PhD Thesis defense by Agustín Miguel Rebollada Merino at the VISAVET Centre of the Complutense University of Madrid

November 14^th, 2023

Brucellosis (Brucella spp.) is a reemerging zoonotic notifiable disease. It is subject to surveillance, control, and eradication programs worldwide. Most Member States of the European Union are considered free of the disease, but brucellosis is endemic in developing countries, limiting their economy, and causing disease in humans. Despite the relevance of many of these Brucella species, there are some unknown aspects. The main objective of this PhD Thesis has been to explore new aspects of the epidemiology, immune response, and pathology of Brucella infection in domestic and wild animals to implement both diagnosis and control of this disease.

Porcine brucellosis (B. suis biovar 2) results in reproductive problems causing economic losses. However, the lesions of intrauterine B. suis infection are not characterized. Understanding them would facilitate diagnosis and the development of control measures. Histological and microbiological studies were performed on placentas (n = 4) and fetuses (n = 8) during an outbreak of abortions in pigs. Brucella was cultured from vaginal swabs, placentas, and fetal tissues; B. suis biovar 2 infection was confirmed by polymerase chain reaction (PCR). Histologically, necrotizing-hemorrhagic placentitis, suppurative hepatitis, broncho-interstitial pneumonia and lymphoid organ depletion were the main lesions observed. Brucella was detected by immunohistochemistry in tissues. In addition, the lymphocytic population in placentas was composed of T cells, but not B cells.

Infected pigs act as carriers of B. suis contributing to its environmental persistence. The role of offspring and the environment was investigated during and after an outbreak of brucellosis on a pig farm. B. suis was diagnosed in sows (n = 1,140) by culture and PCR of vaginal swabs, indirect enzyme-linked immunosorbent assay (ELISA-I), and brucellin skin test. In weaned piglets (n = 899), diagnosis was made by I-ELISA and brucellin skin test. For the environmental monitoring program, sponges (n = 175) were placed in different areas of the facility and on personnel equipment. Weaned piglets reacted to in vivo techniques for brucellosis diagnosis. In addition, those born during the outbreak

showed higher seropositivity and dermoreactivity than those born after the outbreak. Genetic material (deoxyribonucleic acid, DNA) of Brucella was detected in the farm environment, decreasing after the outbreak. These results suggest a role of the environment and piglets in the epidemiology of brucellosis, which should be considered in surveillance programs.

Ticks have been suggested as vectors or carriers of Brucella. Ticks were collected from wild ungulates from hunting grounds in central Spain and analyzed by PCR. In addition, samples of ungulates from the studied areas were analyzed by ELISA. A total of 229 tick pools from wild boar (Sus scrofa, n = 176; 76.8%), deer (Cervus elaphus, n = 40; 17.4%), mouflon (Ovis orientalis musimon, n = 7; 3.06%) and fallow deer (Dama dama, n= 6; 2.62%) were analyzed. It was observed that 3.93% of the tick pools (9/229) from 17,07% of the hunting grounds (7/41) were Brucella positive. All positive ticks were Dermacentor (D. marginatus or D. reticulatus) from wild boar. ELISA showed positive results in three wild boars from two areas (28.5%) with any Brucella-positive ticks. This study indicates that arthropod vectors should be considered in the epidemiology of brucellosis.

B. ovis causes alterations in the reproductive tract of rams. Its negative economic impact on production justifies a thorough understanding of the interactions between B. ovis and the host. Epididymal lesions of B. ovis-infected rams were histologically classified into early and advanced. In both, expression by immunohistochemistry of Brucella, inflammatory cell markers (CD3, CD79cy) and cytokines (IFN-γ, TNF-, TGF-β1) were evaluated. Early lesions were characterized by epithelial changes, interstitial inflammation, and mild fibrosis; while advanced lesions were granulomas with numerous macrophages, multinucleated giant cells, lymphocytes, and plasma cells. Brucella expression was observed in both stages. The cellular response in B. ovis lesions was predominantly T lymphocytes (CD3+), while there were low numbers of B lymphocytes and plasma cells (CD79cy+). Lymphocytes expressed IFN-γ in both lesions, suggesting an adaptive immune response initiated by T helper (Th) cells 1. TNF- was expressed on neutrophils in epithelial microabscesses and intraepithelial T cells in early lesions,

indicating a promotion of neutrophil phagocytosis. On the other hand, in advanced lesions TNF- was poorly expressed, which may allow persistence of B. ovis. TGF-β1 expression was increased in advanced lesions. These results indicate that the immune response in brucellosis differs according to evolution.
Neurobrucellosis occurs in up to 25% of human patients. Interestingly, central nervous system involvement by B. ceti has been described especially in striped dolphins (Stenella coeruleoalba). Twenty-one cases of neurobrucellosis in striped dolphins with confirmed infection by culture and PCR were evaluated. For each case, 19 parameters were evaluated histologically. In addition, immunohistochemical expression of Brucella, inflammatory cell markers and cytokines was investigated. Brucella was localized in the cytoplasm of macrophages. Inflammation of the leptomeninges, ependyma and/or choroid plexus is lymphohistiocytic, containing macrophages/microglia (Iba-1+), T cells (CD3+) and B cells (CD20+) in equal proportion. Immunohistochemical expression of TNF-, IFN-γ and IL-2 indicates an intense proinflammatory humoral response, stimulating both macrophages and T cells. Neurobrucellosis in cetaceans, humans, and in vivo and in vitro models of neurobrucellosis are pathologically and immunologically similar. Disease surveillance in cetaceans may allow to expand the knowledge of the comparative immunology of infectious diseases, in particular brucellosis, under a One Health approach.