A colorimetric assay for quantitating bovine neutrophil bactericidal activity

A colorimetric assay was developed for quantitating bovine neutrophil bactericidal activity against Staphylococcus aureus. The procedure used the tetrazolium compound, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). The assay was conducted by incubating antibody-opsonized S. aureus with neutrophils in microtiter plates for 1 h at a ratio of 10 bacteria per neutrophil. Neutrophils were then lysed with saponin. The MTT was added and samples were incubated for 10 min. Live S. aureus reduced MTT to purple formazan. Dead bacteria and lysed neutrophils did not react with MTT. Bacterially-reduced formazan was solubilized by adding isopropanol and formazan production was quantitated by measuring absorption at 560 nm. Absorption of formazan was directly related to viable bacteria cell number and was used to determine the number of S. aureus not killed by neutrophils. The percentage of bacteria killed by neutrophils was determined by extrapolation from a standard formazan curve that was derived by incubating MTT with known numbers of S. aureus. The colorimetric MTT assay detected suppressed bactericidal activity after in vitro treatment of bovine neutrophils with colchicine, cytochalasin B, or phorbol 12-myristate 13-acetate. In vitro treatment of neutrophils with low levels of recombinant bovine interferon gamma (rBoIFN-gamma) enhanced bactericidal activity, whereas high levels decreased activity. These results suggest the colorimetric MTT bactericidal assay is efficacious in detecting modulation of bovine neutrophil bactericidal activity. Furthermore, the MTT assay has many advantages over traditional bactericidal assays in that it is sensitive, inexpensive, requires less than 3 h to complete, and can analyze many neutrophil samples in a single day.     

A strain of Babesia divergens, attenuated after long term culture

The Weybridge strain of Babesia divergens became less virulent after 18 months in culture and was believed to be attenuated. Inoculations with the attenuated line using as many as 5 x 10(8) infected erythrocytes failed to raise a normal infection in Mongolian gerbils (Meriones unguiculatus) whereas, with the line that had been passaged from culture through gerbils at bimonthly intervals, inoculations of 10(7) infected erythrocytes gave rise to fatal infections. Gerbils were immunised with the attenuated line using doses ranging from 5 x 10(5) to 5 x 10(7) infected erythrocytes. Parasitaemias in the infected gerbils did not exceed 0.7 per cent after immunisation. All animals were protected when challenged with the virulent line four weeks later. Control animals died four days after challenge infection.     

Opsonin-dependent stimulation of bovine neutrophil oxidative metabolism by Brucella abortus

Nonopsonized Brucella abortus and bacteria treated with fresh antiserum, heat-inactivated antiserum, or normal bovine serum were evaluated for their ability to stimulate production of superoxide anion and myeloperoxidase-mediated iodination by neutrophils from cattle. Brucella abortus opsonized with fresh antiserum or heat-inactivated antiserum stimulated production of approximately 3 nmol of O2-/10(6) neutrophils/30 min. Similarly treated bacteria also stimulated the binding of approximately 4.3 nmol of NaI/10(7) neutrophils/h to protein. Significant (P less than 0.05) production of O2- and iodination activity by neutrophils were not stimulated by nonopsonized bacteria or organisms treated with normal bovine serum. Seemingly, B abortus stimulated oxidative metabolism in bovine neutrophils; however, the stimulation was dependent on the presence of bacterial associated opsonins.     

Ammonium chloride prevents lytic growth of reovirus and helps to establish persistent infection in mouse L cells

Ammonium chloride, a lysosomotropic agent that raises intralysosomal pH, reduces the yield of reovirus during infection of mouse L cells. Subsequent removal of ammonium chloride results in the rapid establishment of a persistent infection.