research article frequency and clinical epidemiology...

Hindawi Publishing CorporationJournal of Veterinary MedicineVolume 2013 Article ID 797019 3 pageshttpdxdoiorg1011552013797019

Research ArticleFrequency and Clinical Epidemiology of CanineMonocytic Ehrlichiosis in Dogs Infested withTicks from Sinaloa Mexico

Carolina Guadalupe Sosa-Gutierrez12 Maria Teresa Quintero Martinez2

Soila Maribel Gaxiola Camacho3 Silvia Cota Guajardo3

Maria D Esteve-Gassent4 and Mariacutea-Guadalupe Gordillo-Peacuterez1

1 Unidad de Investigacion Medica en Enfermedades Infecciosas y Parasitarias Hospital de PediatriaCentro Medico Nacional Siglo XXI Instituto Mexicano del Seguro Social 07300 Mexico City Mexico

2Departamento de Parasitologia Facultad de Medicina Veterinaria y Zootecnia Universidad Nacional Autonoma de MexicoMexico City Mexico

3 Departamento de Parasitologia Facultad de Medicina Veterinaria y Zootecnia Universidad Autonoma de Sinaloa SIN Mexico4Department of Veterinary Pathobiology College of Veterinary Medicine and Biomedical Sciences Texas AampM University TX USA

Correspondence should be addressed to Carolina Guadalupe Sosa-Gutierrez mcarososagtzyahoocommx

Received 23 April 2013 Revised 18 July 2013 Accepted 17 September 2013

Academic Editor Masanori Tohno

Copyright copy 2013 Carolina Guadalupe Sosa-Gutierrez et alThis is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Ehrlichia canis is a rickettsial intracellular obligate bacterial pathogen and agent of caninemonocytic ehrlichiosisThe prevalence ofthis disease in veterinary medicine can vary depending on the diagnostic method used and the geographic location One hundredand fifty-two canine blood samples from six veterinary clinics and two shelters from Sinaloa State (Mexico) were analyzed in thisstudy All animals were suspected of having Canine Monocytic Ehrlichiosis (CME) The diagnostic methods used were the ELISA(Snap4Dx IDEXX) together with blood smear and platelet count From all dogs blood samples analyzed 743 were positive to Ecanis by ELISA and 401 were positive by blood smear The sensitivity and specificity observed in the ELISA test were 788 and867 In addition thrombocytopenia was presented in 876 of positive dogs The predominant clinical manifestations observedwere fever anorexia depression lethargy and petechiae Consequently this is the first report in which the morulae were visualizedin the blood samples and E canis-specific antibodies were detected in dogs from Sinaloa Northwest of Mexico

1 Introduction

Ehrlichia canis is the causative agent of canine monocyticehrlichiosis (CME) Moreover CME is an emerging diseasein veterinary medicine and E canis has been considered inthe last decade as a potential zoonotic pathogen [1 2] It is aworldwide disease transmitted by a tick bite The competentvector for its transmission is the Ixodidae ticks Rhipicephalussanguineus and Dermacentor variabilis [3] In dogs the CMEis a multiphase disorder that progresses in three stagesacute subclinical and chronic Each phase is characterizedby several clinical and hematologic abnormalities Thrombo-cytopenia is a common finding in E canis infected dogs and

many clinicians tend to use it as an indication for antibiotictreatment and it is observed in 84 of the cases and itsseverity varies in the different disease phases [4] During thesubclinical stage a moderate thrombocytopenia is observedwhile the chronic phase is characterized by severe leukopeniaand anemia In this stage dogs show other complications suchas hypocellular marrow suppressed splenic sequestrationdecreased life of platelets and an increase of circulatingmigration-factor platelet inhibitor [3 4] The relationshipbetween the magnitude of thrombocytopenia and prevalenceof E canis has been established in countries such as Brazil in2004 where 841 of infected dogs showed thrombocytope-nia [3] Taken together more data is necessary to determine

2 Journal of Veterinary Medicine

the environmental factors and infected vector prevalence inMexico due to high incidence of CME in this region ofthe country The purpose of this study was to evaluate thefrequency and clinical manifestations of dogs from SinaloaMexicowith clinical suspicion ofCMEbyELISA (SNAP4Dx)and blood smears and with a history of tick infestation

2 Material and Methods

21 Location The study was conducted in Culiacan SinaloaMexico located north 27∘ 021015840 south 22∘ 291015840 north latitudeeast 105∘ 231015840 to 109∘ 281015840 west longitude

22 Samples and Serological Analysis Samples were collectedbetween March 2006 and July 2007 and 152 blood samplesfrom dogs were collected in six veterinary clinics and twoshelters from Sinaloa State Mexico Blood samples werechosen by dogs (males and females) with tick infestationand clinical signs suggesting CME (fever anorexia lethargydepression petechiae bruising of the skin or prone tobleeding in mucous membranes and epistaxis) Threemlof blood sample in EDTA tubes was obtained from eachdog by the radial vein All samples were processed usingtwo techniques to detect E canis specific IgG antibodiesthe IDDEX ELISA kit and the Snap test Samples were pro-cessed according to the manufacturerrsquos recommendationsIn addition blood smears were done immediately after theblood was drawn to detect forms of E canis morulae inmonocytes using Wrightrsquos stain Finally platelet count wasperformed by an Automatic Hematology Analyzer (IDEXXQBC VetAutoread) All counts less than 200000 platelets120583Lwere considered as thrombocytopenia [4]

23 Statistical Analysis Theplatelet counts from each samplewere comparedwith the seropositive and seronegative resultsQualitative variables such as sex (female male) age (lt1 year1ndash3 years 3ndash5 years gt5 years) presence of thrombocytopenia(yes no) fever (yes no) anorexia (yes no) lethargy (yesno) depression (yes no) petechiae (yes no) skin ecchymosis(yes no) epistaxis (yes no) bleeding tendency (yes no)and anorexia (yes no) were evaluated and used on the chisquare or Fisher exact tests using EpiInfo 35 which providesregression analysis estimating linear 95 confidence interval(95CI)

3 Results

Among the canine individuals with suspected CME 745(113152) have E canis-specific antibodies In addition 401(61152) were found positive when examined by smear todetect E canis morulae in monocytes (Figure 1) Overall theclinical manifestations observed (Table 1) were fever (912)anorexia (867) depression (850) lethargy (726) andpetechiae (726) The male-female ratio was 1 1 and themost affected age was 1-2 years old (53) followed by under1 year (28) and 3ndash5 years (19) We compare the resultsobtained from the blood smear technique and Snap 4DXof the 152 samples analyzed 60 (382) and 21 (137) were

Table 1 Clinical manifestations presented by dogs positive forantibodies to E canis

Clinicalmanifestation

Positive119873 = 113

Frequency()

Odds ratio(CI) P value

Fever 103 912 36 (13ndash102) lt001Anorexia 98 867 408 (16ndash103) lt001Lethargy 82 726 NS NSDepression 96 850 393 (16ndash97) lt001Petechiae 82 726 25 (11ndash57) lt001Ecchymosis 22 195 NS NSEpistaxis 80 708 NS NSThrombocytopenia 106 938 52 (17ndash129) lt001NS Not Significant Evaluated by chi square test and calculating odds ratio(OR) and 95 confidence intervals (CI)

Figure 1 Observed using light microscopy this is an image createdfrom a peripheral blood smear of a dog infected with CanineMonocytic Ehrlichiosis This image shows the morulae within thecytoplasm of a monocyte Wrightrsquos stain

positive and negative for both methods diagnostically with asensibility of 788 (95CI 698ndash856) and specificity 867(95CI 743ndash991) and were assessed by chi square test bycalculating odds ratios (OR) and 95 confidence intervals(CI) with a 212 (95CI 101ndash445) 119875 lt 005 The 876(106) of the dogs with thrombocytopenia were positive tothe presence of antibodies of E canis and were evaluatedby chi square test by calculating odds ratios (OD) and 95confidence intervals (CI) with a 242 (95CI 89ndash659) 119875 lt001 this parameter is a risk factor for disease and showedhigh rates of exposure consistent with previous reports [5]

4 Discussion

The results obtained in this study were similar to thoseobserved in 2002 in which the authors compared fiveserodiagnostic methods for E canis and found 792 positivesamples using the same ELISA techniques as the one utilizedin our study [6] InMexico (2000) a national study reported a33 of seroprevalence for E canis using the same diagnosticmethod as those presented in here [7] Contrary to what wasfound in this study our results showed the highest ehrli-chiosis case In Yucatan Mexico a 441 seroprevalence forCME has been reported using the same diagnostic methodYucatan is in a climate zone similar to that of Sinaloa Taken

Journal of Veterinary Medicine 3

together our study shows a frequency for CME similar if nothigher than that observed in other regions of the countryas well as in other countries of Center and South Americawhich suggests the increasing importance of this diseasein the canine population E canis has been described as apotential zoonotic pathogen for humans [5] and thereforeour findings and current studies suggest its importance ashuman pathogen in Latin American countries

A positive result for E canis by ELISA indicates that thedog was or is exposed to this bacterial pathogen and does notnecessarily imply that there is a latent infection On the otherhand when using the ELISA test cross-reactivity with otherEhrlichia species may occur since it is not specific for E canisAntibodies against this pathogen decrease approximately 6to 9 months after infection This together with the presenceof subclinical infection in canids causes difficulties in thediagnosis of the disease by the clinicians Consequently thereis a need of tools to support the diagnosis of the diseasewith hematological tests to evaluate thrombocytopenia sincethis sign implies bacterial growth and its effect on antibody-producing organs

Previous E canis experimental studies [3 4] reportedthrombocytopenia in 89 of the infected dogs In additionthrombocytopenia was described in every stage of infectionand it persisted during subclinical stages of the disease Themorulae are easily identified by light microscopy during theacute phase of infection (4-5 days after exposure) but difficultto detect when the disease progressed undetected Somestudies reported that only 4 of the blood samples studiedshowed morulae in blood smears [8ndash10] In this case 401were found positive which indicates that the search for themorulae in peripheral blood was done in the acute phase [11]Moreover dogs in endemic areas may have high IgG titersspecific to E canis without clinical signs and this can causefalse positive serological tests [3] reinforcing the necessity formore specific test to be used in veterinary medicine

5 Conclusions

In this study we have introduced the presence of the CanineMonocytic Ehrlichiosis in Sinaloa Mexico Dogs infestedwith ticks showed high frequency for E canis in dogs whichsuggests the classification of this region of Mexico as anendemic area for the disease Based on our findings andprevious studies done in Mexico and other Latin Americancountries we suggest that in the absence of molecularmethodologies for the diagnostics of CME a serology test todetect E canis-specific antibodies plus the presence of clinicalmanifestations and thrombocytopenia is a basic tool for itsdiagnosis in endemic areas

References

[1] Y Rikihisa ldquoThe tribe Ehrlichieae and ehrlichial diseasesrdquoClinical Microbiology Reviews vol 4 no 3 pp 286ndash308 1991

[2] A Unver M Perez N Orellana H Huang and Y RikihisaldquoMolecular and antigenic comparison of Ehrlichia canis isolatesfrom dogs ticks and a human in Venezuelardquo Journal of ClinicalMicrobiology vol 39 no 8 pp 2788ndash2793 2001

[3] C Bulla R Kiomi Takahira J Pessoa Araujo Jr L A Trinca RSouza Lopes and C E Wiedmeyer ldquoThe relationship betweenthe degree of thrombocytopenia and infection with Ehrlichiacanis in an endemic areardquo Veterinary Research vol 35 no 1 pp141ndash146 2004

[4] T Waner and S Harrus ldquoEhrlichiosis monocytic caninerdquo inRecent Advances in Canine Infectious Diseases L CarmichaelEd 2004

[5] R I Rodriguez-Vivas R E F Albornoz and G M E BolioldquoEhrlichia canis in dogs in Yucatan Mexico Seroprevalenceprevalence of infection and associated factorsrdquo VeterinaryParasitology vol 127 no 1 pp 75ndash79 2005

[6] M Belanger H L Sorenson M K France et al ldquoComparisonof serological detectionmethods for diagnosis of Ehrlichia canisinfections in dogsrdquo Journal of Clinical Microbiology vol 40 no9 pp 3506ndash3508 2002

[7] O L Nunez ldquoEstudio de la seroprevalencia de Ehrlichia canisen Mexicordquo Revista AMMVEPE vol 14 no 3 pp 83ndash85 2003

[8] E Elias ldquoDiagnosis of Ehrlichiosis from the presence of inclu-sion bodies of morulae of E canisrdquo Journal of Small AnimalPractice vol 33 pp 540ndash543 1991

[9] J A Benavides and G F Ramırez ldquoCasos Clınicos Ehrlichiosiscaninardquo Revista Colombiana de Ciencias Pecuarias vol 16 no3 pp 268ndash294 2003

[10] W C Buhles Jr D L Huxsoll and M Ristic ldquoTropical caninepancytopenia clinical hematologic and serologic responseof dogs to Ehrlichia canis infection tetracycline therapy andchallenge inoculationrdquo Journal of Infectious Diseases vol 130no 4 pp 357ndash367 1974

[11] L Bockino P M Krimer S L Kenneth and J B PerryldquoAn Overview of Canine Ehrlichiosisrdquo 2013 httpwwwvetugaeduvppclerkBockino

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Case Reports in Veterinary Medicine

2 Journal of Veterinary Medicine

the environmental factors and infected vector prevalence inMexico due to high incidence of CME in this region ofthe country The purpose of this study was to evaluate thefrequency and clinical manifestations of dogs from SinaloaMexicowith clinical suspicion ofCMEbyELISA (SNAP4Dx)and blood smears and with a history of tick infestation

2 Material and Methods

21 Location The study was conducted in Culiacan SinaloaMexico located north 27∘ 021015840 south 22∘ 291015840 north latitudeeast 105∘ 231015840 to 109∘ 281015840 west longitude

22 Samples and Serological Analysis Samples were collectedbetween March 2006 and July 2007 and 152 blood samplesfrom dogs were collected in six veterinary clinics and twoshelters from Sinaloa State Mexico Blood samples werechosen by dogs (males and females) with tick infestationand clinical signs suggesting CME (fever anorexia lethargydepression petechiae bruising of the skin or prone tobleeding in mucous membranes and epistaxis) Threemlof blood sample in EDTA tubes was obtained from eachdog by the radial vein All samples were processed usingtwo techniques to detect E canis specific IgG antibodiesthe IDDEX ELISA kit and the Snap test Samples were pro-cessed according to the manufacturerrsquos recommendationsIn addition blood smears were done immediately after theblood was drawn to detect forms of E canis morulae inmonocytes using Wrightrsquos stain Finally platelet count wasperformed by an Automatic Hematology Analyzer (IDEXXQBC VetAutoread) All counts less than 200000 platelets120583Lwere considered as thrombocytopenia [4]

23 Statistical Analysis Theplatelet counts from each samplewere comparedwith the seropositive and seronegative resultsQualitative variables such as sex (female male) age (lt1 year1ndash3 years 3ndash5 years gt5 years) presence of thrombocytopenia(yes no) fever (yes no) anorexia (yes no) lethargy (yesno) depression (yes no) petechiae (yes no) skin ecchymosis(yes no) epistaxis (yes no) bleeding tendency (yes no)and anorexia (yes no) were evaluated and used on the chisquare or Fisher exact tests using EpiInfo 35 which providesregression analysis estimating linear 95 confidence interval(95CI)

3 Results

Among the canine individuals with suspected CME 745(113152) have E canis-specific antibodies In addition 401(61152) were found positive when examined by smear todetect E canis morulae in monocytes (Figure 1) Overall theclinical manifestations observed (Table 1) were fever (912)anorexia (867) depression (850) lethargy (726) andpetechiae (726) The male-female ratio was 1 1 and themost affected age was 1-2 years old (53) followed by under1 year (28) and 3ndash5 years (19) We compare the resultsobtained from the blood smear technique and Snap 4DXof the 152 samples analyzed 60 (382) and 21 (137) were

Table 1 Clinical manifestations presented by dogs positive forantibodies to E canis

Clinicalmanifestation

Positive119873 = 113

Frequency()

Odds ratio(CI) P value

Fever 103 912 36 (13ndash102) lt001Anorexia 98 867 408 (16ndash103) lt001Lethargy 82 726 NS NSDepression 96 850 393 (16ndash97) lt001Petechiae 82 726 25 (11ndash57) lt001Ecchymosis 22 195 NS NSEpistaxis 80 708 NS NSThrombocytopenia 106 938 52 (17ndash129) lt001NS Not Significant Evaluated by chi square test and calculating odds ratio(OR) and 95 confidence intervals (CI)

Figure 1 Observed using light microscopy this is an image createdfrom a peripheral blood smear of a dog infected with CanineMonocytic Ehrlichiosis This image shows the morulae within thecytoplasm of a monocyte Wrightrsquos stain

positive and negative for both methods diagnostically with asensibility of 788 (95CI 698ndash856) and specificity 867(95CI 743ndash991) and were assessed by chi square test bycalculating odds ratios (OR) and 95 confidence intervals(CI) with a 212 (95CI 101ndash445) 119875 lt 005 The 876(106) of the dogs with thrombocytopenia were positive tothe presence of antibodies of E canis and were evaluatedby chi square test by calculating odds ratios (OD) and 95confidence intervals (CI) with a 242 (95CI 89ndash659) 119875 lt001 this parameter is a risk factor for disease and showedhigh rates of exposure consistent with previous reports [5]

4 Discussion

The results obtained in this study were similar to thoseobserved in 2002 in which the authors compared fiveserodiagnostic methods for E canis and found 792 positivesamples using the same ELISA techniques as the one utilizedin our study [6] InMexico (2000) a national study reported a33 of seroprevalence for E canis using the same diagnosticmethod as those presented in here [7] Contrary to what wasfound in this study our results showed the highest ehrli-chiosis case In Yucatan Mexico a 441 seroprevalence forCME has been reported using the same diagnostic methodYucatan is in a climate zone similar to that of Sinaloa Taken





5 Conclusions


References



















Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of










5 Conclusions


References



















Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of













Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of






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