Volume 30, Number 10, October 2008 Splenic changes in visceral leishmaniasis Table 1 General characteristics of a sample of dogs, from an endemic area of visceral leishmaniasis, used in this study Parameters Number of animals Spleen culture Leishmanin’ skin test Serologya With emaciationc Groups of animals Infected Potentially Resistant to VL 22 – + 0·55 ± 0·39 6/20 (30%) Potentially susceptible to VL 25 + – 1·54 ± 0·91b 15/25 (60%)d Undeﬁned susceptibility status 11 + + 0·87 ± 0·77 2/11 (18%) Noninfected 14 – – 0·28 ± 0·13 4/13 (31%) a Expressed as mean ± SD of the OD. 490 nm obtained in indirect ELISA for detection of anti-Leishmania antibodies in the serum; bStatistically signiﬁcant from the control group, Kruskal–Wallis test, P < 0·001; cNumber of emaciated animals/total number of animals in which emaciation was investigated (%); dStatistically signiﬁcant from the other groups, χ2-test P = 0·02. peri-arteriolar lymphocyte sheath, germinal centre, man- tle zone and marginal zone (24); slightly disorganized, with either hyperplastic or hypoplastic changes leading to a loss in deﬁnition of any of the regions of the white pulp; moderately disorganized, when the white pulp was evident, but its regions were poorly individualized or indistinct; extensively disorganized, when the follicular structure was barely distinct from the red pulp and T-cell areas (Figure 1d–i). The last two categories were frequently associated with lymphoid atrophy. 6. Frequencies of cell populations in the red pulp, with the frequencies of lymphoblasts, plasma cells, macrophages, granulocytes, and megakaryocytes semiquantiﬁed as absent, rare (seen as isolated cells or small aggregates in a small proportion of the examined ×400-magniﬁed microscopic ﬁelds), few (seen as isolated cells or small aggregates in approximately half of the examined ×400- magniﬁed microscopic ﬁelds), average (seen as single cells or aggregates observed in most of the examined ×400-magniﬁed microscopic ﬁelds), or frequent (consist- ing of single cells or aggregates seen in roughly all of the examined ×400-magniﬁed microscopic ﬁelds). The cells were characterized morphologically according to the following criteria: a. Lymphoblasts: Cells with round nuclei containing condensed chromatin, inconspicuous nucleoli, and scant cytoplasm. b. Plasma cells: Cells with eccentric nuclei, heterochro- matin dispersed around the edge in a pattern similar to the numerals of an analogue clock face, and basophilic cytoplasm with clear, perinuclear vacuoles. c. Macrophages: Large cells with oval or reniform nuclei containing loosely packed chromatin surrounded by a rim of eosinophilic cytoplasm with indistinct edges. © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Parasite Immunology, 30, 515–524 d. Granulocytes: Cells with highly lobulated nuclei with densely packed chromatin. e. Megakaryocytes: Very large cells with wide and clearly deﬁned cytoplasm and large, lobulated nuclei containing condensed chromatin. 7. Parasite burden, where the amount of amastigotes in the spleen sections was estimated by conventional light micros- copy examination of a minimum of 40 and a maximum of 100 nonoverlapping high-power (×1000-magniﬁed) fields per section. The examined ﬁelds were equally distributed between the subcapsular (Figure 1k) and internal red pulp (Figure 1l). The results are expressed as the proportion of independent ﬁelds with amastigotes. Expression and analysis of the results The numerical data shown in the text, table and graphs represent absolute or relative numbers of animals with the corre- sponding condition, unless otherwise stated. The signiﬁcance of the differences between the proportions of the studied variables among the groups was tested using the χ2-test with a Yates’ correction, or a Fishers’ exact probability test when recommended. When appropriate, a paired t-test or the Kruskal– Wallis statistic were used for testing differences involving the means of numerical data obtained from different groups (25). The critical level of signiﬁcance was set at P < 0·05%. RESULTS Perisplenitis Perisplenitis was present in 20 (28%) out of 72 animals (Table 2 and Figure 1a). It was more frequent in animals in the infected and potentially susceptible to VL group (18 out of 25 animals, 72%) than other groups (χ2-test, P < 0·0001). 517