Abstract
The aim of this study was evaluating the association and correlation between the diagnostics tests used for Leishmania spp. detection in dogs and ticks. We evaluated 99 dogs and 990 Rhipicephalus sanguineus. In dogs, we used bone marrow aspirates and lymph node fine-needle aspiration biopsy (FNAB) for direct parasitological examinations and real time-polymerase chain reaction (RT-PCR) and collected blood samples for enzyme-linked immunosorbent assays (ELISA). In ticks, two laboratory techniques [immunohistochemistry to lipophosphoglycan (IHC) and RT-PCR] were performed in the intestine, ovaries and salivary glands. With respect to the measurement of diagnostic performance in dogs, lymph node RT-PCR proved to be the best test followed by ELISA and bone marrow RT-PCR. In ticks, intestine IHC were considered as a gold standard for diagnosis of leishmaniasis with intestinal RT-PCR being the best diagnostic test. To arrive at the correlation between laboratory techniques for dogs and their ticks, we evaluated the diagnostic test used for dogs with tests performed in R. sanguineus, which used lymph node FNAB as the gold standard. The intestine IHC technique showed strongest association. We demonstrated that the best tissue for Leishmania spp. detection in dogs was the lymph node and the intestine in case of ticks. As for laboratory techniques, the isolated analysis of each species presented a strong agreement between immunohistochemistry and RT-PCR when compared to its gold standard. In addition, we concluded that the immunohistochemistry of ticks’ intestines was a better technique for diagnosing Leishmania spp. in R. sanguineus, thereby showing almost perfect correlation with the lymph node FNAB.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
The datasets used and analysed during the current study are available from the corresponding author on request.
References
Andrade HM, de Toledo Vde P, Marques MJ, Franca Silva JC, Tafuri WL, Mayrink W, Genaro O (2002) Leishmania (Leishmania) chagasi is not vertically transmitted in dogs. Vet Parasitol 103:71–81. https://doi.org/10.1016/s0304-4017(01)00552-0
Andreadou M, Liandris E, Kasampalidis IN, Taka S, Antoniou M, Ntais P, Vaiopoulou A, Theodoropoulos G, Gazouli M, Ikonomopoulos J (2012) Evaluation of the performance of selected in-house and commercially available PCR and real-time PCR assays for the detection of Leishmania DNA in canine clinical samples. Exp Parasitol 131(4):419–424. https://doi.org/10.1016/j.exppara.2012.05.012
Aragão H, Fonseca Fd (1961) Notas de ixodologia: VIII. Lista e chave para os representantes da fauna ixodológica brasileira: notas de ixolodologia Memórias do Instituto Oswaldo Cruz 59:115–129
Bilgic HB, Bakirci S, Kose O, Aksulu A, Hacilarlioglu S, Karagenc T (2016) Determination the role of Rhipicephalus sanguineus for transmission of leishmania major to reservoir animals. Turkiye Parazitol Derg 40:179–184. https://doi.org/10.5152/tpd.2016.4911
Blanc G, Caminopetros J (1930) La transmission du Kala-Azar méditerranéen par une tique: Rhipicephalus sanguineus. CR Acad Sci 191:1162–1164
Campos JH, Costa FA (2014) Participation of ticks in the infectious cycle of canine visceral leishmaniasis. Teresina, Piaui, Brazil Rev Inst Med Trop Sao Paulo 56:297–300. https://doi.org/10.1590/s0036-46652014000400005
Chen Z, Liu Q, Liu JQ, Xu BL, Lv S, Xia S, Zhou XN (2014) Tick-borne pathogens and associated co-infections in ticks collected from domestic animals in central China. Parasit Vectors 7:237. https://doi.org/10.1186/1756-3305-7-237
Coutinho MT et al (2005) Participation of Rhipicephalus sanguineus (Acari: Ixodidae) in the epidemiology of canine visceral leishmaniasis. Vet Parasitol 128:149–155. https://doi.org/10.1016/j.vetpar.2004.11.011
Chaouch M, Mhadhbi M, Adams ER, Schoone GJ, Limam S, Gharbi Z, Darghouth MA, Guizani I, BenAbderrazak S (2013) Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Leishmania infantum in canine leishmaniasis based on cysteine protease B genes. Vet Parasitol 198(1):78–84. https://doi.org/10.1016/j.vetpar.2013.07.038
Dabaghmanesh T, Asgari Q, Moemenbellah-Fard MD, Soltani A, Azizi K (2016) Natural transovarial and transstadial transmission of Leishmania infantum by naive Rhipicephalus sanguineus ticks blood feeding on an endemically infected dog in Shiraz, south of Iran. Trans R Soc Trop Med Hyg 110:408–413. https://doi.org/10.1093/trstmh/trw041
Dantas-Torres F (2011) Ticks as vectors of Leishmania parasites. Trends Parasitol 27:155–159. https://doi.org/10.1016/j.pt.2010.12.006
Dantas-Torres F et al (2010a) Detection of Leishmania infantum in Rhipicephalus sanguineus ticks from Brazil and Italy. Parasitol Res 106:857–860. https://doi.org/10.1007/s00436-010-1722-4
Dantas-Torres F, Martins TF, de Paiva-Cavalcanti M, Figueredo LA, Lima BS, Brandao-Filho SP (2010b) Transovarial passage of Leishmania infantum kDNA in artificially infected Rhipicephalus sanguineus. Exp Parasitol 125:184–185. https://doi.org/10.1016/j.exppara.2010.02.003
Dantas-Torres F, Latrofa MS, Otranto D (2011) Quantification of Leishmania infantum DNA in females, eggs and larvae of Rhipicephalus sanguineus. Parasit Vectors 4:56. https://doi.org/10.1186/1756-3305-4-56
Dantas-Torres F et al (2019) Canine leishmaniasis control in the context of one health. Emerg Infect Dis 25:1–4. https://doi.org/10.3201/eid2512.190164
de Almeida RF, Garcia MV, Cunha RC, Matias J, e Silva EA, de Fatima Cepa Matos M, Andreotti R (2013) Ixodid fauna and zoonotic agents in ticks from dogs: first report of Rickettsia rickettsii in Rhipicephalus sanguineus in the state of Mato Grosso do Sul, mid-western Brazil. Exp Appl Acarol 60:63–72. https://doi.org/10.1007/s10493-012-9641-y
de Morais RC et al (2013) Detection of Leishmania infantum in animals and their ectoparasites by conventional PCR and real time PCR. Exp Appl Acarol 59:473–481. https://doi.org/10.1007/s10493-012-9611-4
Edwards KT, Goddard J, Varela-Stokes AS (2009) Examination of the internal morphology of the Ixodid tick, Amblyomma maculatum Koch, (Acari: Ixodidae); a “how-to” pictorial dissection guide. Midsouth Entomol 2:28–39
Feitosa APS, Chaves MM, Veras DL, de Deus DMV (1908) Portela NC Junior, Araújo AR, Alves LC, Brayner FA (2018) Assessing the cellular and humoral immune response in Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) infected with Leishmania infantum (Nicolle. Ticks Tick Borne Dis 9(6):1421–1430. https://doi.org/10.1016/j.ttbdis.2018.06.007
Geisweid K, Weber K, Sauter-Louis C, Hartmann K (2013) Evaluation of a conjunctival swab polymerase chain reaction for the detection of leishmania infantum in dogs in a non-endemic area. Vet J 198:187–192. https://doi.org/10.1016/j.tvjl.2013.07.025
Giraud P, Ranque J, Cabassu H (1954) Epidemiologie de la leishmaniose viscérale humaine mediterranéenne, en particulier dans sés rapports avec la leishmaniose canine. Arch Fr Pediatr 11:337–353
Gondard M et al (2020) Upscaling the surveillance of tick-borne pathogens in the French Caribbean Islands. Pathogens. https://doi.org/10.3390/pathogens9030176
Gomes AH, Ferreira IM, Lima ML, Cunha EA, Garcia AS, Araujo MF, Pereira-Chioccola VL (2007) PCR identification of Leishmania in diagnosis and control of canine Leishmaniasis. Vet Parasitol 144(3–4):234–241. https://doi.org/10.1016/j.vetpar.2006.10.008
Guimarães JH, Battesti DMB, Tucci EC (2001) Ectoparasitos de importância veterinária. Plêiade
Jaeschke R, Guyatt GH, Sackett DL (1994) Users’ guides to the medical literature. III. How to use an article about a diagnostic test. B. What are the results and will they help me in caring for my patients? The evidence-based medicine working group. Jama 271:703–707. https://doi.org/10.1001/jama.271.9.703
Landis JR, Koch GG (1977) The measurement of observer agreement for categorical data. Biometrics 33:159–174
Lima VM, Goncalves ME, Ikeda FA, Luvizotto MC, Feitosa MM (2003) Anti-leishmania antibodies in cerebrospinal fluid from dogs with visceral leishmaniasis. Braz J Med Biol Res 36:485–489
Maia C, Ramada J, Cristovao JM, Goncalves L, Campino L (2009) Diagnosis of canine leishmaniasis: conventional and molecular techniques using different tissues. Vet J 179:142–144. https://doi.org/10.1016/j.tvjl.2007.08.009
Maia C et al (2020) Monitoring Leishmania infection and exposure to Phlebotomus perniciosus using minimal and non-invasive canine samples. Parasit Vectors 13:119. https://doi.org/10.1186/s13071-020-3993-7
Manna L et al (2004) Comparison of different tissue sampling for PCR-based diagnosis and follow-up of canine visceral leishmaniosis. Vet Parasitol 125:251–262. https://doi.org/10.1016/j.vetpar.2004.07.019
Manoj RRS, Iatta R, Latrofa MS, Capozzi L, Raman M, Colella V, Otranto D (2020) Canine vector-borne pathogens from dogs and ticks from Tamil Nadu. India Acta Trop 203:105308. https://doi.org/10.1016/j.actatropica.2019.105308
McGee S (2002) Simplifying likelihood ratios. J General Int Med 17:646–649. https://doi.org/10.1046/j.1525-1497.2002.10750.x
McKenzie KK (1984) A study of the transmission of canine leishmaniasis by the tick, Rhipicephalus sanguineus, and an ultrastructural comparison of the promastigote., Oklahoma State University
MedCalc Software Ltd O, Belgium (2020) MedCalc® Statistical Software version 19.5.3. MedCalc Software Ltd, Ostend, Belgium
Medeiros-Silva V et al (2015) Successful isolation of Leishmania infantum from Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) collected from naturally infected dogs. BMC Vet Res 11:258. https://doi.org/10.1186/s12917-015-0576-5
Mohammadiha A, Mohebali M, Haghighi A, Mahdian R, Abadi AR, Zarei Z, Yeganeh F, Kazemi B, Taghipour N, Akhoundi B (2013) Comparison of real-time PCR and conventional PCR with two DNA targets for detection of Leishmania (Leishmania) infantum infection in human and dog blood samples. Exp Parasitol 133(1):89–94. https://doi.org/10.1016/j.exppara.2012.10.017
Montalvo AM, Alba A, Fraga J, Marzoa A, Torres C, Muskus C (2020) Improving the sensitivity of an hsp20-based PCR for genus detection of Leishmania parasites in cutaneous clinical samples: a proof of concept. Parasitol Res 119:345–349. https://doi.org/10.1007/s00436-019-06520-6
Norton AJ, Jordan S, Yeomans P (1994) Brief, high-temperature heat denaturation (pressure cooking): a simple and effective method of antigen retrieval for routinely processed tissues. J Pathol 173:371–379. https://doi.org/10.1002/path.1711730413
Nzelu CO, Kato H, Peters NC (2019) Loop-mediated isothermal amplification (LAMP): an advanced molecular point-of-care technique for the detection of Leishmania infection. PLoS Negl Trop Dis 13:e0007698. https://doi.org/10.1371/journal.pntd.0007698
Paz GF et al (2009) Evaluation of the vectorial capacity of Rhipicephalus sanguineus (Acari: Ixodidae) in the transmission of canine visceral leishmaniasis. Parasitol Res 106:523. https://doi.org/10.1007/s00436-009-1697-1
Paz GF et al (2010) Association between the prevalence of infestation by Rhipicephalus sanguineus and Ctenocephalides felis felis and the presence of anti-Leishmania antibodies: a case-control study in dogs from a Brazilian endemic area. Prev Vet Med 97:131–133. https://doi.org/10.1016/j.prevetmed.2010.08.006
Paz GF et al (2010) Evaluation of the vectorial capacity of Rhipicephalus sanguineus (Acari: Ixodidae) in the transmission of canine visceral leishmaniasis. Parasitol Res 106:523–528. https://doi.org/10.1007/s00436-009-1697-1
Pennisi MG, Persichetti MF, Serrano L, Altet L, Reale S, Gulotta L, Solano-Gallego L (2015) Ticks and associated pathogens collected from cats in Sicily and Calabria (Italy). Parasit Vectors 8:512. https://doi.org/10.1186/s13071-015-1128-3
Perosso J et al (2014) Alteration of sFAS and sFAS ligand expression during canine visceral leishmaniosis. Vet Parasitol 205:417–423. https://doi.org/10.1016/j.vetpar.2014.09.006
Persichetti MF, Solano-Gallego L, Serrano L, Altet L, Reale S, Masucci M, Pennisi MG (2016) Detection of vector-borne pathogens in cats and their ectoparasites in southern Italy. Parasit Vectors 9:247. https://doi.org/10.1186/s13071-016-1534-1
Rakhshanpour A et al (2017) Transmission of Leishmania infantum by Rhipicephalus sanguineus (Acari: Ixodidae) in Dogs. Iran J Parasitol 12:482–489
Sakamoto CAM, Bresciani KDS, Serrano ACM, Lima VMF, Machado GF, Kanamura CT, Costa AJ (2007) Canine Visceral Leishmaniasis at Jaboticabal - São Paulo State, Brazil - First Case Report. Ars Vet 23:125–128. https://doi.org/10.15361/2175-0106.2007v23n3p125-128
Sangioni LA, Horta MC, Vianna MC, Gennari SM, Soares RM, Galvão MA, Schumaker TT, Ferreira F, Vidotto O, Labruna MB (2005) Rickettsial infection in animals and Brazilian spotted fever endemicity. Emerg Infect Dis 11(2):265–270. https://doi.org/10.3201/eid1102.040656
Santos TR, Carreira VS, Ferrari HF, Moreira MA, Luvizotto MC (2014) Comparison of PCR with stained slides of bone marrow and lymph nodes aspirates with suspect diagnosis for leishmaniasis. Acta Trop 140:137–140. https://doi.org/10.1016/j.actatropica.2014.08.016
Saridomichelakis MN, Mylonakis ME, Leontides LS, Koutinas AF, Billinis C, Kontos VI (2005) Evaluation of lymph node and bone marrow cytology in the diagnosis of canine leishmaniasis (Leishmania infantum) in symptomatic and asymptomatic dogs. Am J Trop Med Hyg 73:82–86
Satta G, Chisu V, Cabras P, Fois F, Masala G (2011) Pathogens and symbionts in ticks: a survey on tick species distribution and presence of tick-transmitted micro-organisms in Sardinia, Italy. J Med Microbiol 60:63–68. https://doi.org/10.1099/jmm.0.021543-0
Solano-Gallego L, Rossi L, Scroccaro AM, Montarsi F, Caldin M, Furlanello T, Trotta M (2012) Detection of Leishmania infantum DNA mainly in Rhipicephalus sanguineus male ticks removed from dogs living in endemic areas of canine leishmaniosis. Parasit Vectors 5:98. https://doi.org/10.1186/1756-3305-5-98
Swets JA (1988) Measuring the accuracy of diagnostic systems. Science 240:1285–1293. https://doi.org/10.1126/science.3287615
Trotta M, Nicetto M, Fogliazza A, Montarsi F, Caldin M, Furlanello T, Solano-Gallego L (2012) Detection of Leishmania infantum babesia canis, and rickettsiae in ticks removed from dogs living in Italy. Ticks Tick Borne Dis 3:294–297. https://doi.org/10.1016/j.ttbdis.2012.10.031
Viol MA et al (2016) Identification of Leishmania spp. promastigotes in the intestines, ovaries, and salivary glands of Rhipicephalus sanguineus actively infesting dogs. Parasitol Res. https://doi.org/10.1007/s00436-016-5111-5
Xu D et al (2015) Molecular detection of vector-borne agents in dogs from ten provinces of China. Parasit Vectors 8:501. https://doi.org/10.1186/s13071-015-1120-y
Author information
Authors and Affiliations
Contributions
TRSD: original draft, formal analysis, validation, visualisation; MAV: data curation, investigation, methodology; VMFL: methodology, validation, writing—review and editing; BCMO: data curation, investigation, methodology; LVS: data curation, investigation, methodology; AJC: supervision, writing—review and editing; JFG: validation, writing—review and editing; KDSB: conceptualisation, project administration, resources, funding acquisition, supervision, writing—review and editing.
Corresponding author
Ethics declarations
Consent for publication
Not applicable.
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Highlights
• The gold standard in diagnostics tests of Leishmania spp. in ticks was intestine IHC.
• The best tissue for Leishmania spp. detection in dogs was the lymph node.
• The intestine was the best tissue for Leishmania diagnosis in ticks.
• There was strong agreement between IHC and RT-PCR for ticks.
• Intestine IHC in ticks showed almost perfect correlation with the lymph node FNAB.
Rights and permissions
About this article
Cite this article
Santos-Doni, T.R., Viol, M.A., Lima, V.M.F. et al. Canine visceral leishmaniasis and Rhipicephalus sanguineus: evaluation and comparison of classical techniques. Vet Res Commun 46, 109–120 (2022). https://doi.org/10.1007/s11259-021-09834-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11259-021-09834-y