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Development of Novel Multiplex PCR for Diagnosis of Co-infected Hemo-parasites in Cattle

DOI: 10.18805/IJAR.B-4695    | Article Id: B-4695 | Page : 1504-1509
Citation :- Development of Novel Multiplex PCR for Diagnosis of Co-infected Hemo-parasites in Cattle.Indian Journal of Animal Research.2021.(55):1504-1509
Pankaj Kumar, Abhay Kumar, Kamal Sarma, Paresh Sharma, Rashmi Rekha Kumari, Manish Kumar pankajvet@gmail.com
Address : Division of Livestock and Fisheries Management, ICAR Research Complex for Eastern Region, Patna-800 014, Bihar, India.
Submitted Date : 26-06-2021
Accepted Date : 31-08-2021

Abstract

Background: A novel, rapid and specific multiplex polymerase chain reaction was developed to diagnose hemo-parasitic infection in bovine blood co-infected with three of the most common hemo-parasites. 
Methods: The diagnostic process relied on the detection of the three different bovine hemoparasites isolated from red blood cells (RBCs) of cattle (N=30) by conventional Giemsa stained blood smear (GSBS) and confirmed by multiplex PCR. The multiplex PCR system was used to diagnose GSBS positive blood samples (N=12) found infected or co-infected with hemoparasites. The designed multiplex primer sets was attempted to amplify 205, 313 and 422 bp fragments of apocytochrome b, sporozoite and macroschizont 2 (spm2) and 16S rRNA gene for Babesia bigemina, Theileria annulata and Anaplasma marginale, respectively.
Result: This multiplex PCR was sensitive with the ability to detect the presence of 150 ng of genomic DNA. The primers used in this multiplex PCR also showed highly specific amplification of specific gene fragments of each respective parasite. Comparing the two detection methods revealed that 58.33% of specimens showed concordant diagnoses with both techniques. The specificity, positive predictive value and kappa coefficient of the agreement was highest for diagnosis of B. bigemina and lowest for A. marginale. The overall Kappa coefficient for diagnosis based on GSBS for multiple pathogens compared to multiplex PCR was 0.56, slightly behind the threshold of 0.6 of agreement. Therefore, confirmation should always be based on PCR to rule out false positives due to differences in subjective observations, stain particles and false negatives due to low parasitemia. The simplicity and rapidity of this specific multiplex PCR method make it suitable for large-scale epidemiological studies and follow-up of drug treatments.
 

Keywords

Anaplasma Babesia Co-infection multiplex PCR Theileria

References

  1. Balakrishnan, N. (2017). Current scenario of tick-borne diseases in India-A review. Journal of Communicable Diseases. 49(2): 6-13 
  2. Bilgiç, H.B., Karagenç, T., Simuunza, M., Shiels, B., Tait, A., Eren, H. and Weir, W. (2013). Development of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bovis and Anaplasma marginale in cattle. Experimental parasitology. 133(2): 222-229. 
  3. El-Sayed, A. and Kamel, M. (2020). Climatic changes and their role in emergence and re-emergence of diseases. Environmental science and pollution research international. 27(18): 22336-22352. doi:https://doi.org/10.1007/s11356-020- 08896-w. 
  4. Ganguly, A., Bisla, R.S., Singh, H., Bhanot, V., Kumar, A., Kumari, S., Maharana, B.R. and Ganguly, I. (2017). Prevalence and haematobiochemical changes of tick borne haemoparasitic diseases in crossbred cattle of Haryana, India. Indian Journal of Animal Sciences. 87(5):552-557. 
  5. Ganzinelli, S., Benitez, D., Gantuya, S., Guswanto, A., Florin Christensen, M., Schnittger, L. and Igarashi, I. (2020). Highly sensitive nested PCR and rapid immunochromatographic detection of Babesia bovis and Babesia bigemina infection in a cattle herd with acute clinical and fatal cases in Argentina. Transboundary and Emerging Diseases. 67: 159-164. doi:10.1111/tbed.13435.
  6. Jain, N.C. (1999). Essentials of Veterinary Hematology. Lea and Febiger, Philadelphia.
  7. Jayalakshmi, K., Sasikala, M., Veeraselvam, M., Venkatesan, M., Yogeshpriya, S., Ramkumar, P.K., Selvaraj, P. and Vijayasarathi, M.K. (2019). Prevalence of haemoprotozoan diseases in cattle of Cauvery delta region of Tamil Nadu. Journal of Parasitic Diseases. 43(2): 308-312. doi:https://doi.org/ 10.1007/s12639-019-01094-7.
  8. Kala, S.K., Deo, B.G. and Kumari, N. (2018). Epidemiological aspects of theileriosis in c attle in and around Patna, Bihar, India. International Journal of Current Microbiology Applied Sciences. 7: 1183-1191 
  9. Knight, P.A., Williamson, S.W., Brown, C.G.D., Bell-Sakyi, L., Kirvar, E., Kinnaird, J., Hussain, K., Hall, R., Boulter, N. and Tait, A. (1998). Identification of a Theileria annulata antigen expressed in multiple stages of the parasite life cycle. Experimental Parasitology. 90(1): 110-121. 
  10. Kumar, B., Mondal, D. and Jithin, M. (2019). Evaluation of reactive oxidative damage on erythrocytic cells due to clinical babesiosis in lactating cows. International Journal of Livestock Research. 9(9): 55-64 
  11. Kumar, P., Kumar, P., Roy, R.K., Kumari, R.R., Kumar, A., Sarma, K., Sharma, P. and Kumar, M. (2021). Mixed infection of tick-borne haemo-parasites in water buffalo and associated pathological responses and treatment. Indian Journal of Animal Research. doi:10.18805/IJAR.B-4450.
  12. Kumari, R.R., Kumar, R., Kumar, P. and Kumar, M. (2019). Emergence and variations in disease ecology of tick-borne bovine theileriosis in East India. International Journal of Livestock Research. 9(11): 12-25. doi: 10.5455/ijlr.201909 2006 1236.
  13. Kundave, V.R., Ram, H., Banerjee, P.S., Garg, R., Mahendran, K., Ravikumar, G.V.P.P.S. and Tiwari, A.K. (2018). Development of multiplex PCR assay for concurrent detection of tick borne haemoparasitic infections in bovines. Acta Parasitologica. 63(4): 759-765.
  14. Landis, J.R. and Koch, G.G. (1977). An application of hierarchical kappa-type statistics in the assessment of majority agreement among multiple observers. Biometrics. 33(2): 363-374. doi:10.2307/2529786.
  15. Larcombe, S., Kolte, S.W., Ponnudurai, G., Kurkure, N., Magar, S., Velusamy, R., Rani, N., Rubinibala, B., Rekha, B., Alagesan, A. and Weir, W. (2019). The impact of tick- borne pathogen infection in Indian bovines is determined by host type but not the genotype of Theileria annulata. Infection, Genetics and Evolution. 75: 103972. doi:https:/ /doi.org/10.1016/j.meegid.2019.103972.
  16. Pain, A., Renauld, H., Berriman, M., Murphy, L., Yeats, C.A., Weir, W., Kerhornou, A., Aslett, M., Bishop, R., Bouchier, C., Cochet, M., Coulson, R.M., Cronin, A., de Villiers, E.P., Fraser, A., Fosker, N., Gardner, M., Goble, A., Griffiths- Jones, S., Harris, D.E., Katzer, F., Larke, N., Lord, A., Maser, P., McKellar, S., Mooney, P., Morton, F., Nene, V., O’Neil, S., Price, C., Quail, M.A., Rabbinowitsch, E., Rawlings, N.D., Rutter, S., Saunders, D., Seeger, K., Shah, T., Squares, R., Squares, S., Tivey, A., Walker, A.R., Woodward, J., Dobbelaere, D.A., Langsley, G., Rajandream, M.A., McKeever, D., Shiels, B., Tait, A., Barrell, B., Hall, N. (2005). Genome of the host-cell transforming parasite Theileria annulata compared with T. parva. Science. 309: 131-133.
  17. Prabhakaran, H.S., Ghosh, K.K., Kumari, R.R., Kumar, P. and Kumar, M. (2021). Evaluation of sporozoite and macroschizont antigen (Spm2) of Theileria annulata for its diagnostic potential. Ticks and Tick-Borne Diseases. 14(4): 101691.
  18. Roy, S., Bhandari, V., Barman, M., Kumar, P., Bhanot, V., Arora, J.S., Singh, S. and Sharma, P., (2021). Population genetic analysis of the Theileria annulata parasites identified limited diversity and multiplicity of infection in the vaccine from India. Frontiers in microbiology. 11: 3471. 
  19. Tian Z, Tian, Z., Du, X., Du, J., Gao, S., Yu, R., Hassan, M.A., Liu, G., Luo, J. and Yin, H. (2018). Development of an indirect ELISA based on the recombinant Spm2 protein for detection of tropical theileriosis. Acta Tropica. 182: 232-236. 
  20. Trevethan, R. (2017). Sensitivity, Specificity and Predictive Values: Foundations, Pliabilities and Pitfalls in Research and Practice. Frontiers in Public Health. 5: 307. 
  21. Turkson, P.K. and Ganyo, E.Y. (2015). Relationship between haemoglobin concentration and packed cell volume in cattle blood samples. Onderstepoort Journal of Veterinary Research. 82(1): 1-5. 

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