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Original Article MOLECULAR DETECTION of ANAPLASMA SPP

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Original Article MOLECULAR DETECTION of ANAPLASMA SPP Bulgarian Journal of Veterinary Medicine, 2019, 22, No 4, 457465 ISSN 1311-1477; DOI: 10.15547/bjvm.2135 Original article MOLECULAR DETECTION OF ANAPLASMA SPP. IN CATTLE OF TALESH COUNTY, NORTH OF IRAN S. SALEHI-GUILANDEH1, Z. SADEGHI-DEHKORDI1, A. SADEGHI-NASAB2 & A. YOUSEFI3 1Department of Parasitology, Faculty of Paraveterinary Medicine, Bu-Ali Sina Uni- versity, Hamedan, Iran; 2Department of Clinical Sciences; Faculty of Paraveterinary Medicine, Bu-Ali Sina University, Hamedan, Iran; 3Young Researchers and Elites club, Science and Research Branch, Islamic Azad University, Tehran, Iran Summary Salehi-Guilandeh, S., Z. Sadeghi-Dehkordi, A. Sadeghi-Nasab & A. Yousefi, 2019. Mo- lecular detection of Anaplasma spp. in cattle of Talesh County, North of Iran. Bulg. J. Vet. Med., 22, No 4, 457465. Anaplasmosis is generally caused by intraerythrocytic rickettsia of Anaplasma genus and transmitted biologically and mechanically. The current study was designed to determine the prevalence of Anaplasma spp. in cattle in Talesh; one of the rainy Iranian counties in Gilan province, Iran. From May to November 2015, one hundred and fifty blood samples of cattle were collected from different regions in Talesh. DNA was extracted from blood samples and subsequently, 16S rRNA and MSP4 genes were analysed by Nested-PCR method for differentiation of Anaplasma spp. The results showed that 40.66% of blood samples were positive for Anaplasma spp. and that 24.66%, 35.33%, 9.33% and 12% of positive samples were infected with A. phagocytophilum, A. bovis, A. marginale and A. centrale respectively. Statistical analysis by Chi-square test did not show any significant rela- tionship between the presence of Anaplasma species and variables sex, age and tick infestation (P˃0.05). The 4 species of Anaplasma reported in this study are of potential importance for animal and public health. Key words: Anaplasma, cattle, tick-borne, Talesh, Iran INTRODUCTION Anaplasmosis is generally caused by in- is important in two respects: first, eco- traerythrocytic rickettsia of genus nomic losses from reduced milk produc- Anaplasma (Dumler et al., 2001). This tion, abortion and weight loss iinduced by bacteria is transmitted biologically by Anaplasma ovis, Anaplasma marginale, ticks and mechanically by Stomoxys and Anaplasma centrale and Anaplasma bovis Tabanus or blood contaminated instru- (Rymaszewska & Grenda, 2008). Second ments (Kocan et al., 2010). Anaplasmosis comes the zoonotic importance of Ana- Molecular detection of Anaplasma spp. in cattle of Talesh County, North of Iran plasma phagocytophilum (HGA) which Sampling causes a variety of clinical syndromes from From May to November 2015, one hun- mild in healthy subjects to severe in im- dred and fifty blood samples (each 2 mL) munocompromised or elderly people (Mur- were collected from the jugular vein of ray et al., 2005; Robinson et al., 2009). cattle (75 male; 75 female) in a sterile test Anaplasmosis generally occurs in sub- tube containing 1 mL 96% ethanol from tropical and tropical areas but as a result twelve different farms in the different re- of global warming, the distribution of dis- gions of Talesh county. ease and particularly anaplasmosis may be All applicable international, national, expected to change, due to the location and institutional guidelines for the care changes of biological vectors (Jonsson & and use of animals were followed. Reid, 2000; Kocan et al., 2010). Hence the present survey was conducted to de- DNA extraction termine the prevalence of Anaplasma spp. DNA of blood samples was extracted us- in cattle in Talesh county, one of the rainy ing the DNA extraction minikit (YTA, Iranian counties in Gilan province, Iran. Iran, Cat No: FABGK001) following the manufacturer protocol. Twenty μL Pro- MATERIALS AND METHODS teinase K and 200 μL BG Buffer were added to 50 mg of fixed blood sample, Study area mixed thoroughly by vortexing and incu- Talesh is located on the southwestern bated for 15 min at 60 °C. In the next coast of the Caspian Sea (37° 21'54" N, step, 200 μL ethanol (96%) was added to 50° 5' 34" E). Talesh County covers an the sample and mixed thoroughly by vor- area of 2,373 square kilometers area of texing for 30 s. The complete volume of Gilan Province. The annual average pre- solution was transferred into the BG co- cipitation and temperature are 1,360 mm lumn and was centrifuged at 8,000 rpm for and 24.9 °C respectively (Fig. 1). 1 min. Then BG column was placed to a new collection tube. The BG column was immediately washed twice with 500 μL and 750 μL, respectively W1 Buffer, cen- trifuged at 14000 rpm and then the flow- through was discarded. Subsequently 100 μl of Elution Buffer was added to the membrane center of BG column and cen- trifuged for 2 min at 14000 rpm to elute the DNA. The extracted DNA was ana- lysed in agarose gel and also by spectro- photometry. The ratio of OD260 to OD280 and the amount of extracted DNA and its purity were measured. PCR of 16S rRNA gene The Anaplasma spp. 16S rRNA gene was Fig. 1. Map of studied areas, sampling re- amplified by PCR in 50 μL reaction vo- gions in Talesh county (Gilan province, Iran). lume, containing 10 mM Tris-HCl 458 BJVM, 22, No 4 S. Salehi-Guilandeh, Z. Sadeghi-Dehkordi, A. Sadeghi-Nasab & A. Yousefi Table 1. Sequence details of primers used in PCR and Nested-PCR of 16S rRNA and MSP4 genes Primer Genbank No. Nucleotide sequences Nucleotide Target PCR 5' → 3' positions organism product P1 M60313 AGAGTTTGATCCTGGCT 1-20 Anaplasma 781 bp (16S rRNA) CAG spp. P2 AGCACTCATCGTTTACA 796-815 GCG P6 M73224 CTTTATAGCTTGCTATA 69-90 A. phago- 509 bp (16S rRNA) AAGAA cytophilum P4 GTTAAGCCCTGGTATTT 575-594 CAC P7 Af283007 CAAATCTGTAGCTTGCT 65-86 A.centrale 513 bp (16S rRNA) ACGGA P4 GTTAAGCCCTGGTATTT 575-594 CAC P8 AB196475 CTCGTAGCTTGCTATGA 68-89 A.bovis 509 bp (16S rRNA) GAAC P4 GTTAAGCCCTGGTATTT 575-594 CAC M-OM F HM640938.1 GGGAGCTCCTATGAATT 1-33 A.margina- 867 bp (MSP4) ACAGAGAATTGTTTAC le/A. ovis M-OM R CCGGATCCTTAGCTGA ACAGGAATCTTGC 839-867 M-MAF HM640938.1 CTGAAGGGGGAGTAAT 113-131 A. margi- 344 bp M-MAR (MSP4) GGG nale GGTAATAGCTGCCAGA 435-456 GATTCC (pH 9.0), 30 mM KCl, 1.5 mm MgCl2, μL reaction volume, containing 10 mM 250 μm of each dNTP, 0.5 μm of each Tris-HCl (PH 9.0), 30 mM KCl, 1.5 mM forward and reverse primers (P1/P2) (Ta- MgCl2, 250 μm of each dNTP, 0.5 μm of ble 1), 1.0 U Taq polymerase (Cinnagen, each forward and reverse primers specific Iran) and 2 μL of DNA in automated ther- for each species (Table 1), 1.0 U Taq po- mocycler (Astec, Germany) for 37 cycles. lymerase (Cinnagen, Iran) and 1 μL (di- Following an initial denaturation step of luted 1:200) of PCR products (781 bp) in 5 min at 95 °C, each cycle consisted of a automated thermocycler (Astec, Germany) denaturing step of 45 s at 94 °C, annealing for 35 cycles. Following an initial denatu- for 45 s at 58 °C and extension for 45 s at ration step of 5 min at 95 °C, each cycle 72 °C and was completed with the final consisted of a denaturing step of 45 s at extension step for 5 min. The PCR ampli- 94 °C, annealing for 45 s at 57 °C, exten- cons (781 bp) were analysed in 1.5% aga- sion step of 45 s at 72 °C and a final ex- rose gel stained with ethidium bromide tension step for 5 min at 72 °C. The PCR and UV condition (Fig.2A). amplicons (A. bovis: 509 bp; Fig. 2B, A. phagocytophilum: 509 bp; Fig. 3A), A. Nested-PCR of 16S rRNA gene centrale: 513bp; Fig. 3B) were analysed This technique was performed for detec- in 1.5% agarose gel stained with ethidium tion of different Anaplasma species in 25 bromide. BJVM, 22, No 4 459 Molecular detection of Anaplasma spp. in cattle of Talesh County, North of Iran PCR of MSP4 gene first step was performed for detection of A. marginale/ A. ovis in 25 μL reaction For primers design limitations that existed volume, containing 10 mM Tris-HCl (PH in the 16s rRNA gene for differentiation 9.0), 30 mM KCl, 1.5 mM MgCl , 250 between A. ovis and A. marginale, we 2 μm of each dNTP, 0.5 μm of each forward used the MSP4 gene. This technique in and reverse primers specific for A. mar- 1 2 3 4 5 6 M 1 2 3 4 5 6 7 8 9 M 10 Fig. 2. A. PCR products of Anaplasma spp. 16S rRNA gene (781 bp), lane 1: negative control, lane 2: positive control, lanes 3 to 6: Anaplasma spp., M: 100 bp DNA ladder B. Nested-PCR pro- ducts of A. bovis in 16S rRNA gene (509bp), Lanes 1 to 8: A. bovis, lane 9: positive control; lane 10: negative control. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 M A 1 2 3 4 5 6 7 8 9 10 11 12 M 13 B Fig. 3. A. Nested-PCR products of A. phagocytophilum in 16 srRNA gene (509 bp), lanes 2–5, 7–17, 20 and 21: A. phagocytophilum, lane 22: positive control, lane 23: negative control, M: 100 bp DNA ladder. B. Nested-PCR products of A. centrale in 16 srRNA gene (513 bp), lanes 2–11: A. centrale, lane 12: positive control, lane 13: negative control. 460 BJVM, 22, No 4 S. Salehi-Guilandeh, Z. Sadeghi-Dehkordi, A.
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