Angiostrongylus vasorum is a nematode that is part of the Metastrongyloidea superfamily that parasitizes do- mestic dogs (Canis familiaris) and wild carnivores. Disease caused by Angiostrongylus vasorum is increasingly diagnosed in dogs, as the geographic range of the parasite increases along with awareness among clinicians. A determinant factor in the pathology of canine angiostrongylosis seems to be related to the location of the parasite in the definitive host. The presence of the parasite inside the arteries and branches of the host promotes a me- chanical and metabolic action on the vessels walls, which may alter its homeostasis [1] [2] resulting in pneumo- nia, loss of racing performance, coughing and anemia [3] .

Flow cytometry is a technique widely used in human medicine that is also becoming a helpful tool in veteri- nary medicine, being applied to a greater variety of conditions for diagnosis and prognosis of diseases and par- ticularly in small animal practice [4] [5] .

The bronchoalveolar lavage (BAL) is an accurate technique for the diagnosis of canine angiostrongylosis, es- pecially in the situations when the feces’ parasitological is negative and the clinical symptomatology matches the infection [6] [7] . Considering that BAL is a technique that allows the retrieval of cells and other elements that line the lung surface (airway) for cytological evaluation, the goal of this experimental study was to perform flow cytometric analyses of cells retrieved using BAL to determine CD4+/CD8+ profile during the infection.

The phenotypic analysis of recovered cells in the BAL was performed through the identification and demarca- tion of a R1 region, excluding debris (Figure 2(a)). Figures 2(c)-(e) show examples of marks of different cell populations recovered with the BAL of dogs and marked with primary ACMOS against cell surface receptor (CD4, CD8).

The absolute number and percentage of total, helper and cytotoxic T cells were estimated based on expression of CD4+ (helper T-cell) and CD8+ (cytotoxic T-cell) on the gated lymphocyte population. As shown outlined in Figure 1(b), the cells population in region R1 showed no type 1 fluorescence, when not marked with specific cell surface markers MoAb.

The TCD4+ cell population was more representative in infected group from 30 to 120 DAI (p < 0.05) (Figure 3(a)). The same profile was observed in relation to CD8+ T-cell population. Fluctuation on these T-cell markers was apparent as a significant difference in the absolute number of CD8+ T-cells detected between infected and non-infected animals (p < 0.05) (Figure 3(b)).

There were significant differences in CD4+ (p < 0.05) and CD4:CD8 ratio (p < 0.05) infected group in rela-

tion to non-infected one from 30 and 120 DAI (Figure 3(c)).

The gut and lung phases of helminth parasite invasion represent particular sites where the migrating larvae can be relatively easily eliminated from the host. Although this, tissue-migrating helminth parasites of many species have found that a relatively easy way to use mammalian hosts for their survival is to gain access to the blood circulation, often via the lung, where they mature, reproduce, and use the host excretory processes for dispersal and reinfection, as Angiostrongylus vasorum [10] .

It has long been recognized that increasing host age and exposure to infections result in a state of Th2- mediated immunity which protects the host from reinfection [10] . Through the evaluation of lymphocites surface markers by flow cytometry was be possible identify the phenotype of blood lymphocites at different periods of infection. It was found that after 30 DAI the population of T lymphocytes (CD4+ and CD8+) was higher in infected animals than control animals.

The identification of subpopulations of T lymphocytes by evaluating the co-expression of CD4 and CD8 re- ceptor proteins has shown that despite the increase in both populations, there was a predominance of CD4+ T- cells of CD8+ T-cells. These increases of CD4+ T-cells associated with the increase of the ratio between CD4+/ CD8+ suggest polarization of a Th2 response.

Profound blood and tissue eosinophilia are among the hallmark features of parasitic helminth infection, ob- served in response to activation of CD4+ Th2 lymphocyte at specific stage of parasite life cycle [10] . Helminths infections generates a dominant type 2 response among both adaptive (Th2) and innate (macrophage, eosinophil, and innate lymphoid) immune cell types [12] .

Although, at the present study, have not been evaluated the profile of cytokines produced during infection by A. vasorum, the cellular phenotype may suggest, indirectly, the action of certain cytokines in the activation of some specific cell types, such as eosinophils.

According to Barçante et al. (2012), absolute cell counts recovered from lungs of infected dogs revealed that eosinophils showed a significant increase in number from 30 reaching peaks at 30 and 60 DAI .The evaluation of lung cellularity in infected animals suggests that the phase of greatest antigenicity of A. vasorum happens in definitive host, when occurs rapid growth and migration to the heart and lungs [13] . In this way, the increase of eosinophils could represent a consequent augment of a Th2 immune response, which agrees with the increase in the number of CD4 + cells and the CD4/CD8 ratio from infection, with significant changes to the corresponding period of 30 to 120 DAI. Still this focus, Harvie et al. (2010) who pointed the lung how an important site for priming immune protection. Furthermore, the lung-initiated, CD4 T-cell-dependent, and IL-4.

This study shows that the immunophenotyping of cells retrieved using BAL providing additional information about inflammatory diseases during infection by A. vasorum. However, the immune cells, signaling factors, and cytokines that mediate such immunity and how and where they act within the body remain largely undefined during angiostrongylosis

The work was partially supported by FAPEMIG and CNPq—Brazil. The authors wish to thank Eduardo Luiz de Oliveira, Hudson Andrade dos Santos and Edna Maia for technical assistence.