Volume 30, Number 10, October 2008 Splenic changes in visceral leishmaniasis Figure 2 Association between changes in the follicles and marginal zones of the spleen of dogs from an endemic area of visceral leishmaniasis. (a) association between follicle size and marginal zone size; (b) association between germinal centre size and marginal zone size. Each circle on the graph represents data from one animal. emaciation and disorganization of the lymphoid tissue was observed even in animals with negative splenic culture. Out of 10, 8 (80%) animals who were undernourished in this group had loss of white pulp architecture deﬁnition, whereas only 9 (36%) out of 25 without emaciation exhibited this altera- tion. In addition, being underweight was associated with perisplenitis. This condition was present in 12 (46%) out of 26 animals with emaciation, but in only 6 (15%) out of 41 animals with normal nutritional status (χ2-test, P = 0·0107). DISCUSSION Changes in lymphoid organs associated with VL have been previously reported in various studies. However, the current study presents a systematic view of changes in different compartments of the spleen in naturally infected dogs from an endemic area of VL, and describes different patterns of immunological responses to the parasite. In a previous study, we have shown that negative LST results were associated with high levels of serum antibody activity and emaciation in L. chagasi-infected dogs. We have now shown that infected animals with negative LST also have (i) increased frequencies of perisplenitis, granuloma, and parasites in the red pulp (ii) a variety of white pulp alterations, such as atrophy and loss of structural deﬁnition (iii) lymphoid follicles that are reduced in number and size, and (iv) germinal centres and marginal zones that are frequently small or absent. These animals, with loss of lymphoid follicle deﬁnition, perisplenitis, and a high parasite density in the spleen (in which parasites were seen by conventional histology), were also more frequently underweight; this association likely reﬂects the status of the clinical disease. Furthermore, we observed that the distribution of parasites in the spleen was not homogeneous. In heavily infected animals, parasites were distributed almost continu- ously in the red pulp. In animals with a less intense parasite burden, a discontinuous distribution of infected cells was observed, predominating in the subcapsular area. Perisplenitis is a common ﬁnding in canine VL (26,27). Although the increase in spleen size may predispose that organ to trauma or mechanical stress, the actual genesis of peris- plenitis in leishmaniasis remains unknown. It is interesting, however, that the perisplenitis was associated with the presence of parasite-containing macrophages in subcapsular areas. The presence of such macrophages in capsular and subcap- sular inﬂammatory inﬁltrates has also been observed in other studies (26,27). These ﬁndings may indicate a role for inﬂammation in parasite dissemination (28,29), or that the parasite elicits an inﬂammatory reaction. In light of previously published data regarding immunity to Leishmania, it seems contradictory that splenic granulo- mas were more frequently found in the group of infected animals with negative LST than in infected animals with positive LST. In effect both, granuloma and positive LST, may result from DTH reactions to Leishmania antigens. The granulomas observed in the spleen of the animals in this study varied from poorly structured aggregates, consisting of at least four large epithelioid macrophages (Figure 1b), to the barely organized structure shown in Figure 1(c). Although parasites were not usually seen in the interiors of epithelioid cells, amastigotes were more frequently observed in the spleen of animals with granulomas. Hence, as observed in humans with long duration VL (30), these granulomas appear to reﬂect a persistent chronic inﬂammation in response to uncontrolled infection, rather than protection against the disease. Although granulomas may constitute a step towards infection control, the conditions in their interior may be © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Parasite Immunology, 30, 515–524 521