Occurrence of ticks and tick-borne mixed parasitic microbiota in cross-bred cattle in District Lahore, Pakistan

Hasan, M.; Roohi, N.; Rashid, M. I.; Ali, S.; Ul-Rehman, Z.

doi:10.1590/1519-6984.266721

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Brazilian Journal of Biology

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Original Article • Braz. J. Biol. 82 • 2022 • https://doi.org/10.1590/1519-6984.266721 copy

Occurrence of ticks and tick-borne mixed parasitic microbiota in cross-bred cattle in District Lahore, Pakistan

Ocorrência de carrapatos e microbiota parasitária mista transmitida por carrapatos em bovinos mestiços no distrito de Lahore, Paquistão

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

The present study was focused on the incidence of ticks and tick-borne diseases (TTBD) in cross-bred cattle (Friesian x Sahiwal) of two farms (n = 2548) in district Lahore, Pakistan. We collected total of 572 ticks (adults and nymphs) and blood samples (10 ml) for microscopic i.e., blood smear test - Giemsa Stain (BST) and molecular analysis; Reverse Line Blot-General Primer-PCR (RLB-PCR) and Specie Specific Primer PCR (SP-PCR) from infested cattle (n = 100) from months of April to September. Results: The tick specie identified was Rhipicephalus microplus at both farms, with significant difference in infestations rate amongst both farms (p< 0.0001). The cross-bred cattle having higher ratio of Friesian blood and lower ratio of Sahiwal blood were mostly infested by ticks (p < 0.0458) and haemoparasites (p <0.474) and vice versa. The SP-PCR showed higher number of haemoparasites infection than BST, which revealed 16% T. annulata (p < 0.0001 and k value 0.485, 0.0001), 51% B. bigemina (p < 0.0001 and k value 0.485, 0.0001) and 15% A. marginale (p < 0.001 and k value 0.207, 0.001), respectively. The single infection with B. bigemina was 34% (n = 34/100) and A. marginale 6% (n = 6/100). The double infection with T. annulata/B. bigemina was 8% (n = 8/100) and B. bigemina/A. marginale 1% (n = 1/100). Whereas the triple infection with T. annulata/B. bigemina/A .marginale was 8% (n = 8/100). The phylogenetic study of isolated sequence of T. annulata revealed close homology to isolates from Iran (87%), B. bigemina to isolates from Cuba (94 to 100%) and A. marginale with isolates from Pakistan (99 to 98%).

Keywords:
Rhipicephalus microplus; polymerase chain reaction; Theileria annulata; Babesia bigemina; Anaplasma marginale

Primer	Primer Sequence	Target Genome	Predicted amplicon size	References
Primer Set-A Forward Reverse	Reverse Line Blot (RLB) General primer 5ˊ- GAC ACA GGG AGG TAG TGA CAA G - 3ˊ 5ˊ- CTA AGA ATT TCA CCT CTG ACA GT - 3ˊ	18s rRNA gene	430 bp	Gubbels et al., 1999GUBBELS, J.M., DE VOS, A., VAN DER WEIDE, M., VISERAS, J., SCHOULS, L., DE VRIES, E. and JONGEJAN, F., 1999. Simultaneous detection of bovine Theileria and Babesia species by reverse line blot hybridization. Journal of Clinical Microbiology, vol. 37, no. 6, pp. 1782-1789. http://dx.doi.org/10.1128/JCM.37.6.1782-1789.1999. PMid:10325324. http://dx.doi.org/10.1128/JCM.37.6.1782-...
Primer Set-B Forward Reverse	T. annulata specific 5ˊ- CAA ATG AGC TTC TGG GGA GC - 3ˊ 5ˊ- TTC CTG CCA TTG CCA AAA GTC - 3ˊ	Cytochrome b gene	475 bp	Bilgic et al., 2010BILGIC, H.B., KARAGENÇ, T., SHIELS, B., TAIT, A., EREN, H. and WEIR, W., 2010. Evaluation of cytochrome b as a sensitive target for PCR based detection of T. annulata carrier animals. Veterinary Parasitology, vol. 174, no. 3-4, pp. 341-347. http://dx.doi.org/10.1016/j.vetpar.2010.08.025. PMid:20880635. http://dx.doi.org/10.1016/j.vetpar.2010....
Primer Set-C Forward Reverse	B. bigemina specific 5ˊ- GAC GAA TCG GAA AAG CCA CG - 3ˊ 5ˊ- AGA GGG ACT CCT GTG CTT CA - 3ˊ	18s rRNA gene	321 bp	Umber et al., 2020UMBER, R., SHAFQAT, S., MATIULLAH, K., IMRAN, R., HAROON, A. and ANEELA, D., 2020. Identification of 23 kD immunogen from native antigens of Babesia bigemina in splenectomized calf. International Journal of Agriculture and Biology, vol. 24, pp. 1788-1794.
Primer Set-D Forward Reverse	B.bovis specific 5ˊ- AAT ATG GGT TGG GCA ATG CG - 3ˊ 5ˊ- CCA CCC AAA ACA AGA GCA ACT - 3ˊ	Cytochrome b gene	269 bp	Umber et al., 2020UMBER, R., SHAFQAT, S., MATIULLAH, K., IMRAN, R., HAROON, A. and ANEELA, D., 2020. Identification of 23 kD immunogen from native antigens of Babesia bigemina in splenectomized calf. International Journal of Agriculture and Biology, vol. 24, pp. 1788-1794.
Primer Set-E Forward Reverse	A. marginale specific 5ˊ- CCT TAT GGG GTG GGC TAC AC - 3ˊ 5ˊ- CCC GAG AAC GTA TTC ACC GT - 3ˊ	16s rRNA gene	178 bp	Designed in current study

PCR Protocols for Heamoparasites
Primers	Cycles Number	Initial Denaturing	Denaturing	Annealing	Extension	Final Extension
RLB	40	95°C for 4 min	94°C for 35 seconds	51°C for 35 seconds	72°C for 35 seconds	72°C for 10 min
T.annulata	35	95°C for 4 min	95°C for 30 seconds	58°C for 30 seconds	72°C for 1 min	72°C for 10 min
B.bigemina	37	95°C for 1 min	95°C for 30 seconds	57°C for 30 seconds	72°C for 30 seconds	72°C for 5 min
B.bovis	40	94°C for 5 min	94°C for 5 seconds	60°C for 45 seconds	72°C for 45 seconds	72°C for 10 min
A.marginale	35	95°C for 5 min	94°C for 30 seconds	55°C for 30 seconds	72°C for 1 min & 30 seconds	72°C for 5 min

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[1] *e-mail: imran.rashid@uvas.edu.pk