Enterospora nucleophila (Microsporidia) in Gilthead Sea Bream (Sparus aurata): Pathological Effects and Cellular Immune Response in Natural Infections

Enterospora nucleophila is a microsporidian responsible for an emaciative disease in gilthead sea bream ( Sparus aurata). Its intranuclear development and the lack of in vitro and in vivo models hinder its research. This study investigated the associated lesions, its detection by quantitative polymerase chain reaction, and the cellular immune response of naturally infected fish. The intensity of infection in the intestine was correlated with stunted growth and reduced body condition. At the beginning of the outbreaks, infection prevalence was highest in intestine and stomach, and in subsequent months, the prevalence decreased in the intestine and increased in hematopoietic organs and stomach. In heavy infections, the intestine had histologic lesions of enterocyte hypercellularity and proliferation of rodlet cells. Infected enterocytes had E. nucleophila spores in the cytoplasm, and a pyknotic nucleus, karyorhexis or karyolysis. Lymphocytes were present at the base of the mucosa, and eosinophilic granule cells were located between the enterocytes. In intestinal submucosa, macrophage aggregates containing spores were surrounded by lymphocytes and granulocytes, with submucosal infiltration of granulocytes. Macrophage aggregates appeared to develop into granulomata with necrotic areas containing parasite remnants. Immunohistochemistry revealed mast cells as the main type of granulocyte involved. Abundant IgM+ and IgT+ cells were identified by in situ hybridization in the submucosa when intracytoplasmic stages were present. This study describes the lesions of E. nucleophila in gilthead sea bream, an important aquaculture species.

DO SPLEEN MACROPHAGE AGGREGATES UNDERGO SEASON-DEPENDENT CHANGES? A STEREOMICROSCOPIC INVESTIGATION IN WILD OHRID TROUT (SALMO LETNICA KAR.)

In past decades macrophage aggregates (MACs) were being used in many monitoring studies as useful immu-nological biomarkers of environmental pollution and they are still encountered in the so-called “future biomarkers”, since quantitative studies in aquaculture and/or field of their normal fluctuations as a result of the influence of natural factors are extremely rare. Our investigation was designed to quantitatively evaluate the influence of the seasons on MAC's relative volume, number and diameter in the Ohrid trout (Salmo letnica Kar.) spleen. Data showed that all investigated parameters had significantly higher values in fish collected during spring and winter. Correlation anal-yses revealed that the increasing of the relative volume of MACs is a result of the increasing of the relative volume of melanin loaded MACs, as well as the increasing of their number and diameter. These results confirmed that the sea-son can affect MACs variations and should be taken into consideration, especially when spleen MACs are used as pollution biomarkers in monitoring studies.

Effects of Unleaded Petroleum on the Macrophage Aggregates (MA) formation in Red King tilapia (Oreochromis sp.) Fingerlings

The Philippines is one of the major producers of tilapia, the most cultured fish and widely consumed in the world. Although fishes in general is said to be adapted to various stressful conditions, the effect on several cellular immune parameters may be of interest to determine the capacity of the organism to withstand stressors. In this paper, the effect of unleaded petroleum on the splenic macrophage aggregate (MA) formation was studied. This was done to have an overview of the immune response of Tilapia or fishes in general when an oil spill, which almost occur annually at different parts of the world, happen. Histological analysis assessed the area occupied by splenic MA 24 hours after introduction of unleaded petroleum to the aquatic system. To determine whether Mabuhay balls, a technology that claims to be beneficial in terms of improving water quality, was added to one tank (T1) to be able to compare it with another tank (T2). There is a strong statistically significant difference between the groups at day1 (p=0.000) opposite the result of day 6 (p=0.155). Thus, unleaded petroleum increased MA formation, a sign that may indicate a high immune activity as an initial positive response to stress. Mabuhay ball have lessen the mortality but has no effect on splenic MA formation.

Morphometric Evaluation of Interrenal Gland and Kidney Macrophages Aggregates in Normal Healthy Rainbow Trout (Oncorhynchus mykiss) and after Bacterial Challenge withYersinia ruckeri

Macrophage aggregates (MA) occur in various organs of fish as discrete aggregations of pigmented macrophages. The study presented herein investigates the quantitative modifications from normal anatomical condition, of interrenal gland (IG) and kidney MA in six treatment groups of adult rainbow trout submitted to either specific or aspecific immune stimulation and subsequently challenged withYersinia ruckeri. Routinely stained tissue sections from both IG and kidney were analysed. The percentage of tissues occupied by MA and the MA density (number/mm2) were calculated on at least 10 randomly selected nonoverlapping fields taken from each tissue section. MA morphometric findings from challenged fish were compared to those from a control group. Results showed that fish from control group displayed a statistically significant (P<0.05) higher percentage of tissue occupied by MA and MA density. Among different treatment groups, anti-Yersinia ruckeriimmunized fish, which did not show clinical signs of disease after bacterial challenge, displayed higher values of morphometric parameters compared with symptomatic fish from other groups. Our study demonstrates that the quantification of the area occupied by MA might be an efficient parameter to evaluate the general condition of a salmonid population since it positively correlates with the health status and negatively with stress factor such as the acute bacterial infection.