Clinico-therapeutic Studies on Haemonchosis in Goats

Haemonchosis spp. infection is most common and economically important strongyle nematode infection in sheep and goats (Brahma et al., 2015). The disease is more rampant and has been reported from all over the world where sheep and goats are reared (Odeniran et al., 2016). Goats may become carriers once they acquire the infection, therefore, act as a potential source of pasture contamination (Shrivastava et al., 2018). In goats and sheep, the prevalence may be as high as 100 per cent over a period in monsoon season (Futagbi et al., 2015). Acute haemonchosis is characterized by dark-coloured faeces with blood and sudden death of affected animals (Kandasamy et al., 2013). Diagnosis is mainly based on clinical signs, faecal examination and molecular techniques. A novel system called FAMACHA was developed in South Africa for screening of anaemia in sheep and goat. This technique can be applied at farm level to mitigate the problems related to treatment like, drug resistance in Haemonchus contortus (Kaplan et al., 2004). There are several classes of anthelmintic drugs effective against Haemonchus spp. like benzimidazoles (e.g., albendazole), imidazothiazoles, macrocyclic lactones, salicylanides (e.g., closantel) etc. but resistance to all classes of anthelmintic drugs have been seen (Sager et al.,  2009). Thus, the treatment should be carried out with some recent anthelmintic because they have advantages over traditional anthelmintic, also a new indigenous anthelmintic should be tried with the idea that it will be cheaper easily available and less anthelmintic resistant.

In-vitro evaluation of anthelmintic activity of plant extract residues
 
Aqueous herbal extract of papaya seed was prepared and the dilution of extract was made in different concentrations viz. 2.5 mg/ml, 5 mg/ml, 10 mg/ml, 15 mg/ml in NSS for evaluation of their anthelmintic activity (Sharma et al., 1971). Adult Haemonchus worms were collected from the abomasum of freshly slaughtered goats and transferred in beaker containing NSS at 37°C (Bhatnagar et al., 1961). Anthelmintic efficacy was observed by keeping 30 worms in different dilutions of test extract in NSS. Number of live and dead adult worms were counted at 1 hr, 2 hr and 3 hr. intervals. The corrected mortality for each extract was calculated by considering the mortality of worms, if any, in the NSS.
       
Corrected mortality was calculated as per the formula given by Sangwan and Sangwan (1988).  
Therapeutic trials
 
Therapeutic trials were conducted to determine the efficacy of herbal anthelmintic (Carica papaya) in comparison to chemical anthelmintic to control the nematode worms of goats. FAMACHA technique was applied for the screening of the cases for faecal, haematological and biochemical examination by FAMACHA score card. Adult goats naturally infected with nematodal worms without any previous history of use of anthelmintic was randomly selected for this study.
 
Collection and examination of faecal sample
 
Faecal samples were collected from the animals showing clinical signs and symptoms of haemonchosis. The samples were examined by using direct and indirect methods as described by Soulsby (1982). The speciation of parasites was done as per egg and larval morphology.
 
Experimental protocol
 
Following faecal examination, 12 positive goats were selected and were divided into two groups- T₁ and T₂. Another Group T was kept as positive control. The group T₁ was treated by closantel (10 mg/kg body wt.) as single dose and group T₂ was treated by Aq. Extract of Carica papaya seed extract. All the clinical parameters were studied at 0-, 7- and 14-day interval periods.
 
Collection of blood samples for hematology and biochemical study
 
Blood samples were collected for the study of different haemato biochemical parameters from all the goats on day 0, 7 and 14. The parameters studied were Hb, TEC, TLC, total protein, albumin, globulin, serum glucose, ALT, GGT, Na, K, calcium and phosphorus.
 
Efficacy of the drugs
 
It was assessed based on percent reduction in faecal egg count (EPG), restoration of haemogram and different bio-chemical parameters, absence of clinical signs and improvement in general condition after treatment.
 
Statistical analysis
 
Statistical analysis was conducted to determine the difference among the groups at the same sampling time by using ANOVA, post- hoc Tukey’s test with general linear models in SPSS 16.

In vitro trial of anthelmintic activity of Carica papaya extract and closantel drug
 
Corrected mortality of adult worms with Closantel and aq. Extract of Carica papaya (2.5, 5, 10, 15 mg/ml) has been shown in the Table 1. In vitro trial revealed that closantel @ 10 mg/ml had corrected mortality 26, 28 and 25 at 1 hr, 2 hr, and 3 hrs respectively with respect to control 0, 2 and 5 in the same interval period. Among different concentration the corrected mortality of aq. Extract 15 mg/ml were found to be 29, 27 and 25 at 1 hr, 2 hrs and 3 hrs of trial with respect to control. Thus, based on in vitro trial and corrected mortality percentage the herbal extract of 15 mg/ml concentration was selected for further in vivo study along with closantel @ 10 mg/ml b.wt. Similar in vivo and in vitro studies on the efficacy of anthelmintic against Haemonchus in goats were done by Akhter et al., (2014). Unlike corrected mortality of adult worm of Haemonchus contortus, they used Egg Hatch Assay (EHA). Present findings were in accordance with Hoste et al., (2006) who reported the effect of tannin-rich plants on internal nematodes in ruminants. 
 

 
Haematological observations
 
The present study revealed that goats suffering from Haemonchosis had significantly (p<0.05) lower Haemoglobin, PCV and TEC (Table 2). Rapid blood loss in Haemonchosis affected goats was due to blood sucking nature of the worms (Besier et al., 2016). Significant increase in Hb, PCV and TEC in both groups T₁ and T₂ indicated Carica papaya extract efficacy in treatment and increase in haematological parameters was comparable to closantel in this study. Significant decrease in total leucocytic count (TLC) was found on 7^th and 14^th day and it might have occurred due to antioxidant property of Carica papaya (Farida and Iswahyani, 2018). Similar observation was also made by (Qamar and Makbool, 2012).
 

 
Biochemical observations
 
Decreased total serum protein (Table 3) in the infected goats in present study may be attributed to haemodilution, a compensatory mechanism for the abomasal haemorrhages. The mean albumin levels were also significantly lower that might be due to abomasal damage, selective loss of albumin which is smaller in size and osmotic sensitivity to fluid movement (Tanwar and Mishra, 2001). After treatment with closantel and Carica papaya extract non- significant improvement in total protein level while significant improvement in albumin level was observed (Bandyopadhya and Dasgupta, 2000).
 

       
Lower blood glucose (Table 3) finding in Haemonchosis affected goats was in accordance with Arora et al., (2001). This might be due to reduction in food intake and absorption from injured gut and due to elevation of blood gastrin level (Nicholls et al., 1988). Non-significant improvement was noticed in treatment group on 14^th day post treatment.
       
Serum enzyme ALT concentration in goats varied indifferently while GGT had significant variation (Table 4). Findings of Kumar et al., (2013) supported increased levels of ALT in the present study. Non - significant lowering of mean ALT level was observed in both treatment group on 7^th and 14^th days post treatment. Significant decreases in the mean GGT levels were also observed in group T₂ on 14^th day of treatment. The elevation of serum ALT and AST level indicated some disruptive activities in organs or altered membrane permeability or may be due to lack of excretion (Bordoloi et al., 2012). High level of ALT and AST also occur due to traumatic damage to the abomasal mucosa caused by larvae and adult (Al-Zubaidy et al., 1987).
       

 
Major electrolytes Na and K and macro minerals Ca and P (Table 5) were significantly decreased from the unaffected control level. Murad et al., (2018) also reported 7.2% drop in calcium level in blood during experimental Haemonchus spp. infection. Gilani, (1981) reported hypophosphatemia after conducting the trial on haemonchosis. Significant improvement in mean Na, K, Ca, and P post treatment in both Closantel and Carica papaya group might be due to elimination of parasites, improvement in digestion and absorption.
 

 
EGG Per gram (EPG)
 
The mean value of EPG (Table 6) showed variable results. Al Omar et al., (2016) also reported variability in faecal egg count of Haemonchus contortus infection in their study. This might be due to individual animal’s genetic makeup and the environment to which the animals are exposed (Gadahi et al., 2009). It was found that significant improvement in faecal egg count was reported in both treatment groups on 7_th and 14_th day post treatment with slightly better Faecal egg count Reduction Test (FECRT) in group T₂ in comparison to T₁. This signifies that the Carica papaya seed extract works satisfactorily in reducing the faecal egg count and can be a good alternative to chemical anthelmintic.
 

 
FAMACHA
 
Mean±S.E of FAMACHA score in different groups have been shown in Table 7. FAMACHA score system measuring from 1 to 5 was used to assess the degree of anaemia in goats in all three groups. The post treatment values with each of the drug decreased significantly on 7^th and 14^th day of treatment with lowest FAMACHA score found in T₂ group (Carica papaya group). As per Pugh and Baired (2012) plants that contains tannin have been found to reduce Faecal Egg Count and worm burden in goats. Jaiswal et al., (2008) and Iliev et al., (2017) also reported the anthelmintic property of papaya seed extract. Reduction in FAMACHA Score in both the treatment group suggest that closantel is a preferred choice of anthelmintic for Haemonchosis in goats with rotational use of Carica papaya seed extract is alternative and economical choice for the treatment.
 

 
FAMACHA score, EPG and haematological parameters
       
Haematological correlation with FAMACHA Score and EPG has been shown in Table 8. Strongly negative correlation was found between FAMACHA vs Hb, PCV and TEC. Highly significant negative correlation between EPG and PCV, EPG and Hb and positive correlation between FAMACHA score and EPG was observed. These findings are in accordance with the findings of Bala et al., (2015) and Baihaqi et al., (2020).
 

The present study concluded that FAMACHA technique can be used for detecting the severity of anaemia in Haemonchus infected goats. Strong negative correlation was found between Hb and EPG, FAMACHA score and Hb and strong positive correlation was found between FAMACHA score and EPG. Closantel and Carica papaya seed extract (aq.) were found to be effective in in vitro and in vivo trial. Strong negative correlation was found between Hb and EPG, F.S and Hb and strong positive correlation was found between F.S and EPG. Based on biochemical and haematological examination, it was found that herbal anthelmintic (Carica papaya seed extract) can be a good alternative for controlling Haemonchosis in goats. Planned grazing, high plain of nutrition, early diagnosis by FAMACHA technique and use of anthelmintic can prevent drug resistance and help in preventing mortality in goats from Haemonchus.