Enhancement of Pasteurella Haemolytica A1 Leukotoxin Activity by Bovine Serum Albumin�

Growth of Pasteurella haemolytica A 1 in media containing fetal bovine serum has been observed to enhance leukotoxin (LKT) activity, but the mechanism of this increase is not understood. We found that bovine serum albumin in the absence of other serum components also enhances LKT activity. During the logarithmic growth phase, LKT activity was 30,700 � 12,900 Toxic Units (TU)/ml for P. haemolytica grown in RPM I medium containing 0. 5% bovine serum albumin (BSA) (BSA-LKT), compared to 120 � 40 TU/ml in medium containing RPMI alone (RPMILKT). In other experiments, addition of 0.5% BSA to RPMI-LKT culture filtrate(RPMI-LKT/BSA) resulted in LKT activity intermediate ( 13,000 � 1,600 TU/ml) between that of BSA-LKT (55,600 � 11,500 TU/ml) and RPMI-LKT (2,200 � 900 TU/ml). The activity of RPMI-LKT, BSA-LKT and RPMI-LKT /BSA decreased when the toxin preparations were incubated at room temperature (25�C) for 2 hours; however, the decrease in RPMI-LKT activity was more pronounced. Concentrated toxin from logarithmic growth phase RPMI-LKT culture supernatants contained a single LKT activity peak (Peak I) on Sephacryl HR-400 which had a K of 0.01 and estimated av molecular weight > > 669,000. In contrast, gel filtration of concentrated toxin from logarithmic growth phase BSA-LKT culture supernatants had three activity peaks with K values of 0.04 (Peak 1), 0.66 (Peak II), and av 0.87 (Peak Ill). Gel filtration of RPMI-LKT/BSA also consisted of Peaks I, II, and Ill. Peaks I, II, and Ill contain a prominent 97000 band on SDS-PAGE which was identified as LKT by Western blot analysis with an monoclonal antibody (MAB) to LKT. The identity of Peaks I, II, and Ill were further confirmed as LKT by target cell specificity (lysed bovine lymphoma cells and not equine leukocytes) and neutralization with a MAB toP. haemolytica LKT. It is concluded that LKT produced in RPMI medium alone is a large aggregate which undergoes conformational changes in the presence of BSA resulting in at least two additional LKT forms which may be partially disaggregated forms of the large aggregate LKT produced in RPMI.Veterinary Patholog

Antiretroviral Spermicide WHI-07 Prevents Vaginal and Rectal Transmission of Feline Immunodeficiency Virus in Domestic Cats

WHI-07 [5-bromo-6-methoxy-5,6-dihydro-3′-azidothymidine-5′-(p-bromophenyl)-methoxy alaninyl phosphate] is a novel dual-function aryl phosphate derivative of zidovudine with potent anti-human immunodeficiency virus (HIV) and spermicidal activities. WHI-07 was active against the feline immunodeficiency virus (FIV). This study evaluated whether topical application of WHI-07 as a single agent and in combination with an organometallic vanadium complex, vanadocene dithiocarbamate (VDDTC), via a nontoxic gel microemulsion can block vaginal as well as rectal transmission of feline AIDS (FAIDS) by chronically FIV-infected feline T cells in the natural host model. Genital transmission of FIV was monitored in recipient cats by the appearance of viral antibodies to FIV Gag proteins and by virus isolation of blood leukocytes as measured by FIV reverse transcriptase activity and FIV-specific PCR. Microbicidal activity was considered effective when the treated cats did not show evidence of FIV infection for up to 18 weeks postchallenge. An aggregate analysis of 46 specific-pathogen-free cats revealed that a single dose of the infected cell inoculum efficiently transmitted FIV infection when delivered into the vagina (100%) or rectum (66%). Pretreatment of the vagina or rectum with 2% WHI-07 alone or in combination with 0.25% VDDTC significantly (P = 0.004) protected cats from genital transmission by the highly infectious inoculum (7 million FIV(Bangston)-infected feline T cells). Collectively, using the vaginal and rectal transmucosal model for FAIDS, our studies demonstrated that WHI-07 either alone or in combination with a vanadocene has clinical potential for the development of a dual-function anti-HIV microbicide for sexually active women