www.ijasvm.com IJASVM InternationalJournalofAgricultural SciencesandVeterinaryMedicine ISSN:2320-3730 Vol.5,No.1,February2017 E-Mail:editorijasvm@gmail.com oreditor@ijasvm.comm@gmail.com
Int. J. Agric.Sc & Vet.Med. 2017 Arthi A and Tresamol P V, 2017 Research Paper ISSN 2320-3730 www.ijasvm.com Vol. 5, No. 1, February 2017 2017 www.ijasvm.com. All Rights Reserved MOLECULAR IDENTIFICATION OF BABESIA CANIS VOGELI AND ITS THERAPEUTIC MANAGEMENT WITH IMIDOCARB Arthi A 1* and Tresamol P V 2 *Corresponding Author: Arthi A, arthivet@gmail.com A one and a half year old female Labrador was presented with a history of anorexia and vomiting. Hematological abnormalities were thrombocytopenia and monocytosis. Babesia canis piroplasms were detected on blood smear examination and the dog was treated with injection imidocarb at a dose of 6 mg/ kg body weight subcutaneously (SC) twice, with a two week interval. Hematology was performed on day s three and fourteen to assess the improvement. The present communication deals with the identification of the species/subspecies B. canis vogeli by Polymerase Chain Reaction (PCR) and the therapeutic management of the same. Keywords: B. canis vogeli, Imidocarb, Polymerase chain reaction, Thrombocytopenia INTRODUCTION Babesiosis is a tick borne hemoprotozoal disease and two species of babesia affect dogs. Babesia canis the large piroplasm is mainly transmitted by the brown dog tick Rhipicephalus sanguineous and has three subspecies. The three subspecies (currently claimed to be distinct species) of B. canis: B. rossi, B. canis and B. vogeli are morphologically identical but vary in geographic distribution, vector specificity, genetic characteristics and the clinical signs which they induce in dogs (Matjila et al., 2004). Transmission is also possible through iatrogenic means (contaminated needles and surgical instruments) and in utero from dam to puppies (Ayoob et al., 2006). Following is the molecular detection of babesiae subspecies and therapeutic management of canine babesiosis with injection imidocarb. MATERIALS AND METHODS A one and a half year old female Labrador was presented to the University Veterinary Hospital, Kokkalai, Thrissur with a history of anorexia and vomiting since few days. Clinical examination revealed lymphadenopathy, pyrexia (102.8 o F) and 1 M.V.Sc Scholar, Department of Veterinary Epidemiology and Preventive Medicine, College of Veterinary and Animal Sciences, Mannuthy, Thrissur 680651, Kerala Veterinary and Animal Sciences University, Kerala, India. 2 Professor and Head, Department of Veterinary Epidemiology and Preventive Medicine, College of Veterinary and Animal Sciences, Mannuthy, Thrissur 680651, Kerala Veterinary and Animal Sciences University, Kerala, India. 87
Int. J. Agric.Sc & Vet.Med. 2017 Arthi A and Tresamol P V, 2017 palor of mucous membranes. Other vital parameters like pulse, heart rate and respiration rate were within the normal ranges. On abdominal palpation there was splenomegaly. Figure 3: Ethidium Bromide Stained Agarose Gel Electrophoresis of PCR Products from B canis vogeli (Lane 2) Peripheral blood smear examination revealed the presence of B.canis piroplasms within the red blood cells (Figure 1). Anemia, severe thrombocytopenia and monocytosis were evident on hematology. Ultrasonography of the abdomen revealed moderate splenomegaly (Figure 2) and Figure 1: B. canis piroplasms Within the Red Blood Cells Figure 2: Ultrasonography Showing Moderate Splenomegaly the liver and kidneys retained their normal size and architecture. DNA was extracted from 100 µl EDTA-anticoagulated whole blood using the commercial QIAGEN blood and tissue DNA extraction kit as prescribed by the manufacturer. Using the specific forward primer BAB1 (5 0 -GTG AAC CTT ATC ACT TAA AGG-3 0 ) and reverse primer BAB4 (5 0 -CAA CTC CTC CAC GCA ATC G-3 0 ) PCR was performed to amplify an approximately 590 bp B.c. vogeli fragment of the 18S rrna gene of babesiae (Duarte et al., 2008). Amplification conditions were: 2 min at 94 C, 35 cycles each of 94 C for 30 s, 56 C for 30 s, and 72 C for 1 min, with the addition of a final extension period of 6 min at 72 C. The PCR amplicons were visualized by electrophoresis in 88
Int. J. Agric.Sc & Vet.Med. 2017 Arthi A and Tresamol P V, 2017 1.5% agarose gel stained with ethidium bromide under UV transillumination (Figure 3). Results were captured and recorded using a digital documentation system. RESULTS AND DISCUSSION The dog was treated with Inj. Imicarb (Imidocarb) at a dose rate of 6 mg/kg SC two doses with a two week interval. Blood for hematology was collected periodically and remarkable improvement was noticed in the platelet counts. A slight reduction in the erythrocyte count and hemoglobin values was noticed. Blood smear examination was repeated after two weeks and four weeks to check for parasitemia. No piroplasms could be detected after treatment. The dog did not show any adverse effects towards the drug. The dog s appetite gradually improved and after third day of treatment remarkable improvement was noticed in the condition. The subspecies was confirmed to be B. canis vogeli which usually causes a mild or moderate clinical disease with hemolytic regenerative anemia (Solano-Gallego et al., 2011). Thrombocytopenia and monocytosis were observed in the current case which correlates with the reports of Salem et al. (2014) as they were the most commonly observed hematological abnormalities. Moderate to marked thrombocytopenia is a common finding as in this case which is mostly immune-mediated or due to splenic sequestration of platelets. The sequestration and destruction of infected and non-infected erythrocytes takes place in the spleen resulting in splenomegaly. Hence the spleen acts as the defense system against babesia infection. Elevations in the platelet count post treatment in our case suggest that the therapeutic response to imidocarb is faster when compared to other anti-babesial drugs. This is also supported by the findings of Mathe and coworkers (Mathe et al., 2006) where the urine colour of hemoglobinuric dogs changes to normal, forty eight hours after imidocarb administration. CONCLUSION The hemolytic syndrome which is an important feature of babesiosis is due to intravascular and extravascular hemolysis caused by parasite destruction and also by auto antibodies directed towards both infected and non-infected red cells. Imidocarb at the above mentioned dose offers prophylaxis for a period of two weeks against B. canis infection. Even though the side effects of imidocarb like hypersalivation, tachycardia, dyspnea, vomiting and diarrhea were reported (Abdullah et al., 1984), no such findings were encountered in the present case. Side effects of imidocarb can be controlled by giving atropine at a dose of 0.02-0.04 mg/kg SC prior to imidocarb administration. REFERENCES 1. Abdullah A S, Sheikh-Omar A R, Baggot J D and Zamri M (1984), Adverse Effects of Imidocarb dipropionate (Imizol ) in a Dog, Vet Res Comm., Vol. 8, pp. 55-59. 2. Ayoob A L, Hackner S G and Prittie J (2010), Clinical Management of Canine Babesiosis, J. Vet. Emergency and Critical Care, Vol. 20, pp. 77-89. 3. Duarte S C, Linhares G F C, Romanowsky T N, da SilveiraNeto O J and Borges L M F (2008), Assessment of Primers Designed for the Subspecies-Specific Discrimination among Babesia canis canis, Babesia canis vogeli and Babesia canis rossi 89
Int. J. Agric.Sc & Vet.Med. 2017 Arthi A and Tresamol P V, 2017 by PCR Assay, Vet. Parasitol, Vol. 152, pp. 16-20. 4. Matjila P T, Penzhorn B L, Bekker C P J, Nijhof A M and Jongejan F (2004), Confirmation of Occurrence of Babesia canis vogeli in Domestic Dogs in South Africa, Vet. Parasitol., Vol. 122, pp. 119-125. 5. Mathe A, Voros K, Papp L and Reiczigel J (2006), Clinical Manifestations of Canine Babesiosis in Hungary (63 Cases), Acta Veterinaria Hungarica, Vol. 54, pp. 367-385. 6. Salem N Y and Farag H S (2014), Clinical, Hematologic, and Molecular Findings in Naturally Occurring Babesia canis vogeli in Egyptian Dogs, Vet. Med. Intern. 7. Solano-Gallego L and Baneth G (2011), Babesiosis in Dogs and Cats Expanding Parasitological and Clinical Spectra, Vet. Parasitol., Vol. 181, pp. 48-60. APPENDIX Table 1: Hematology on Day 0, 3 and 14 Parameter Day 0 Day 3 Day 14 Ref. Range Hb (g/dl) 13.2 10.2 11.3 12-18 RBC/mm 3 4.38 3.21 4.03 5.50-8.80 W BC/mm 3 10.7 13 8.7 6-17 VPRC (%) 26.4 19.6 26.2 37-55 M CV (fl) 60.3 61.1 65 60-77 M CH (pg) 30.1 31.8 28 19-25 MCHC (%) 50 52 43.1 32-36 Platelet count (lacs/mm 3 ) 44 215 171 160-525 Lymphocytes 2.7 2.4 1.7 0.7-5.1 M onocytes 1.2 0.7 0.6 0.2-1.7 Granulocytes 6.8 9.9 6.3 4.4-12.6 90