PCR Based Diagnosis and Clinical Management of Ehrlichiosis in a Dog.
Canine Monocytotropic Ehrlichiosis (CME) is a tick borne rickettsial disease caused by obligate intracellular pathogen, Ehrlichia canis, transmitted by brown dog tick (Rhipicephalous sanguineus). Ehrlichiosis is associated with severe mortality and morbidity among the members of the family Canidae in almost all parts of the world (Harrus et al., 2012). In India, the favourable ecological conditions for abundant tick population favours high prevalence of E canis among dogs (Lakshman et al., 2007; Abd Rani et al., 2011). E canis infections elicit wide range of clinical manifestations ranging from uncontrolled pyrexia to severe hemorrhagic disease. Pancytopaenia, elevated ALT, AST and amylase activities, hypoproteinemia associated with hypoalbuminemia and hyperglobulinemia and thrombocytopaenia were predominantly observed hemato-biochemical alterations in E canis infected dogs (Greig et al., 1996). Acute ehrlichiosis may be life threatening due to severe bleeding tendencies, resulting in hemorrhagic shock and hence requires early diagnosis. Whereas, routine diagnostic methods like blood smear examination are least sensitive due to peculiar factors of ehrlichial lifecycle.
History and Diagnosis
A fourteen month old male Labrador retriever dog was presented with history of recurrent pyrexia, anorexia, vomiting and weakness for last seven days. The vaccination and deworming history was proper and regular. Owner reported history of tick infestation observed three months back, which was cleared by regular use of Cypermethrin 1% shampoo (Clinar (a)). On clinical examination, the animal was dull, depressed, dehydrated, lethargic and rectal temperature evinced pyrexia (104.3[degrees]F). Physical examination revealed popliteal lymphadenopathy, spleenomegaly and hepatomegaly. Conjunctival, oral and penile mucous membranes were pale. Hemato-biochemical analysis showed microcytic hypochromic anaemia, immune mediated thrombocytopenia, monocytopenia, elevated ALP and ALT activities, hyperglobulinemia and hypoalbuminemia (Table 1). Blood smear and buffy coat were examined after Giemsa method of staining and no parasites were observed, monocytes were also not observed. But, on PCR evaluation of peripheral blood buffered with EDTA, presence of E. canis organisms was detected.
Treatment was initiated using the antiricketsial drug; Doxycycline (5mg/kg BID per orally for 21 days). Supportive therapy included antacid (Ranitidine, 1mg/kg BID per orally for 21 days), antihistamine (Cetrizine, 0.5mg/kg BID per orally for first 7 days), anti inflammatory steroid (Prednisolone acetate, 1mg/kg SID per orally for two weeks followed by tapering dose for next 1 week), hematinic (Dexorange (b) syrup, 10ml BID for 21 days), hepato protectant (Silybon (c) syrup, 10ml BID for 45 days) antioxidants (Ascorbic acid, 20mg/kg OD per orally for 21 days) and B-complex vitamin (Neurobion forte (d), one tablet SID for 21 days). During the course of therapy, the animal was examined for clinical and parasitological improvement weekly once. Eventhough, the animal showed significant improvement by one week of therapy and cleared from E canis infection by 21st day, complete recovery towards normal clinico-hemato-biochemical parameters were evident by 70th day.
Canine monocytotropic ehrlichiosis is caused by obligate intracellular organism E canis, transmitted by arthropod vector brown dog tick (Rhipicephalus sanguineus) which can be found indoors also. In 1935, Donetein and Lestoquard in Algeria initially identified E canis in dog blood. E canis can be detected intracytoplasmically within monocytes and macrophages as clusters of organisms called morulae in blood smears stained with any Romanowsky stain (Harrus et al., 2012). Previous studies on prevalence of E canis infection among dogs reported prevalence rate ranging from 3.1- 88.0 percent worldwide (Murphy et al., 1998; Dagnone et al., 2003; Bulla et al., 2004; Macieira et al., 2005; Diniz et al., 2007; Carvalho et al., 2008; Alexandre et al., 2009; Dagnone et al., 2009; Faria et al., 2010; Silva et al., 2012). In Indian scenario, a few published reports are available on the prevalence of CME, but, are least reliable due to inappropriate sampling; 50 percent in Chennai and 20.6 percent from four different regions of India were reported (Lakshmanan et al., 2007; Abd Rani et al., 2011). Ehrlichiosis could be observed in dogs of all age groups, whereas, German shepherd breed is more susceptible. Incubation period of acute CME may range from 8-20 days, eventhough, it may prolong upto several months in chronic cases (Harrus et al., 2012).
Clinical signs of ehrlichiosis include fever, weakness, lethargy, anorexia, lymphadenomegaly, splenomegaly, hepatomegaly, weight loss, edema (in hind legs, tail, scrotum), pale mucous membranes due to anemia and epistaxis, petechiae, ecchymoses, prolonged bleeding during estrus, hematuria or melena associated with thrombocytopenia (Troy and Forrester, 1990; Hoskins, 1991; Das and Konar, 2013). In acute disease, hematologic abnormalities like thrombocytopenia, mild anemia, and panleukopenia may be observed (Davoust et al., 1991). Thrombocytopenia occurs due to increased platelet consumption and decreased platelet half life, probably as a result of immune mediated splenic sequestration and destruction (Harrus et al., 2012). Spleen is most likely to harbour E. canis organisms during subclinical phase of CME and last organ to accommodate parasite before elimination. The chronic form of CME is characterized by bone marrow hypoplasia and impairment of all bone marrow cells, thus resulting in pancytopenia. Serum chemistry abnormalities will include hyperproteinemia, hyperglobulinemia, hypoalbuminemia and elevated alanine aminotransferase and alkaline phosphatase activities. Occular signs like anterior uveitis chorioretinitis, neuromuscular signs like seizures, ataxia, cerebellar dysfunction, tremor and polyarthritis may also occur in complicated cases (Harrus et al., 1999).
The diagnosis of ehrlichiosis is based on anamnesis, clinical signs, hemato-biochemical analysis and serologic findings (Harrus et al., 2012). The routine diagnostic test performed is direct screening of peripheral blood smear for presence of E. canis morula in monocytes, which can be detected only for short period (in acute phase), but, may not be observed during subclinical and chronic stages of infection (Hibbler et al., 1986; Harrus et al., 1999; Mylonakis et al., 2003; Nakaghi et al., 2008). The morulae could be observed in blood smear only in about 4-6% of clinical cases (Waner et al., 2001). In some cases, the immune mediated pancytopenia will results in absence of monocytes in peripheral blood, which again makes difficult to find morulae. The highest likelihood of detecting morulae was observed in examination of buffy coat smear (Harrus et al., 2012). Even so, the search for morulae in circulating monocytes is still the routine diagnostic method for ehrlichiosis but, in most cases unrewarding (Moreira et al., 2005). Since, the direct detection method has low sensitivity, further diagnostic tests such as serology or molecular techniques must be conducted for confirmation. The high sensitivity and specificity of molecular techniques like PCR in diagnosing canine ehrlichiosis has been reported already (Iqbal et al., 1994; Mcbride et al., 1996; Nakaghi et al., 2008). The quantification of bacterial load and possibility of investigating specific gene fragments makes PCR a superior technique among others (Sainz et al., 2015).
CME can be successfully treated with antibiotics belong to tetracycline family. Doxycycline at 5 mg/kg twice daily or 10 mg/kg once daily for prolonged periods (upto 3-4 weeks) is found to be effective in eliminating parasitemia (Breitschwerdt et al., 1998). Recovered dogs may remain sub-clinically infected carriers after shorter treatments with doxycycline, even at the recommended doses. Chloramphenicol also can be used in dogs under one year of age, but is not usually recommended. Imidocarb dipropionate has also been described as a potential treatment for ehrlichiosis in dogs (Price et al., 1980). In addition to antimicrobial therapy, supportive therapy with fluids and blood transfusion can be insisted in severe cases. Short term therapy with low immunosuppressive doses of glucocorticoids (1 to 2 mg/kg Prednisolone, PO) may be beneficial early in the treatment period when severe or life-threatening thrombocytopenia is present. There are high chances for re-infection with E canis, because no immunity will develop after an active clinical infection. So PCR should be repeated after discontinuing treatment. Nested PCR for the detection of E canis may be useful for laboratory diagnosis and assessment of the efficacy of antibiotic therapy for E canis infection; hence, it provides more reliable and quick diagnosis (Wen et al., 1997).
Prevention of ehrlichiosis can be achieved by early diagnosis, appropriate therapy, hygienic management practices and proper control of ticks. The use of highly sensitive diagnostic methods like PCR will help in rapid as well as reliable detection of early stages of disease and monitoring the efficacy of chemotherapeutic agent in eliminating the organisms from the blood and other tissue aspirates.
The authors are thankful to the Director, ICAR-IVRI for providing facilities for conducting this work.
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S.G. Sangeetha (1), Y. Ajith (2), S.K. Dixit (3) and K.K. Reena (4)
Division of Medicine
Indian Veterinary Research Institute (IVRI)
Bareilly - 243122 (Uttar Pradesh)
(1.) Post Graduate Scholar
(2.) Ph.D. Scholar and Corresponding author.
(3.) Principal Scientist
(4.) Ph.D. Scholar, Division of Parasitology
(a) - Brand of Virbac India Ltd., Mumbai
(b) - Brand of Franco Indian Pharma Pvt. Ltd.
(c) - Brand of Micro labs Ltd., Mumbai
(d) - Brand of Merck India, Mumbai
Table 1: Hemato-biochemical parameters of the patient before and after therapy. Parameter Day 0 Day 21 Day 70 Reference values# Total Erythrocyte count 4.07 4.95 5.73 5-7.9 (106/mm3) PCV (%) 21 29 39.9 35-57 Haemoglobin (g/dl) 9.7 11.2 12.8 12-19 MCV (fL) 42 56 69.7 66-77 MCH (pg) 13.2 19.37 22.3 21-26.2 MCHC (%) 24.9 28.4 32 32-36.3 Total Leukocyte Count ([10.sup.3]/[mm.sup.3]) 9.1 7.7 12.6 5-14.1 Neutrophil (%) 76 66 69 58-85 Lymphocyte (%) 16 30 22 8-21 Eosinophil (%) 8 4 7 0-9 Monocyte (%) 0 0 2 2-10 Basophil (%) 0 0 0 0-1 Platelets (Lakhs/mm3) 0.6 2.23 2.56 2.11-6.21 Total Protein (g/dL) 5.4 5.7 6.4 5.4-7.5 Albumin (g/dL) 1.8 2.1 3.0 2.3-3.1 Globulin (g/dL) 3.6 3.4 3.4 2.4-4.4 A:G ratio 0.5 0.62 0.88 0.6-1.3 ALT or SGPT (IU/L) 164 528 54 10-109 ALP (IU/L) 228 356 24 1-114 Serum creatinine (mg/dL) 1.3 1 1.1 0.5-1.7 Parameter Key findings Total Erythrocyte count Immune mediated (106/mm3) haemolytic anaemia PCV (%) Microcytic hypochromic Haemoglobin (g/dl) anaemia MCV (fL) MCH (pg) MCHC (%) Total Leukocyte Count ([10.sup.3]/[mm.sup.3]) Neutrophil (%) Lymphocyte (%) Eosinophil (%) Monocyte (%) Monocytopaenia Basophil (%) Immune mediated Platelets (Lakhs/mm3) thrombocytopenia Total Protein (g/dL) Hypoalbuminemia Albumin (g/dL) Globulin (g/dL) Hyperglobulinemia A:G ratio ALT or SGPT (IU/L) Acute hepatic injury ALP (IU/L) Serum creatinine (mg/dL) # March 2012: Reference ranges, 10th edn. The Merck Veterinary Manual
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|Title Annotation:||Short Communication; polymerase chain reaction|
|Author:||Sangeetha, S.G.; Ajith, Y.; Dixit, S.K.; Reena, K.K.|
|Date:||Jan 1, 2017|
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