C.C. Santana et al. the infective inoculum is very likely identical to that causing human infection in endemic areas. Observations made in canine VL may therefore conﬁrm and reﬁne conclusions drawn from studies using murine experimental models. Recently, we observed that a positive leishmanin skin test (LST) was negatively associated with emaciation and speciﬁc antibody activity in Leishmania-infected dogs (16). In the present study, we examined the spleen histology of these animals to identify alterations potentially associated with disturbances in the immune response to Leishmania and other pathogens that occur at late stages of the disease. We decided to examine the spleen because it plays a central role in VL. The spleen is infected in all cases of the disease. In contrast to other organs like the liver, the spleen main- tains the infection during the entire course of VL (11,12). In fact, a splenic index of parasitism is used as a clinical criterion of therapeutic response in human VL (17). The spleen is also a lymphoid organ with cellular diversity and compartmentalized microenvironments, suitable for the deﬁnition of immunologic processes that may inﬂuence the outcome of infections (18,19). During the course of VL, the spleen becomes an evident site of interaction between the immune system and the Leishmania, because all of the obligatory participants in the immune response against the parasite are present in large quantities. These include the antigen (live parasites or their debris), antigen-presenting cells, and lymphocytes capable of responding to these antigens. Changes in the splenic microenvironment have been identiﬁed following the experimental infection of mice by a variety of pathogens, including Leishmania (20,21). Our hypothesis is that such alterations are also present in the course of natural infection by L. chagasi in dogs and that they reﬂect a status of susceptibility to the disease. MATERIALS AND METHODS Animals Spleen samples from 72 dogs were used in this study. The specimens were obtained from the archive of histological samples of the Gonçalo Moniz Research Center, FIOCRUZ, Salvador, Brazil. These animals were stray dogs of different breeds and estimated ages collected from the streets of Jequié (Bahia State, Brazil, an endemic area for visceral leishmaniasis) at different periods between 1998 and 2001 in collaboration with the Endemic Diseases Surveillance Program of the State Health Service. The nutritional status of the animals was recorded into two categories: showing evidence of emaciation (when prominences of bones from the vertebral column, iliac crest and ribs were present), or normal (when bone prominences were absent). The presence of anti-Leishmania antibodies in the serum were investigated 516 Parasite Immunology by ELISA, and a cellular immune response against L. chagasi antigens was detected by LST. The details of these tests are reported elsewhere (16,22,23). The animals were kept for at least 48 h in the municipality kennel. When not claimed by their owners, the animals were culled, as recommended by the Brazilian program for the control of zoonotic diseases. Spleen specimens were then collected for both Leishmania, detection by in vitro culture, and histology. All procedures were conducted in accordance with the Oswaldo Cruz Foundation guidelines for animal experimentation. All of the animals that had detectable Leishmania in their spleen cultures or a positive LST, with available spleen samples, were included in the study. The animals were distributed into the following categories: (i) Infected and potentially resistant to VL – 22 animals with a positive LST and negative spleen culture for Leishmania; (ii) Infected and potentially susceptible to VL – 25 animals with a negative LST and positive spleen culture for Leishmania; (c) Infected with undeﬁned susceptibility status – 11 animals with both a positive LST and positive spleen culture for Leishmania. As a control group, specimens from 14 animals with negative spleen cultures, a negative LST, and the absence of anti-Leishmania antibody activity in the serum as measured by ELISA were randomly selected; we referred to this group as noninfected. Table 1 summarizes the characteristics of the groups used in the study. Spleen samples and histological analysis Three to 4 mm-thick slices of spleen tissue were cut transversally to the capsule and ﬁxed in 10% formalin. After ﬁxation, tissue slices were embedded in parafﬁn. Four to 5 μm-thick sections were cut and stained with haematoxylin and eosin (H&E). These sections were examined by at least two of the authors who were blind to previous knowledge of the identities of the animals. The authors observed the follow- ing parameters: 1. Presence of perisplenitis, as evidenced by a focal thickening of the splenic capsule with an inﬂammatory inﬁltrate frequently associated with incipient ﬁbrosis (Figure 1a). 2. Presence of granuloma, with focal aggregates of four or more epithelioid macrophages (Figure 1b,c). 3. Lymphoid follicle frequency, in which the density of lym- phoid follicles in the splenic tissue sections was classiﬁed as rare (follicles were infrequently found), low (follicles were easily found but sparse in the red pulp), average (follicles were uniformly distributed and easily found in the section) and high (follicles were frequent and large). 4. Size of lymphoid follicles, germinal centre and marginal zone, in which the lymphoid follicles and the marginal zone were classiﬁed as tiny, small, average and large. 5. Degree of structural organization of the white pulp, in which the white pulp was classiﬁed as: well organized, with distinct © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Parasite Immunology, 30, 515–524