Clinical value of anti-Leishmania (Leishmania) chagasi IgG titers detected by flow cytometry to distinguish infected from vaccinated dogs

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Abstract

Leishmune® vaccination covers a broader number of endemic areas of canine visceral leishmaniasis (CVL) and therefore the development of new serological devices able to discriminate CVL from Leishmune® vaccinees becomes an urgent need considering the post-vaccine seroconversion detected throughout conventional methodologies. Herein, we have described the establishment of a flow cytometry based methodology to detect anti-fixed L. (L.) chagasi promastigotes antibodies (FC-AFPA-IgG, FC-AFPA-IgG1 and FC-AFPA-IgG2) in sera samples from Leishmania (Leishmania) chagasi infected dogs and Leishmune® vaccinees. The results of FC-AFPA were reported along the sera titration curve (1:128–1:524,288), as percentage-of-positive-fluorescent-parasite (PPFP). The use of PPFP = 20% as a cut-off edge to segregate negative and positive results at sera dilution 1:2048 revealed outstanding performance indexes that elect FC-AFPA-IgG and IgG2 (both detected by polyclonal FITC-labeled second step reagent) applicable to the serological diagnosis of CVL, with 100% of specificity for both IgG and IgG2 and 97 and 93% of sensitivity, respectively. Moreover, FC-AFPA-IgG, applied at sera dilution 1:2048, also appeared as a useful tool to discriminate L. chagasi infected dogs from Leishmune® vaccinees, with 76% of specificity. Outstanding likelihood indexes further support the performance of FC-AFPA-IgG for exclusion diagnosis of CVL in Leishmune® vaccinees. Analysis of FC-AFPA-IgG at sera dilution 1:8192 revealed the most outstanding indexes, demonstrating that besides the ability of PPFP ≤20% to exclude the diagnosis of CVL, a PPFP values higher 80%, mostly observed for infected dogs (INF) have a minimal change to come from a non-infected animal (NI) or Leishmune® vaccinees (VAC). Together, our findings showed the potential of both anti-L. chagasi FC-AFPA-IgG and IgG2 to distinguish the serological reactivity of L. chagasi infected dogs from Leishmune® vaccinees, which will further contribute for the differential diagnosis in the context of CVL immunoprophylaxis.

Introduction

Canine visceral leishmaniasis (CVL) is a debilitating and often fatal disease, caused by Leishmania (Leishmania) infantum syn. Leishmania (L.) chagasi that affects wild and domestic animals as well as humans in several parts of the Old and New World (WHO, 2000). The disease is widespread in tropical and subtropical areas of Latin America, Europe, Africa and Asia. In Brazil, CVL becomes a serious public health problem with occurrence in rural and several urban areas of metropolitan cities (Arias et al., 1996, Tesh, 1995). From the epidemiological standing point, the canine visceral leishmaniasis is considered to be more important than the human disease, due to its higher prevalence and the fact that both asymptomatic and symptomatic dogs are equally infectious to the vectors (Molina et al., 1994). Moreover, the infected dogs, even those with asymptomatic disease display high frequency of Leishmania amastigotes in their skin (Reis et al., 2006a, Reis et al., 2006b).

The major prophylactic practice to control the spread of the urban human visceral leishmaniasis (VL), recommended by the World Health Organization, involves a systematic treatment of human cases besides vector control by insecticide and elimination of seropositive dogs, the main domestic reservoir of VL (Tesh, 1995). However, it is important to mention that the serologic tests recommended by the Brazilian Healthy Ministry (Indirect Immunofluorescence Antibody Test, IFAT and conventional crude antigen enzyme-linked immunosorbent assay, ELISA) display a highly variable efficiency to detect canine L. chagasi infection, mainly due to the cross-reactivity of samples from dogs infected with other parasitic diseases and the low ability of those methods to detect seropositivity in asymptomatic dogs (Lira et al., 2006, Mancianti et al., 1995, Dye et al., 1993; Palatnik-De-Sousa et al., 2001). In clinical practice, the veterinarian is usually confronted with cases of positive serology not compatible with or suggestive of CVL according to lack of specific symptoms (Francino et al., 2006). Despite the specificity of the parasitological methods, including direct microscopic demonstration, culture isolation of Leishmania parasites from tissue aspirates, its use in clinical laboratory have some limitation, mainly due to the variable and relatively low sensitivity of such procedures (Schnur and Jacobson, 1987, Osman et al., 1997, Reale et al., 1999), besides the time-consuming and requirement of experienced personnel (Mettler et al., 2005). In the last decade, the implementation of molecular devices, based on the polymerase chain reaction (PCR) has contributed to the increment of sensitivity with confirmed specificity to detect Leishmania DNA in biological specimens (Pirmez et al., 1999). However, the development of new immunobiological tools to control CVL in endemic areas incited new challenges for the scientific community, considering that vaccines derived from whole crude Leishmania antigens as well as third generation DNA vaccines may lead to false positive results even when applying molecular based diagnosis. Therefore, the search toward the development of alternative serological devices still represents an important challenge for many investigators.

Besides these facts, the dog removal based on seropositivity, has been pointed out as drastic tool to reduce cases of VL mainly due to its impact in general society. Therefore, it has been considered that a protective vaccine for dogs against canine visceral leishmaniasis would represent the most effective control tool in eradication of the disease (Marzochi et al., 1985, Dye, 1996).

In this context, Palatnik-De-Sousa et al. (2001) have described the protective effect of the fucose–mannose ligand (FML)-vaccine on canine visceral leishmaniasis. This formulation had already shown to be safe, immunogenic and protective for dogs against L. chagasi infection. Recently, the FML-vaccine (Leishmune®, Fort Dodge, EUA) has been registered by the Brazilian Ministry of Agriculture and legally authorized for use by veterinary clinicians.

As expected for the use of saponin adjuvants, the Leishmune® canine vaccine induces a strong humoral immune response, soon after the complete vaccination (Cabrera et al., 1999). Therefore, an indistinct IgG mediated anti-L. chagasi immune response is detected in Leishmune® vaccinees, indistinct from that due to natural infection with L. chagasi. As serological devices are still recommended as a tool for epidemiological surveys, the screening of canine population based on conventional serological approaches (IFAT or crude antigen ELISA), have become unconfident to identify naturally infected ones, reservoirs of Leishmania, that should be normally removed for sacrifice (Mendes et al., 2003). While some investigations have been able to demonstrate a dichotomous response to Leishmania antigens in the seropositive naturally infected dogs and Leishmune® vaccinees (Mendes et al., 2003), the development of new techniques still represent a broad field for investigations aiming to develop alternative methodologies of high specificity and sensitivity for an accurate differential diagnosis of CVL in a population that include vaccinated animals.

Since 1995 our groups have been working on flow cytometry based serological approaches applicable in clinical laboratories (Martins-Filho et al., 1995, Rocha et al., 2002, Bicalho et al., 2004, Reis et al., 2005). Flow cytometry is a well-established methodology in clinical laboratories with a large number of applications been available (Jaroszeski and Radcliff, 1999). While the term flow cytometry refers to the measurement of cells, the approach of making sensitive multiparameter optical measurements in a flowing sample stream is a very general analytical approach (Nolan and Mandy, 2006). The past few years we have worked to establishment of several applications of flow cytometry technology for molecular analysis in human and canine diseases and adapted the flow cytometry measurements using microparticles as solid supports (Martins-Filho et al., 1995, Rocha et al., 2002, Rocha et al., 2006, Bicalho et al., 2004, Reis et al., 2005).

In this context, it is possible that IgG subclasses responses may provide a useful marker for disease underlying the post-vaccine seroconversion. Four IgG subclasses (IgG1, IgG2, IgG3 and IgG4) have been described in canine sera samples (Mazza et al., 1993). Despite monoclonal antibodies have been previously used (Quinnell et al., 2003), they are not yet commercially available as fluorochrome-labeled second step reagent neither validated for flow cytometric use.

Herein, we evaluate the performance of the flow cytometry using pre-fixed promastigotes as tool for detection of anti-L. (L.) chagasi immunoglobulin G and subclasses to as the basis for the development of new diagnostic tools for canine visceral leismaniasis that allowed the discrimination between sera sample from Leishmune® vaccinees from L. chagasi infected dogs. Our findings support the use of FC-AFPA-IgG and IgG2 (both detected by polyclonal FITIC-labeled second step reagent) as a helpful tool to elucidate seropositivity during large-scale epidemiological screenings of canine visceral leishmaniasis. Moreover, these methodologies would support the use of Leishmune® to control canine kala-azar despite the seroconversion detected by conventional serological approaches following vaccination.

Section snippets

Study population

Twenty-nine breed adult dogs of both genders, aging from 2 to 6 year-old naturally infected with Leishmania (L.) chagasi, presenting IFAT titer ≥1:40 and positive parasitological diagnosis to Leishmania in tissue smears (bone marrow, ear, skin, spleen, liver or popliteous lymph node), provided by Control Zoonosis Center at Belo Horizonte (Minas Gerais State, Brazil) were enrolled into the group of infected dogs (INF).

Twenty-one healthy German shepherd dogs, age ranging from 2 to 6 years,

Performance indexes elect FC-AFPA-IgG and IgG2 applicable to the serological diagnosis of CVL

Sera samples from 29 L. chagasi naturally infected dogs (INF) and 15 non-infected healthy animals (NI) were tested by FC-AFPA-IgG, FC-AFPA-IgG1 and FC-AFPA-IgG2 in parallel experiments using FITC-labeled sheep (anti-IgG and anti-IgG2) and goat (anti-IgG1) polyclonal antibodies.

The mean values for the percentage of positive fluorescent parasites (PPFP) observed along the FC-AFPA-IgG, IgG1 and IgG2 titration curves are presented in Fig. 1. Data analysis demonstrated that the mean PPFP values

Discussion

The accomplishment of CVL immunoprophylaxis with the availability of Leishmune® for veterinary uses in clinics surrounding the CVL endemic areas has prompted out many issues regarding the value of positive serological reactivity to distinguish CVL from Leishmune® vaccinees. In this context, the RIFI and conventional ELISA have been considered unable to discriminate Leishmania-infected from Leishmune® vaccinees causing serious problems for the public health domain. Despite the cumulative

Acknowledgements

Authors would like to thanks the members of the Chagas Diseases Laboratory for technical assistance. This work was supported by FAPEMIG/BR/grant: EDT-236903. We are thankful to the Polícia Militar de Minas Gerais and Fundação Nacional da Saúde, Ministério da Saúde, Distrito Regional de Belo Horizonte, Minas Gerais, Brazil for their support with dog management. We also thank Professor Leonardo Hortmann for the critical reading of the manuscript, English editorial suggestions and changes.

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