PRELIMINARY HISTOPATHOLOGICAL AND IMMUNOPHENOTIPIC CHARACTERIZATION OF TISSUES FROM SARDINIAN CATTLE INFECTED BY ECHINOCOCCUS GRANULOSUS S.S.

The mechanisms of immune evasion, host-parasite interplay and immune pathogenesis of Echinococcus granulosus (EG) in cattle are poorly characterized, and the scientific literature lacks information on the local inflammatory response. Cystic echinococcosis (CE) caused by EG is the most widespread zoonotic disease in both developed and developing countries. In Italy, CE is considered endemic in Sardinia where the prevalence rates are of 75% in sheep and 41.5% in cattle [1]. The aim of this study was to identify the immune reaction surrounding cysts in livers and lungs from naturally infected bovines slaughtered in Sardinia between 2015-2017. In this study, a total of 70 hydatids, 55 from lungs and 15 from livers, were collected from 21 bovines. Each cyst was measured during macroscopic examination, processed by routine histology and stained with both haematoxylin/eosin and Masson’s trichrome. Fertility was assessed by microscopic examination of protoscoleces’ presence and vitality. Germinal layer (GL) was used for molecular characterization by polymerase chain reaction (PCR), carried out by amplifying fragments within 2 mitochondrial genes, NADH dehydrogenase 1 (ND1) and cytochrome C oxidase subunit 1 (cox1). Cysts were classified according to the degree of inflammatory infiltrate into four categories: absent, mild, moderate and severe. The evaluation of the immune response was carried out by indirect immunohistochemistry (IHC) using the following antibodies: CD3, Cd79α, MAC387 and FoxP3, to identify T and B lymphocytes, macrophages and Treg cells, respectively. Stained tissue sections were analyzed at 200X magnification. CD3 and CD79 positive cells were scored in 5 random fields of the adventitial layer of the cyst. Two fertile pulmonary cysts did not show any inflammation, and the remaining 68 cysts were classified as infertile: of these, 6 cysts (2 lungs, 4 liver) showed mild inflammation, 44 (40 lungs, 4 livers) moderate and 18 (11 lungs, 7 livers) severe inflammatory reaction. PCR results demonstrated that all isolates belonged to E. granulosus sensu stricto (former G1 or sheep strain). IHC showed a majority of T lymphocytes vs B lymphocytes in the 68 samples analyzed. Presence of macrophages and Treg cells was negligible. Furthermore, in the infertile cysts there was a cellular layer, adjacent to the capsule wall, probably derived from macrophages. Our results are in agreement with observations in sheep [2] with a lower prevalence of fertile cysts in cattle. To better understand the parasitic pathogenesis, our future goal will be to carry out proteomic analysis to investigate the molecular cross-talk between host and parasite and to identify novel markers with potential applications in clinical diagnostics. [1] Garippa G. Updates on cystic echinococcosis (CE) in Italy, Parassitologia, 48, 57-59, 2006. [2] Vismarra et al. Immuno-histochemical study of ovine cystic echinococcosis (Echinococcus granulosus) shows predominant T cell infiltration in established cysts, Veterinary Parasitology, 209: 285-288, 2015.