Trop Anim Health Prod DOI 10.1007/s11250-015-0835-2
SHORT COMMUNICATIONS
First molecular evidence of the transplacental transmission of Theileria annulata Vikrant Sudan 1 & Shanker Kumar Singh 1 & Amit Kumar Jaiswal 1 & Rahul Parashar 1 & Daya Shanker 1
Received: 5 January 2015 / Accepted: 15 April 2015 # Springer Science+Business Media Dordrecht 2015
Abstract Bovine tropical theileriosis (BTT) is a serious hindrance in the cattle upgradation programme using the exotic germplasm. There is a wide range of variations in the pathobiology alongside clinical symptoms of the animals suffering from BTT. The present paper communicates the first report about the transplacental transmission of T. annulata in a cross bred 2-day old calf. T. sergenti, T. lestoquardi and T. equi are known to undergo transplacental transmission, but baring a single citation in literature, there are no records about the transplacental transmission of T. annulata.
2015). The global annual losses due to BTT are estimated to be of US$800 million (Brown 1997) while in India alone, the disease accounts for an approximate annual loss of US$384.3 million (Minjauw and McLeod 2003). The present paper describes the first molecular evidence regarding the transplacental transmission of T. annulata in a 2-day-old cross bred calf from its immune carrier dam.
Keywords Bovine tropical theileriosis . Calf . Theileria annulata . Transplacental transmission
A crossbred female calf, weighing about 25 kg, was born with normal size, vitality and physiological functions. Anamnesis revealed that the dam recovered from acute clinical course of BTT during the gestation period of the calf. The calf was reported to be in good health initially but later developed sudden pyrexia, laboured breathing, anorexia and adopted lateral recumbence within 36 h of birth. The peripheral blood smear of the calf was made, and blood of both the dam and the calf were taken in EDTA for DNA isolation. Alongside the ticks present on the body of both dam and calf were also taken for salivary glands isolation.
Introduction Bovine tropical theileriosis (BTT) is an important vectorborne protozoan disease affecting the health and productivity of domestic cattle in tropical and subtropical regions of the world. The disease is caused by an apicomplexan parasite Theileria annulata and involves Hyalomma anatolicum anatolicum as its vector in the Indian subcontinent (Gill et al. 1978). BTT is responsible for high rates of mortality in dairy cattle and about 200 million animals are supposed to be exposed to its risk (Radostits et al. 1994; Sudan et al. 2014,
* Vikrant Sudan
[email protected] 1
College of Veterinary Sciences and Animal Husbandry, U. P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan (DUVASU), Mathura 281001, India
Materials and methods
DNA isolation DNA was isolated using the standard phenol chloroform method with minor modifications (Pruvot et al. 2013). Briefly, 100 μl of blood was added into a 1.5-ml tube containing 500 μl of denaturing solution (guanidinium thiocyanate) and vortexed at high-speed for 5 min. A total of 150 μl each of chloroform and phenol were added to it and vortexed for 5 min. The microtube was then centrifuged at 13,000 rpm (15,493×g) for 5 min, and the supernatant was collected. This process was repeated twice. A total of 400 μl of supernatant was collected and 1 ml of absolute ethanol was added to
Trop Anim Health Prod Table 1
Primer sequences along with expected amplicon size and the thermal cycling profile
Primer name
Primer sequence
TAMS F (5′)-GTAACCTTTAAAAACG-(3′) TAMS R (5′)-GTTACGAACATGGGTT-(3′) TASP F (5′)-GCGAATGTGGTCCATTTCTTCC-(3′) TASP R (5′)-GAAGAATGATCCACAACATTGCG-(3′) Thermal cycling profile Initial denaturation Denaturation TAMS primer 94 °C; 60 s 94 °C; 45 s × 30 cycles TASP primer 94 °C; 60 s 94 °C; 45 s × 30 cycles
it, and the sample was left overnight at −20 °C. After that, the sample was centrifuged 13,000 rpm for 10 min, and the pellet was washed with 75 % alcohol twice. The pellet was finally air dried and later re-suspended into 50 μl of TE buffer (Tris, EDTA). Thus, the final prepared sample was concentrated 2:1 compared to the initial blood sample. The ticks were dissected and the salivary glands were taken out. Later on, the salivary glands were minced and 50 μl of nuclease-free water was added to them. The mixture was boiled for 5 min and the supernatant was used as a DNA template.
Product size (bp)
Reference
721
d’Oliveira et al. 1995
561
Habibi et al. 2008
Hybridization 55 °C; 60 s
Extension 72 °C; 60 s
Termination 72 °C; 300 s
57 °C; 60 s
72 °C; 60 s
72 °C; 300 s
PCR primers and conditions Two sets of primer one encoding the 30-kDa major merozoite surface antigen of T. annulata (TAMS) (d’Oliveira et al. 1995) and the other for T. annulata surface protein of schizont stage (TASP) (Habibi et al. 2008) were custom synthesized. The sequences of the primers, the expected amplicon size alongside the thermocyclic profile of the primers, are given in Table 1. PCR reactions were set up into 25 μl volume containing 12.5 μl PCR Master Mix (0.05/μl Taq DNA polymerase in
Lane M: 100bp plus DNA ladder Lane 1: Known positive T. annulata sample Lane 2: Known negative T. annulata sample
Lane M: 100bp plus DNA ladder
Lane 3: Salivary gland of tick of dam
Lane 1: Known positive T. annulata sample
Lane 4: Salivary gland of tick of calf
Lane 2: Known negative T. annulata sample
Lane 5: Blood from dam
Lane 3: Blood from dam
Line 6: Blood from calf Fig. 1 PCR amplification using TAMS primer set. Lane M: 100 bp plus DNA ladder; lane 1: known positive T. annulata sample; lane 2: known negative T. annulata sample; lane 3: salivary gland of tick of dam; lane 4: salivary gland of tick of calf; lane 5: blood from dam; line 6: blood from calf
Line 4: Blood from calf Fig. 2 PCR amplification using TASP primer set. Lane M: 100 bp plus DNA ladder; lane 1: known positive T. annulata sample; lane 2: known negative T. annulata sample; lane 3: blood from dam; line 4: blood from calf
Trop Anim Health Prod
reaction buffer, 4 mM Mgcl2, 0.4 mM dATP, 0.4 mM dCTP, 0.4 mM dGTP, and 0.4 mM dTTP), 1.5 μl of each primer set (15 pmol), 2 μl of the DNA template and total volume was made up to 25 μl using Nuclease free water. DNA from the blood as well as from the salivary glands of ticks of both calf and dam were used. The amplified PCR amplicons were analyzed by agarose gel electrophoresis in 1.5 % agarose gel.
Results and discussion Clinical inspection of affected calf revealed dull appearance of hair coat along with infestation of H. anatolicum anatolicum ticks on its body. The conjunctival mucous membrane was icteric with a few petechial haemorrhages. Ample nasal and ocular discharge was visible. Palpation of lymph nodes revealed normal size and consistency besides the absence of lymphadenopathy. Microscopic examination of Giemsastained blood smear revealed characteristic piroplasms of Theileria spp. in majority of erythrocytes (>60 %). Agarose gel electrophoresis of both blood and the salivary gland samples yielded desired amplicons (Figs. 1 and 2). The normal incubation period of T. annulata varies from 7 to 24 days (Taylor et al. 2007), and the affected animal start showing symptoms of BTT after 7 days post tick bite (Taylor et al. 2007; Godara et al. 2009). The animal in the present study developed signs of BTT within few hours after birth; hence, the possibilities of transmission of T. annulata by ticks are very limited. Interestingly, Godara et al. 2009 reported BTT in a day-old calf infected with T. annulata based on the morphology of the parasite. The selected TAMS primers are highly specific for T. annulata and do not show cross reactivity with T. parva, T. mutans, T. sergenti, T. buffeli, T. velifera, and T. taurotragi (d’Oliveira et al. 1995). However, they show cross reactivity with T. lestoquardi. In order to rule out the possibility of T. lestoquardi, TASP primers were used which amplify T. annulata and but at the same time do not show any amplification with T. lestoquardi (Habibi et al. 2008). Reports of vertical transmission of various Theileria species are well-documented in literature. Theileria equi in mare (Phipps and Otter 2004; Sudan et al. 2013), T. lestoquarti in ewes (Zakain et al. 2014) and T. sergenti in cows (Baek et al. 2003) all undergo vertical transmission. Baring a single citation, regarding vertical transmission of T. annulata (Godara et al. 2009), no valid documentation exists about the transplacental transmission of BTT. In conclusion, this is the first report of molecular validation of transplacental transmission of T. annulata in a neonatal cross bred from India. Further detail studies are thereby warranted to undermine the possible factors attributing to this type of unusual transmissions. Acknowledgments The authors are highly thankful to the Honb’le Vice Chancellor DUVASU for the facilities provided.
Conflict of interest The authors declare that they have no conflict of interest.
References Baek, B.K., Soo, K.B., Kim, J.H., Lee, B.O., Jung, J.M., Onuma, M., Oluoch, A.O., Kim, C.H. and Kakoma, J., 2003. Verification by polymerase chain reaction of vertical transmission of Theileria sergenti in cows. Canadian Journal of Veterinary Research, 67, 278–282. Brown, C.G.D., 1997. Dynamics and impact of tick borne diseases of cattle. Tropical Animal Health and Production, 29, 15–35. d’Oliveira, C., Van-der Weide, M., Habela, A., Jacquiet, P. and Jongejan, F., 1995. Detection of Theileria annulata in blood samples of carrier cattle by PCR. Journal of Clinical Microbiology, 33(10), 2665– 2669. Gill, B.S., Bhayyacharyulu, Y. and Singh, A., 1978. Chemoprophylaxis with oxytertracycline drug in the immunization of cattle against Theileria annulata. International Journal of Parasitology, 8, 467– 469. Godara, R., Sharma, R.L. and Sharma, C.S., 2009. Bovine tropical theileriosis in a neonate calf. Tropical Animal Health and Production, 42(4),551–553. Habibi, G., Bozorgi, S., Esmaeil-Nia, K., Najjar, E. and Mohammadipour, A.R. 2008. Detection and Discrimination of Theileria annulata and Theileria lestoquardi by using a single PCR. Archives of Razi Institute, 63(1), 47–52. Minjauw, B. and McLeod, A., 2003. Tick-borne diseases and poverty. Theimpact of ticks and tick-borne diseases on the livelihood andmarginal livestock owners in India and Eastern and SouthernAfrica. Research Report, DFID Animal Health Programme,Centre of Tropical Veterinary Medicine, University of Edinburgh. Phipps, L., P. and Otter, A., 2004. Transplacental transmission of Theileria equi in two foals born and reared in the United Kingdom. Veterinary Record, 154, 406–408. Pruvot, M., Kamyingkird, K., Desquesnes, M., Sarathapan, N. and Jittapalapong, S., 2013. The effect of the DNA preparation method on the sensitivity of PCR for the detection of Trypanosoma evansi in rodents and implications for epidemiological surveillance efforts. Veterinary Parasitology, 191, 203–208. Radostits, O.M., Blood, D.C. and Gay, C.C., 1994. Veterinary Medicine- A text book of the diseases of Cattle, Sheep, Pigs, Goats and Horses. 8th edn. Bailliere Tindall, London, UK. pp 1210–1212. Sudan,V., Jaiswal, A.K., Srivastava, A., Saxena, A. and Shanker, D., 2013. A rare clinical presentation of transplacental transmission and subsequent abortion by Babesia (Theileria) equi in a mare. Journal of Parasitic Diseases DOI 10.1007/s12639-013-0337-y. Sudan, V., Sharma, R.L., Yadav, R., and Borah, M. K., 2014. Turning sickness in a riverine buffalo naturally infected with Theileria annulata and its successful therapeutic management. Comparative Clinical Pathology, 23, 39–42. Sudan,V., Jaiswal, A.K., Parashar, R. and Shanker, D., 2015. A duplex PCR-based assay for simultaneous detection of Trypanosoma evansi and Theileria annulata infections in water buffaloes. Tropical Animal Health and Production, DOI 10.1007/s11250-015-0808-5. Taylor, M.A., Coop, R.L., Wall, R.L., 2007. Veterinary Parasitology. 3 rd edn. Blackwell Publishing Ltd, UK. pp 109–113. Zakian, A., Nouria, M., Barati, F., Kahroba, H., Jolodar, A. and Rashidi, F., 2014. Vertical transmission of Theileria lestoquardi in sheep. Veterinary Parasitology, 203, 322–325.
