A. Arthi1, P.V. Tresamol2, K. Vinodkumar3, Lobo Andrea Robin1 and Sukanya Sukumaran1

1M.V.Sc Student, 2Professor and Head, 3Assistant Professor, Department of Veterinary Epidemiology and Preventive
Medicine; College of Veterinary and Animal Sciences; KVASU, Mannuthy, Thrissur – 680651 (Kerala).
[Received: 11.4.2017; Accepted: 24.10.2017]

A one year old male German shepherd dog was presented with fever, anorexia and lameness. Peripheral blood
smear and buffy coat examinations were negative for hemoparasites. Hematology showed no derangements in blood values. Polymerase Chain Reaction using primer pairs Ehr521 and Ehr790 amplified a 293bp fragment of 16S gene of Anaplasma phagocytophilum. The animal showed recovery after treatment with Inj. Oxytetracycline and Inj.
Prednisolone followed by tablet Doxycycline. But the symptoms relapsed after a period of three months. Inj. Imidocarb @ 4 mg/kg b.wt. was given S/C. In this study anaplasmosis diagnosed by molecular methods and successfully treated with Inj. Imidocarb is discussed.
Key words: Anaplasma phagocytophilum, Canine Granulocytic Anaplasmosis, Imidocarb, PCR.

Anaplasma phagocytophilumis an obligate intracellular Gram-negative bacterium and the causative agent of
granulocytic ehrlichiosis in human, horses, dogs, cattle and sheep (Lillini et al., 2006). The primary vectors are Ixodes spp.Canine granulocytic anaplasmosis occurs with an array of non- specific signs like anorexia, fever, lethargy and lameness. Occasionally non-productive cough has been reported owing to interstitial infiltration of infected neutrophils (Carrade et al., 2009). Anaemia, thrombocytopenia and elevated liver enzymes are the commonly encountered laboratory abnormalities.

Case History and Observations
A one year old male German shepherd dog was presented to the Teaching Veterinary Clinical Complex, Mannuthy, with a history of sudden reluctance to stand or walk. The dog had a history of lameness at the age of six months and oral calcium supplements were prescribed to treat the same. Radiography was done to rule out hip dysplasia. The animal was generally hand fed. On clinical examination there was elevated rectal temperature (105.90F), congested mucous membranes, popliteal lymphadenopathy and splenomegaly evident on abdominal palpation and ultrasonography. The animal evinced pain while trying to palpate the limbs. No orthopedic or neurological abnormalities could be detected.

Materials and Methods
No moving parasites could be detected on wet film examination. No blood parasites could be detected on examination of smears made from peripheral blood or buffy coat. Blood picture showed granulocytosis (Table-
1). Five microliters of the PCR product was analysed by 1.5% agarose gel electrophoresis followed by ethidium bromide staining. DNA was isolated from whole blood by using Qiagen-Dneasy-Tissue and Blood DNA
extraction Kit according to the manufacturer’s instructions. Polymerase chain reaction using primer pair (Ehr521
TGTAGGCGGTTCGGTAAG TTAAAG) and Ehr790 (CTTAAC GCG TTA GCT ACA ACA CAG) was performed to amplify a 293- bp fragment of 16S gene of Anaplasma phagocytophilum. Cycling conditions were initial denaturation at 95oC for 5min, followed by 40 amplification cycles (95oC for 1min, 55oC for 1min, and 72oCfor 1min), and a final extension step at 72oC for 5min (Fig.1).
Treatment was initiated with Inj.
Oxytetracycline @ 10 mg/kg b.wt. I/V, Inj. Pantoprazole @ 1 mg/kg b.wt., fluid therapy with DNS, Inj. Prednisolone @ 2 mg/kg b.wt. I/M and B-complex supplements orally for three days. This was followed by Tab. Doxycycline @ 10 mg/kg b.wt. orally once daily for three weeks. The signs gradually subsided after treatment with oxytetracycline
and doxycycline, and the animal was discharged as cured. But the dog was presented after three months with similar signs. After recurrence Inj. Imidocarbdipropionate @ 4 mg/kg b.wt. S/C was given and Inj. Atropine sulphate @ 0.025 mg/kg b.wt. S/C was given ten minutes befor it.

Results and Discussion
The dog had an uneventful recovery post treatment. No recurrence was reported until six months after therapy with Inj. Imidocarb. The affinity of A. phagocytophilum towards neutrophils and disabling neutrophil bactericidal functions there by causing immunosuppression as was also reported by Sykes (2010). Leukopenia and impairment of neutrophil function will lead to secondary opportunistic infection. Lameness is associated with neutrophilic
polyarthritis. Reactive lymphoid hyperplasia could result in splenomegaly and lymphadenopathy. Platelet estruction with  an increased bone marrow megakaryocytes resulting in thrombocytopenia was also
mentioned by Little (2010). Since Imidocarb has the added advantage of persistant activity it can be considered for treatment when there is recurrence of disease as seen in this case or in the case of antimicrobial failure. Treatment
failure can also occur when there are concurrent infections. PCR is considered to be the most specific tool to diagnose this disease as the appearance of morulae within the granulocytes in peripheral blood smears is
not specific to this species as also reported by Domingos et al (2011). The absence of morulae in granulocytes was consistent with the findings of Carrade et al. (2009) where the morulae appeared only after 4 days of experimental inoculation and persisted for 4 to 8 days.

Carrade, D.D., Foley, J.E., Borjesson, D.L. and Sykes, J.E. (2009). Canine granulocytic anaplasmosis: a review. J.
Vet. Intern. Med., 23: 1129-1141.

Domingos, C.M., Trotta, M., Briend‐Marchal, A. and Medaille, C. (2011). Anaplasmosi -s in two dogs in France and molecular and phylogenetic characterization of Anaplasma phagocytophilum. Vet. Clin. Pathol., 40: 215-221.

Lillini, E., Macri, G., Proietti, G. and Scarpulla, M. (2006). New findings on anaplasmosis caused by infection with
Anaplasma phagocytophilum. Ann. the New York Acad. of Scien., 1081: 360-370.

Little, S.E. (2010). Ehrlichiosis and anaplasmosis in dogs and cats.Vet. Clinics of North America: Small Anim. Pract., 40: 1121-1140.

Sykes, J.E. (2010). Immunodeficiencies caused by infectious diseases.Vet. Clinics of North America: Small Anim. Pract., 40(3): 409-423.


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