Vero cells were inoculated with the six species of Brucella (B. abortus, B. melitensis, B. suis, B. neotomae, B. canis, and B. ovis) and examined by fluorescence and electron microscopy. All Brucella spp. were internalized by Vero cells. In all cells except those inoculated with B. canis, the numbers of intracellular brucellae increased with time after inoculation. Intracellular brucellae were first seen within phagosomes and phagolysosomes. Subsequent localization within cisternae of the rough endoplasmic reticulum was seen with all species of Brucella, except B. canis, which was restricted to phagolysosomes. Although rough brucellae were more adherent and entered a greater number of Vero cells, intracellular replication occurred in a larger percentage of cells with smooth rather than with rough brucellae. These results suggest that phagocytosed Brucella spp. are transferred 1) to cisternae of the rough endoplasmic reticulum, where unrestricted bacterial replication takes place; or 2) to phagolysosomes in which Brucella spp. fail to replicate. The various strains of Brucella spp. differ in their ability to induce their own transfer to the rough endoplasmic reticulum.

Abstract. Ligated ileal loops of calves were inoculated with Brucella abortus and examined at 2, 4, 6, 10, and 24 hours post-inoculation. B. abortus was identified by light and electron microscopy using immunoperoxidase and antibody-coated colloidal gold techniques. B. abortus was detected in vesicles, phagolysosomes, and large vacuoles of lymphoepithelial cells. Numbers of intracellular bacteria decreased with time after inoculation. B. abortus was also seen between and below lymphoepithelial cells and free in the dome interstitiurn and intestinal lymph vessels. Neutrophils and macrophages in both epithelium and lamina propna contained intact or degraded bacteria within phagosomes, phagolysosomes, and multivesicular bodies. These studies showed that (1) transepithelial migration of B. abortus occurred principally by dome lymphoepithelial cell endocytosis and transport, and (2) B. abortus was degraded by macrophages and neutrophils of the gut-associated lymphoid tissue.The alimentary tract is generally accepted as a major route in the transmission of Brucella abortus in cattle.Licking of aborted fetuses and placental membranes or ingestion of contaminated milk by calves introduces brucellae onto the oral mucosa, tonsils, and gastrointestinal mucosa. Passage of B. abortus through epithelial barriers results in an acute regional lymphadenitis and bacteremia.21Epithelium covering domes of ileal Peyer's patches (IPP) is an important site of entry for several bovine pathogens that traverse the intestinal mucosa. 9J5,22,28,31 Ileal dome epithelium of calves and lambs are composed of a uniform layer of lymphoepithelial cells which have short, irregular microvilli and considerable endocytic a~t i v i t y .~J~. *~,~' Recently, two distinct types of Peyer's patches have been described in young ruminants (lambs and calves): 10~11,23-25 1) Ileal Peyer's patches (IPP), which were examined in this study, are a continuous group of lymphoid tissue in the terminal ileum that involute at sexual maturity and function as a 1" lymphoid organ in lambs for the production of B cells; and 2) Jejunal Peyer's patches (JPP), which correspond to the common concept of Peyer's patches and persist throughout life. Since IPP's are a site of contact for the immune system and antigen, they also have characteristics of 2" lymphoid organs for the production of antibody secreting ~e l l s .~O ,~~The purpose of this study was to characterize the uptake and processing of B. abortus in the ileum of colostrum-deprived calves using light and transmission electron microscopy. Our primary interests were the possible entry of bacteria through the lymphoepithelial cells of the Peyer's patches and the cellular response to bacterial infection in the gut-associated lymphoid tissue (GALT). Materials and MethodsFive colostrum-deprived calves from brucellosis-free herds were fed 500 ml of heat inactivated (56 C A 30 minutes) bovine serum and kept in isolation throughout the experiment (Table 1). All calves were within 24-96 hr of age at the time of inoculation. ...

In pregnant ruminants, Brucella abortus localizes and replicates within the rough endoplasmic reticulum of trophoblastic epithelial cells. In this study, Vero cells were exposed to B. abortus to investigate its internalization and intracellular growth in nonphagocytic cells. A new double-fluorescence staining procedure to discriminate between extracellular and intracellular bacteria was developed. Studies with the doublefluorescence staining procedure and quantitative bacteriologic culture of disrupted host cells showed that various B. abortus strains replicated within Vero cells, including smooth virulent (strains 2308S and 544), smooth attenuated (strain 19), and rough (strains 45/20 and 2308R) strains. Rough brucellae were more adherent and entered a greater number of Vero cells. Intracellular replication occurred in a larger percentage of cells with smooth virulent (2308S and 544) strains than with smooth attenuated (19) or rough (45/20 and 2308R) strains. Differences in adhesiveness and invasiveness were correlated to hydrophobicity of the organism, as measured by hydrocarbon adherence. Ultrastructurally, intracellular smooth (2308S) and rough (45/20) brucellae were consistently found within cisternae of the rough endoplasmic reticulum and nuclear envelope. The results suggest that transfer to the rough endoplasmic reticulum is the limiting step in the infection of nonphagocytic cells by B. abortus.

Invasion of the gravid bovine uterus by Brucella abortus often leads to placentitis and abortion. Within the placenta, brucellae replicate to high numbers in the trophoblastic epithelium. 1.7.8 Minimal degenerative changes in brucellae-filled trophoblasts suggest a unique bacterium-host cell relationship. In experimentally infected goats* and in chick embryo^,^ B. abortus replicates in the rough endoplasmic reticulum (RER) of trophoblasts and in nonphagocytic cells, respectively. Localization in the RER is unique among bacterial pathogens and may be critical for replication of B. abortus in certain tissues and in the pathogenesis of bovine brucellosis. Before this host-parasite relationship is further investigated, it is necessary to determine if B. abortus replicates in the RER of trophoblasts in the natural host. In this study, we report the intracellular location of B. abortus in the gravid bovine uterus as determined by immunohistochemical and electron microscopic examination.Six cows in midgestation were infected topically onto the conjunctivae by instillation of 1 x lo7 or 1 x lo8 organisms of B. abortus strain 2308. Cows were euthanatized with intravenous pentobarbital at 199 to 253 days of gestation (36 to 80 days post-exposure). Time of necropsy was based on onset of premature delivery. Brucella abortus was isolated from uterine exudate, chorioallantoic placenta, and fetal tissues by direct smears of tissues onto tryptose agar from all cows. For light microscopic examination, tissues from placenta and uterus were fixed for 24 hours in 10% neutral buffered formalin, processed by routine paraffin embedding techniques, sectioned, and stained with hematoxylin and eosin and a Brucella-specific avidin-biotin irnmunoperoxidase technique.6 Tissues from placenta and placentomes were fixed in 2.5% glutaraldehyde in 1.0 M cacodylate buffer and processed, and thin sections were stained with uranyl acetate and lead citrate and examined with transmission electron microscopy (Philips 4 10).Microscopic lesions were moderate to severe in all cows. Purulent exudate containing intra-and extracellular brucellae was in the inter-and periplacentomal uterochorionic spaces and at the base of cotyledonary villi. Chorioallantoic membranes had marked edema, several macrophages and neutrophils in the interstitium, segmental fibrinous vasculitis, and multifocal erosion of periplacentomal chorionic epithelium. Trophoblasts lining the base of cotyledonary villi and periplacentomal regions were filled with brucellae (Fig. 1). Numbers of infected trophoblasts varied among placentomes collected from the same cow; there were individual to confluently infected trophoblasts. In three cows, brucellae were present in neutrophils and macrophages and were also present extracellularly in the placental interstitium. Large colonies of extracellular brucellae were in the interstitium and capillary lumens in the chorionic villi of one cow (Fig. 2). Brucellar antigen was most abundant in trophoblasts; lesser quantities were in phagocytic cells and w...

Goats, both in late pregnancy and soon after parturition, were inoculated intravenously with Brucella abortus, and mammary glands and supramammary lymph nodes were examined by light and electron microscopy at 2 to 55 days post-inoculation. After 7 days, lymphoplasmacytic, histiocytic interstitial mastitis with a lobular and periductal distribution were detected microscopically. Brucellae, identified in tissues with immunoperoxidase staining and antibody-coated colloidal gold stain, were first seen in macrophages and neutrophils throughout mammary parenchyma, but most commonly in mammary alveoli. In subsequent samples, infected phagocytes progressively increased in number, especially in ductal and alveolar lumina, and adjacent parenchyma. B. abortus was in phagosomes and phagolysosomes in macrophages and neutrophils; degenerate and necrotic phagocytes were often filled with brucellae. Extracellular brucellae were associated with ruptured necrotic infected phagocytes. Supramammary lymph nodes draining infected mammary glands were enlarged. Lymphofollicular hyperplasia, medullary plasmacytosis, and sinus histiocytosis were seen microscopically. Brucellae were seen exclusively in macrophages, which were most often located in subcapsular and cortical sinuses. This study suggests that phagocytic leukocytes 1) transport brucellae into mammary glands; 2) provide a site for intracellular replication in mammary secretions; and 3) transport brucellae from mammary glands to supramammary lymph nodes.