Printer Friendly

PCR Based Diagnosis and Clinical Management of Ehrlichiosis in a Dog.

Introduction

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

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.

Discussion

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.

Acknowledgements

The authors are thankful to the Director, ICAR-IVRI for providing facilities for conducting this work.

References

Abd Rani, P.A.M., Irwin, P.J., Coleman, G.T., Gatne, M. and Traub, R.J. (2011). A survey of canine tick-borne diseases in India. Parasites and Vectors 4: 141-48.

Alexandre N., Santos A.S., Nuncio M.S., Sousa R., Boinas F., Bacellar F., (2009). Detection of Ehrlichia canis by polymerase chain reaction in dogs from Portugal. Vet. J. 181: 343-44.

Breitschwerdt, E.B., Hegarty, B.C. and Hancock, S.I., (1998). Doxycycline Hyclate Treatment of Experimental Canine Ehrlichiosis Followed by Challenge Inoculation with Two Ehrlichia canis Strains. Antimicrob. Agents Chemoth. 42: 362-68.

Bulla, C, Takahira, R.K., Araujo, J.P., Trinca, L.A., Lopes, R.S. and Wiedmeyer, C.E. (2004). The relationship between the degree of thrombocytopenia and infection with Ehrlichia canis in an endemic area. Vet. Res. 35: 141-46.

Carvalho F.S., Wenceslau A.A., Carlos R.S.A. and Albuquerque G.R. (2008). Epidemiological and molecular study of Ehrlichia canis in dogs in Bahia, Brazil. Genetics and Molecular Res. 7: 657-62.

Dagnone A.S., Morais H.A.S., Vidotto M.C., Jojima F.S. and Vidotto O. (2003). Ehrlichiosis in anemic, thrombocytopenic, or tick-infested dogs from a hospital population in South Brazil. Vet. Parasitol. 117: 285-90.

Dagnone A.S., Souza A.I., Andre M.R. and Machado R.Z. (2009). Molecular diagnosis of Anaplasmataceae organisms in dogs with clinical and microscopical signs of ehrlichiosis. Revista Brasileira de Parasitologia Veterinaria 18: 20-25.

Das, M. and Konar, S. (2013). Clinical and hematological study of canine Ehrlichiosis with other hemoprotozoan parasites in Kolkata, West Bengal, India. Asian Pacific J. Tropical Biomed. 3: 913-15.

Davoust, B., Boni, M. and Parzy, D. (1999). Apport du laboratoire au diagnostic de l'ehrlichiose monocytaire canine. Revue francaise des laboratories 310: 25-32.

Diniz P.P.V.P., Schwartz D.S., Morais H.A.S., Breitschwerdt E.B. (2007). Surveillance for zoonotic vector-borne infections using sick dogs from southeastern Brazil. Vector Borne Zoonotic Diseases 7: 689-97.

Faria, I.L., Dagnone, A.S., Munhoz T.D., Joao C.F., Pereira W.A., Machado R.Z., Tinucci-Costa, M. (2010). Ehrlichia canis morulae and DNA detection in whole blood and spleen aspiration samples. Revista Brasileira de Parasitologia Veterinaria 19: 98-102.

Greig, B., Asanovich, K.M., Armstrong, P.J. and Dumler, J.S. (1996). Geographic, clinical, serologic, and molecular evidence of granulocytic ehrlichiosis, a likely zoonotic disease, in Minnesota and Wisconsin dogs. J Clin. Microbiol. 34: 44-48.

Harrus, S., Waner, T., Bark, H., Jongejan, F. and Cornelissen, A.W. (1999). Recent advances in determining the pathogenesis of canine monocytic ehrlichiosis. J Clin. Microbiol. 37: 2745-49.

Harrus, S., Waner, T., Neer, T.M. and Greene C.E. (2012). Infectious Diseases of the Dog and Cat. 4th Edn. WB Saunders: Philadelphia. p. 227-37'.

Hibbler, C.E. et al. (1986). Rickettsial infections in dogs part II: Ehrlichiosis and infectious cyclic trombocytopenia. Comp Cont Educ Pract Vet. 8: 106-13.

Hoskins, J.D. (1991). Ehrlichial diseases of dogs: diagnosis and treatment. Canine Practice (USA).

Iqbal, Z., Chaichanasiriwithaya, W. and Rikihisa, Y. (1994). Comparison of PCR with other tests for early diagnosis of canine ehrlichiosis. J Clin. Microbiol. 32: 1658-62.

Lakshmanan, B., John, L, Gomathinayagam, S. and Dhinakarraj, G. (2007). Molecular detection of Ehrlichia canis from blood of naturally infected dogs in India. Veterinarski Arhiv. 77: 307.

Macieira, D.B., Messick, J.B., Cerqueira, A.D.E., Freire, I.M., Linhares, G.F., Almeida, N.K. and Almosny, N.R. (2005). Prevalence of Ehrlichia canis infection in thrombocytopenic dogs from Rio de Janeiro, Brazil. Vet. Clin. Pathol. 34: 44-48.

McBride, J.W., Corstvet, R.E., Gaunt, S.D., Chinsangaram, J., Akita, G.Y. and Osburn, B.I., (1996). PCR detection of acute Ehrlichia canis infection in dogs. J. Vet. Diagnos. Invest. 8: 441-47.

Moreira, S.M., Bastos, C.V., Araujo, R.B., Santos, M. and Passos, L.M.F. (2003). Retrospective study (1998-2001) on canine ehrlichiosis in Belo Horizonte, MG, Brazil. Arquivo Brasileiro de Medicina Veterinaria e Zootecnia 55: 141-47.

Murphy G.L., Ewing S.A., Whitworth L.C., Fox J.C. and Kocan A.A. (1998). A molecular and serologic survey of Ehrlichia canis, E. chaifeensis, and E. ewingii in dogs and ticks from Oklahoma. Vet. Parasitol. 79: 325-39.

Mylonakis, M.E., Koutinas, A.F., Billinis, C, Leontides, L.S., Kontos, V., Papadopoulos, O., Rallis, T. and Fytianou, A. (2003). Evaluation of cytology in the diagnosis of acute canine monocytic ehrlichiosis (Ehrlichia canis): a comparison between five methods. Vet. Microbiol. 91: 197-204.

Nakaghi, A.C.H., Machado, R.Z., Costa, M.T., Andre, M.R. and Baldani, CD. (2008). Canine ehrlichiosis: clinical, hematological, serological and molecular aspects. Ciencia Rural 38: 766-70.

Price, J.E. and Dolan, T.T. (1980). A comparison of the efficacy of imidocarb dipropionate and tetracycline hydrochloride in the treatment of canine ehrlichiosis. The Veterinary Record 107: 275-77.

Sainz, A., Roura, X., Miro, G., Estrada-Pena, A., Kohn, B., Harrus, S. and Solano-Gallego, L. (2015). Guideline for veterinary practitioners on canine ehrlichiosis and anaplasmosis in Europe. Parasites and Vectors 8: 1.

Schaefer, J.J., Needham, G.R., Bremer, W.G., Rikihisa, Y., Ewing, S.A. and Stich, R.W., (2007). Tick acquisition of Ehrlichia canis from dogs treated with doxycycline hyclate. Antimicrobial Agents and Chemo. 51: 3394-96.

Silva G.C.F., Benitez A.N., Girotto A., Taroda A., Vidotto M.C., Garcia J.L., Freitas J.C, Headley S.A. and Vidotto, O. (2012). Occurrence of Ehrlichia canis and Anaplasma platys in household dogs from northern Parana. Revista Brasileira de Parasitologia Veterinaria 21: 379-85.

Troy, G.C and Forrester, S.D. (1990). Canine Ehrlichiosis. In: Greene, C.E., Infectious Diseases of the Dog and Cat. W.B. Saunders, Philadelphia, p. 48-59.

Waner, T., Harrus, S., Jongejan, F., Bark, H., Keysary, A. and Cornelissen, A.W. (2001). Significance of serological testing for ehrlichial diseases in dogs with special emphasis on the diagnosis of canine monocytic ehrlichiosis caused by Ehrlichia canis. Vet. Parasitol. 95: 1-15.

Wen, B., Rikihisa, Y., Mott, J.M., Greene, R., Kim, H.Y., Zhi, N., Couto, G.C, Unver, A. and Bartsch, R. (1997). Comparison of nested PCR with immunofluores cent-antibody assay for detection of Ehrlichia canis infection in dogs treated with doxycycline. J. Clin. Microbiol. 35: 1852-55.

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.

E-mail: ajithy123456789@gmail.com

(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
COPYRIGHT 2017 Intas Pharmaceuticals Limited
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2017 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:Short Communication; polymerase chain reaction
Author:Sangeetha, S.G.; Ajith, Y.; Dixit, S.K.; Reena, K.K.
Publication:Intas Polivet
Article Type:Report
Date:Jan 1, 2017
Words:2787
Previous Article:Therapeutic Management of Ehrlichiosis and Hepatozoonosis in a Dog.
Next Article:Diagnosis and Management of Haemotropic Mycoplasmosis in Dogs.
Topics:

Terms of use | Privacy policy | Copyright © 2020 Farlex, Inc. | Feedback | For webmasters