab	36.00^ab	40.60^ab	38.00^ab	43.00^a	44.60^a	31.40^c	39.40^ab	42.80^a	39.20^ab	35.80^ab	37.60^ab	2.022	0.005	0.037	0.036
TLC (×10^3/µl)	21.99	13.78	24.2	20.40	16.98	17.55	17.18	17.12	19.62	13.80	14.42	15.25	3.181	0.258	0.206	0.632
Protein (g/dl)	7.91^ab	7.49^abc	7.54^abc	7.56^abc	7.91^ab	7.80^ab	7.69^ab	6.80^bc	6.35^c	8.38^a	7.97^ab	7.09^bc	0.261	0.002	0.000	0.035
Albumin (g/dl)	3.81	4.04	4.20	3.97	4.09	4.29	3.17	3.15	3.54	3.92	3.96	4.13	0.128	0.003	0.000	0.941
Globulin (g/dl)	4.10	3.46	3.34	3.59	3.83	3.51	4.52	3.64	2.81	4.46	4.01	2.97	0.325	0.000	0.889	0.190
AG ratio	0.93	1.18	1.28	1.11	1.11	1.28	0.77	0.95	1.29	0.91	1.01	1.44	0.112	0.000	0.324	0.544
Glucose(mg/dl)	50.52	57.64	51.04	54.89	55.47	54.39	61.35	63.32	63.14	76.38	72.50	68.27	3.441	0.468	0.000	0.657
Creatinine (mg/dl)	1.65	1.55	1.73	1.22	1.22	1.34	1.00	0.99	1.12	1.50	1.52	1.73	0.082	0.020	0.000	0.978
AST (IU/l)	321.4^a	318.5^a	210.3^b	157.6^bc	173.9^bc	145.9^bc	202.0^bc	213.0^b	314.0^a	148.5^bc	149.5^bc	141.5^c	14.116	0.556	0.000	0.000
ALT (IU/l)	49.48^a	21.00^bc	41.00^ab	32.05^abc	36.55^abc	32.35^abc	26.50^abc	26.60^abc	25.10^abc	28.41^abc	29.93^abc	10.81^c	5.302	0.165	0.007	0.008
Note: Means having different superscript letters in a row differ significantly with respect to treatment and period interaction. T, Treatments; P, Periods.

Saponins of deoiled mahua seed cake supplementation did not alter plasma glucose concentration (Table 1). Ferre et al. (1994) reported decrease in plasma glucose values from 40 day post infection in lambs infected with F. hepatica. It was due to depression in voluntary feed intake and hepatic glycogenic pathways (Phiri et al. 2007). Total protein and globulin (g/dl) fraction were significantly (P<0.001) higher in group C and M5, whereas albumin and Albumin:Globulin (AG) ratio were higher in group M10. Over 24 week study period, significantly (P<0.001) lower concentration of total protein and albumin was observed at 16 WPI. F. gigantica was reported to cause hypoalbuminemia and hyperglobuminemia with a resultant decrease in A:G ratio (Swarup 1986). The current findings were in accordance with above observation. Since the liver is of considerable importance in protein metabolism, any damage to its cells caused by migrating juvenile flukes will be reflected on the total serum proteins and the decrease in the level will depend on the size of the inoculum and duration of infection (Mbuh and Mbwaye 2005).

Plasma creatinine (mg/dl) concentration peaked at 24 WPI. Creatinine concentration in group C and M5 was significantly (P<0.05) lower compared to group M10. The same finding was observed in F. hepatica infected sheep. A significantly decreased creatinine concentration points to muscle loss caused by a deficiency of protein and is valuable in the prognosis of recovery from anaemia (Matanovic et al. 2007).

The plasma enzymes AST and ALT were not significantly altered due to infection/supplementation of CT and saponins. However, towards the end of the study both hepatic enzymes were elevated indicating liver damage. Previous research work indicated an increase of AST was a common feature in fasciolosis (Mbuh and Mbwaye 2005). The increase in plasma enzyme may be related to the inflammatory state of liver and to tissue destruction provoked by the parenchymal migration of immature flukes during the first stages of fasciolosis (Mbuh and Mbwaye 2005). The magnitude and duration of elevated serum enzymes during experimental infection is proportional to the extent of liver damage. Serum level of enzyme ALT was significantly higher in infected goats than control up to 15 WPI. However, AST level remained unaffected due to infection (Mbuh and Mbwaye 2005).

The fluke eggs appeared in the faeces of all animals by 102 days post infection (DPI). The lowest egg count was observed in animals of group M10 followed by group M5 (Figure 1). Comparatively lower egg output in animals supplemented with 10% DMSC is an indication that secondary compounds of DMSC exerted their effects on metacercaria of F. gigantica in the gastro intestinal tract and thus, reduces the infestation of F. gigantica. The changes in antibody levels in the sera of F. gigantica infected and DMSC supplemented animals is depicted in (Figure 2). In all the groups, antibody titer at 492 nm rose at 8 WPI and in group C and M5, it remained almost constant thereafter and peaked at 24 WPI. Animals of group M10 showed the lowest OD values indicating establishment of lower intensity infection compared to those of group C and M5. This is yet another indication that dietary CT and saponins have helped the animal to combat the adverse effect of fasciolosis. Zhang et al. (2006) reported a significant increase in production of specific antibodies against F. gigantica from 3 WPI which peaks at 13 WPI and decrease, which is an indicative of development of flukes into adults and reached the bile ducts at about 13 WPI. Once the flukes were in bile ducts, immune response of the host to flukes appears to diminish. Specific antibodies were detected earlier than eggs, suggesting that serology could be used in the diagnosis of fasciolosis in buffaloes.

Figure 1.  Pattern of excretion of F. gigantica eggs by different groups of experimental animals.

Figure 2.  Antibody response against affinity purified F. gigantica conventional antigen in different experimental groups.

It is concluded that the supplementation of DMSC was effective to combat the adverse effect of fasciolosis in buffaloes, which was observed from changes in some serological parameters, humoral antibody response and eggs per gram (EPG) count in buffaloes fed on diet containing 10% DMSC. Further, the study paved the way for development of nutritional amelioration to reduce the deleterious effect of fasciolosis and economic losses of the farmers.

Acknowledgements

Authors are thankful to the Director, Indian Veterinary Research Institute for providing required facilities to carry out the research work and Indian Council of Agricultural Research for financial assistance as AP Cess fund scheme.