Experimental Infection of Calves with Escherichia coli O104:H4 outbreak strain.

In 2011, a severe outbreak of hemolytic-uremic syndrome was caused by an unusual, highly virulent enterohemorrhagic E. coli (EHEC) O104:H4 strain, which possessed EHEC virulence traits in the genetic background of human-adapted enteroaggregative E. coli. To determine magnitude of fecal shedding and site of colonization of EHEC O104:H4 in a livestock host, 30 (ten/strain) weaned calves were inoculated with 10(10) CFU of EHEC O104:H4, EHEC O157:H7 (positive control) or E. coli strain 123 (negative control) and necropsied (4 or 28 d.p.i.). E. coli O157:H7 was recovered until 28 d.p.i. and O104:H4 until 24 d.p.i. At 4 d.p.i., EHEC O104:H4 was isolated from intestinal content and detected associated with the intestinal mucosa. These results are the first evidence that cattle, the most important EHEC reservoir, can also carry unusual EHEC strains at least transiently, questioning our current understanding of the molecular basis of host adaptation of this important E. coli pathovar.

Evaluation of immune functions of rainbow trout (Oncorhynchus mykiss)--how can environmental influences be detected?

In fish, the first line of defense against infectious microorganisms is based on a broad range of nonspecific humoral and cellular immune mechanisms ("innate immunity") which without prior specific activation can act in forming a more static barrier (Fish Shellfish Immunol. 10 (2000) 243; Dev. Comp. Immunol. 25 (2001) 827). This natural resistance is normally effective enough to protect fish from infectious diseases until specific immune responses are being induced (Fig. 1; Dev. Comp. Immunol. 25 (2001) 841). Healthy fish exhibit both nonspecific and specific immune responses depending directly on environmental temperature. Pollution of the natural aquatic environment with industrial or agricultural sewage is an important immunosuppressing factor resulting in higher susceptibility to infectious diseases. To date, the possible immunotoxicity of a substance is evaluated using quantification of humoral factors like lysozyme, complement, C-reactive protein or total immunoglobulins but less often using functional assays. Furthermore, most of the functional assays (phagocytosis, respiratory burst, proliferative response) are based on the measurement of the response of resting but not of specific activated immune cells. However, the physiological responses of the immune system to an infection are based on a complex, stepwise activation and proliferation, especially of the specific immune functions after first contact to the microorganisms. In this report we describe in vitro methods for the evaluation of cellular immune functions of different leukocyte populations after specific in vivo triggering of the immune system. Parameters to be evaluated are activation and proliferation of leukocyte populations, phagocytosis and respiratory burst, secretion of antigen-specific antibodies and specific cell-mediated cytotoxicity. Furthermore, challenge models with bacterial (Aeromonas salmonicida) and viral pathogens (Viral Haemorrhagic Septicemia Virus, VHSV) are presented.

Temperature dependent activation of leucocyte populations of rainbow trout, Oncorhynchus mykiss, after intraperitoneal immunisation with Aeromonas salmonicida.

The temperature dependence of in vivo activation of rainbow trout Oncorhynchus mykiss, leucocyte populations after intraperitoneal injection (i.p.) of fish with a T-cell independent antigen Aeromonas salmonicida (strain MT423) was investigated using a proliferation assay and flow cytometric analysis with mab specific for trout leucocyte surface markers. In trout kept at 15-17 degrees C a prominent activation of blood and spleen leucocytes was found. Also, drastic changes of the percentage of the leucocyte populations in blood and spleen occurred: the amount of monocytes in the blood increased between day 2 and day 7 post injection (p.i.), whereas in spleen the amount of monocytes stayed at a high level (approximately 35%) after a depression between day 4 and day 7 p.i. The percentage of B-lymphocytes was increased first in spleen and then in blood. The percentage of granulocytes in blood was elevated during the whole experiment compared to control fish. In trout kept at 10-12 degrees C only blood leucocytes showed a weak activation after i.p. injection of A. salmonicida, whereas spleen leucocytes showed nearly no reaction. Only the percentage of granulocytes in the blood (day 2-14 p.i.) and of monocytes in the spleen (day 2 and day 8 p.i.) was changed compared to phosphate buffered saline (PBS)-injected fish. However, the development of A. salmonicida specific antibodies was contrary to the cellular reaction. Whereas antibodies could first be detected after 16-18 days p.i. in both groups the amount of antibodies was significantly higher in sera of trout kept at 10-12 degrees C at day 22 and day 28 p.i. than in sera of trout kept at 15-17 degrees C. These results indicate stronger A. salmonicida induced activation of monocytes, granulocytes and B-lymphocytes at higher temperature. However, the development of a specific antibody response against A. salmonicida seemed to be more effective at lower temperatures.

A monoclonal antibody recognising a surface marker on rainbow trout (Oncorhynchus mykiss) monocytes.

The characterisation of a monoclonal antibody (mab 45) reacting with phagocytic leucocytes isolated from blood and spleen of rainbow trout (Oncorhynchus mykiss, Walbaum) is described. The surface marker labelled by this mab is expressed at relative low levels on the membrane of large, nearly nongranulated trout leucocytes, and having the typical morphology of monocytes in flow cytometry (Kfoury et al., 1999, Fish Pathology, 34, 1-6). No reaction of mab 45 with granulocytes, lymphocytes or thrombocytes was detected. In spleen and head kidney, large, polymorphonuclear leucocytes were immunostained. The mab most strongly recognised an antigen of 48 kDa prepared from trout leucocytes of different organs, but not in trout plasma. In an in vitro phagocytosis assay trout monocytes were stained with mab 45 after phagocytosis of Aeromonas salmonicida labelled with the lipophilic fluorescent cell surface linker PKH26. However, previous binding of mab 45 on trout leucocytes did not inhibit the phagocytosis of A. salmonicida particles. Using mab 45, the dynamics of monocytes in blood, spleen and peritoneal cavity could be demonstrated after intraperitoneal injection of trout with inactivated A. salmonicida. The described mab serves as a useful tool to investigate the involvement of monocytes/macrophages in immune reactions of trout to a variety of pathogens.