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Correlation of Monoclonal Antibody Based Latex Agglutination Test with Haematological and Physiological Parameters for Diagnosis of Bubaline Trypanosomosis

Pradeep Kumar Sukhdeep Vohra Krutanjali Swain Arun K. Sangwan
Vol 9(2), 25-31
DOI- http://dx.doi.org/10.5455/ijlr.20180803031953

Around 400 blood samples of buffaloes belonging to thirty villages in seven blocks of district Hisar, Haryana were screened for the presence of Trypanosoma evansi using Giemsa stained thin blood smears and monoclonal antibody based latex agglutination test (mAb-LAT). The haemoglobin (Hb; g/dL), packed cell volume (PCV; %) and the body temperature (oF) were also examined. Blood examination did not reveal any sample positive for T. evansi infection, however, mAb-LAT showed 95 (23.7%), 101 (25.2%), 84 (21.0%), 38 (9.5%) and 82 (20.5%) samples showing strong, moderate, weak positive, doubtful and negative reactivity, respectively. Significant differences were observed in Hb and PCV values between positive samples when compared to suspected and negative samples inmAb-LAT test, while for temperature significant difference was seen between positive and negative samples only. These results pointed towards lower sensitivity of blood smear examination over mAb-LAT test for diagnosis of bubaline trypanosomosis.


Keywords : Buffaloes Haryana mAb-LAT Trypanosoma evansi

The water buffalo (Bubalus bubalis) have been well domesticated in the Indian sub-continent by the era of medieval period. They are an important source of milk supply in today’s period and yield nearly three times milk when compared to cows (TNAU Agritech Portal, 2014). More than half of the total milk produced (55%) in the country is contributed by 47.22 million milch buffalo heads. Among the various diseases reported in buffaloes, trypanosomosis caused by Trypanosoma evansi commonly known as ‘surra’ is of foremost importance. It is an important vector borne disease of tropical and sub-tropical regions of the world (Prasad, 2010). It is mainly transmitted mechanically by various biting flies like Tabanus, Stomoxys, Lyprosia and Haematopota species (Reid, 2002). The disease is pathogenic and widely distributed in Africa, South and Central America and Asia (Luckins, 1998; Pathak and Khanna, 1995; Wernery and Kadeen, 1995). The disease is often fatal in the absence of treatment and exhibits specific clinical signs (anaemia, loss of weight, abortion, stretching of neck, head pressing and death) which are variable from one host and one place to another (Desquesnes et al., 2013). Bubaline trypanosomosis has often been observed as a chronic entity, however, under stress conditions it may become sub-acute or acute (Ray et al., 1992). Recently, a case of surra in human has also been reported in India (Joshi et al, 2005), thereby, assuming its significance as a possible zoonotic threat in near future (Laha and Sasmal, 2007). In India, T. evansi infection is widely prevalent in different parts and is of significant economic importance in livestock production (Juyal et al., 2007). The disease is more common in Rajasthan, Punjab, Haryana, Uttar Pradesh, and Gujarat (Pathak et al., 1995).

The conventional parasitological examinations frequently fail to detect any patent infections and often scanty parasitaemia is observed in peripheral blood even in chronic cases (Killick, 1968).The biochemical tests viz., formol-gel test, mercuric chloride test, flocculation test, turbidity test and concentration techniques like haematocrit centrifugation and Diethyl amino ethyl (DEAE) cellulose purification are reported to be less sensitive than animal inoculation methods which too are laborious, time consuming and unsuitable for large scale use in the field (Pegram and Scott, 1976). In the past, Rayulu (2007) developed a monoclonal antibody based latex agglutination test (mAb-LAT) to detect the circulating antigens of T. evansi in sera of domestic animals at LUVAS, Hisar, Haryana. The mAb-LAT test was also employed by Shyma (2009) and Verma (2010) giving high prevalence of 60.23% and 32.7%, respectively. Thus, the present study was designed to assess the prevalence of trypanosomosis in buffaloes in Hisar district using blood smear and mAb-LAT and correlate its results with haematological and physiological parameters for the confirmation of the disease.

Materials and Methods

Area of Study                                                                         

The district Hisar is located in Haryana, India with 29° 5’5″N latitude and 75° 45’55″E longitudes in western Haryana with the average elevation of 215 m (705 ft) above mean sea level. The region is part of the alluvial Ghaggar-Yamuna plain.

Experimental Design

The study was divided into two groups i.e. group 1 comprising of 300 blood samples collected from randomly selected buffaloes and group 2 of 100 blood samples from suspected cases reported at Teaching Veterinary Clinical Complex (TVCC), Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar or Government Veterinary Hospitals (GVH) at Hisar district from November, 2015 to May, 2016. The blood samples were collected from thirty villages located in seven blocks of district Hisar i.e. Hisar I, Hisar II, Hansi I, Hansi II, Naranaund, Barwala and Agroha of Hisar district. The rectal temperature (oF) was recorded from the presented animal before the collection of blood. The blood samples were collected aseptically and examined for the presence or absence of T. evansi by Giemsa staining of thin blood smears. The haemoglobin (Hb; g/dl) and packed cell volume (PCV; %) were estimated using Sahli’s method and micro-haematocrit method, respectively as per Jain (1986). The serum was also isolated from the collected blood samples and monoclonal antibody based latex agglutination test (mAb-LAT) was performed as per method of Rayulu et al. (2009). The result was interpreted as per the time of agglutination i.e. time of agglutination is 0-5 min it was interpret as strong positive; 5-10 minutes as moderate positive; 10-13 minutes as weak positive; 13-15 min as suspected or doubtful and more than 15 min was negative result.

The other parameters (or risk factor) pertaining to the host like sex (male and female) and age i.e. heifer (upto 3 year), adult (age between 3-8 years) and old (above 8 years of age) were also recorded. The results of mAb-LAT were also compared with Hb, PCV and temperature values of the animals, to draw conclusions, if any. The results were analyzed using student’s T-test and Dunken’s Multiple Range Test (DMRT).

Results and Discussion

For the detection of Trypanosoma evansi  in buffaloes, blood samples were collected from block Hisar I (villages: Balawas, Chirod, Dhansu, Dubeta, Gangwa, Harita, Ladwa), Hisar II (villages: Arya Nagar, Balsmandh, Dhiranwass, Dobhi, Kharia, Ludas), Hansi I (villages: Umra, Kanwari), Hansi II (villages: Bhaklana, Bhatoljatan, Bhatol Rangran, Jeetpara, Dharam Kheri, Khanda Kheri), Narnaund (villages: Rajpura, Shapura, Sulchani, Majra, Milakpur), Agraha (villages: Agroha, Kalirawan) and Barwala (villages: Bhabalpur, Barwala). A total of 400 blood samples which include 51 male, 349 female and 75 heifers, 272 adults and 53 old animals were examined. Maximum of 42 blood samples were collected from village Ludas, Hisar II. The blood smear examination using Giemsa staining did not reveal any case positive for T. evansi infection. Similar were the results of Haque et al. (2012) who found no blood sample positive by Giemsa stained blood smears out of 104 blood samples examined at different districts of Punjab. The lower efficacy of blood smear was also found by Dhami et al. (1999) from Punjab and Swarnkar et al. (1993) from Rajasthan. The results of mAb-LAT test for detection of T. evansi antigen on isolated serum samples of buffaloes from group 1 and group 2 are given in Table 1.

 

 

Table 1: Results of mAb-LAT test for detection of T. evansi antigen

Group mAb-LAT
Strong Moderate Weak Suspected Negative
Group 1 (n=300 60 (20.0) 69 (23.0) 60 (20.0) 34 (11.3) 77 (25.6)
Group 2 (n=100) 35 (35.0) 32 (32.0) 24 (24.0) 4 (4.0) 5 (5.0)
T-test value 3.05260** 1.79406NS 0.85049 NS 2.16594* 4.43344**
Total 95(23.7) 101 (25.2) 84(21.0) 38 (9.5) 82(20.5)

*Significant at p<0.05; **Significant at p<0.01; NS: Non-significant; Figure in parenthesis shows per cent

The mAb-LAT show 95 (23.7%) sample as strong positive, 101 (25.2%) moderate positive, 84 (21%) weak positive, 38 (9.5%) doubtful and 82 (20.5%) negative for trypanosomosis. A total of 280 (70%) samples were found positive for T. evansi as per mAb-LAT revealing a higher prevalence rate in buffaloes at Hisar. Similar findings have been reported in the past by Rayulu (2007) and Shyma (2009) who reported 37.19% and 78.2% prevalence of trypanosomosis, respectively in buffaloes at Hisar using mAb-LAT. As regards the physiological and haematological parameters, the haemoglobin progressively increased from strong (9.87) to negative (12.59) results in group 1 (randomly selected) and from strong (9.9) to negative (12.7) results of mAb- LAT in group 2 (suspected cases) (Table 2).

Table 2: Physiological and haematological parameters in Group 1 (randomly selected) and Group 2 (suspected) buffaloes of Hisar, Haryana

mAb-LAT results Haemoglobin (Mean ± S. E.)
Group 1 Group 2 t test value
Strong 9.87d± 0.21 9.90d±0.21 0.0839 NS
Moderate 10.97c±0.11 10.87c±0.18 0.4726 NS
Weak 11.75b±0.13 12.27a±0.20 2.2022*
Suspected 12.38a±0.15 12.36a±0.28 0.0785 NS
Negative 12.59a±0.07 12.70a±0.49 0.2202 NS
  PCV
Strong 31.57d±0.41 32.57d±0.0.66 1.2925 NS
Moderate 34.94c±0.35 34.61d±0.52 0.5279 NS
Weak 36.78b±0.35 37.55c±0.65 1.0358 NS
Suspected 38.26a±0.49 39.43c±0.95 1.0930 NS
Negative 39.17a±0.27 39. 00c±1.14 0.1441 NS
  Body Temperature
Strong 103.42a±0.13 103.63a±0.09 1.3832NS
Moderate 102.73b±0.11 102.92b±0.07 1.4853 NS
Weak 101.95c±0.13 101.83c±0.10 0.7694 NS
Suspected 101.69cd±0.18 102.21c±0.26 1.6464 NS
Negative 101.52d±0.11 101.32d±0.26 0.7190 NS

Mean bearing different superscripts in a column differ significantly (p<0.05).

The PCV values increased progressively from strong (31.57) to negative (39.17) results in Group 1 and from strong (32.57) to suspected (39.4) and remained almost constant in negative (39) results in Group 2 (suspected cases) (Table 2). In the past, Verma et al. (1976) and Singla et al. (1997) also revealed lower values of Hb and PCV in T. evansi infected calves. Regarding the temperature, there was decrease in the body temperature from strong (103.42oF) to negative (101.52oF) mAb- LAT in group 1and from strong (103.63oF) to negative (101.32oF) results in group 2 (Table 2). The values of body temperature in positive samples were significantly different from negative results in both samples collected from field as well as from TVCC/GVH. Payne et al. (1991) in the past have also reported marked effect on the temperature profiles of the buffaloes with T. evansi infection.

The statistical analysis of the results of Hb, PCV and temperature between Group 1 and Group 2 showed non-significant difference except the value of Hb in weak mAb-LAT results which was significant at P<0.05 by using ‘t test’ (Table 2). Comparison between the values of Hb of animals at different results of mAb-LAT using Dunken’s Multiple Range Test (DMRT) at 5% level showed significant difference of positive (strong, moderate and weak) results in comparison to suspected and negative results, however, the difference between suspected and negative results was non-significant. Similarly, values of PCV at different result of mAb-LAT using DMRT at 5% level showed significant difference between positive (strong, moderate and weak) when compared to suspected and negative results. The body temperature of animals with different results of mAb-LAT showed significant difference between the values of strong, moderate, weak and negative results using DMRT at 5% level showed significant.

Conclusion

In conclusion, the study revealed a higher prevalence (70.0%) of sub-clinical trypanosomosis among buffaloes of Hisar, Haryana by mAb-LAT test. The decreased values of Hb and PCV with increase in the temperature further supported the utilization of mAb-LAT test for diagnosis of bubaline trypanosomosis. The study confirmed the poor efficacy of blood smear examination in diagnosing bubaline trypanosomosis in field hospitals and suggested the use of other techniques for the diagnosis of trypanosomosis in domestic animals.

Acknowledgments

The authors are thankful to the Department of Veterinary Parasitology, College of Veterinary Science, LUVAS, Hisar for providing the necessary facilities.

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