Structure-based design and subsequent optimization of 2-tolyl-(1,2,3-triazol-1-yl-4-carboxamide) inhibitors of p38 MAP kinase.

A computer-aided drug design strategy leads to the identification of a new class of p38 inhibitors based on the 2-tolyl-(1,2,3-triazol-1-yl-4-carboxamide) scaffold. The tolyl triazole amides provided a potent platform amenable to optimization. Further exploration leads to compounds with greater than 100-fold improvement in binding affinity to p38. Derivatives prepared to alter the physicochemical properties produced inhibitors with IC(50)'s in human whole blood as low as 83 nM.

Pyrazinoindolone inhibitors of MAPKAP-K2.

Optimization of pyrazinoindolone inhibitors of MAPKAP-K2 (MK2) provides a reasonable balance of cellular potency and physicochemical properties. Mechanistic studies support the inhibition of MK2 which is responsible for the sub-micromolar cellular efficacy.

Antibodies against Pasteurella multocida in snow geese in the western Arctic.

To determine if lesser snow geese (Chen caerulescens caerulescens) are a potential reservoir for the Pasteurella multocida bacterium that causes avian cholera, serum samples and/or pharyngeal swabs were collected from > 3,400 adult geese breeding on Wrangel Island (Russia) and Banks Island (Canada) during 1993-1996. Pharyngeal swab sampling rarely (> 0.1%) detected birds that were exposed to P. multocida in these populations. Geese with serum antibody levels indicating recent infection with P. multocida were found at both breeding colonies. Prevalence of seropositive birds was 3.5% at Wrangel Island, an area that has no recorded history of avian cholera epizootics. Prevalence of seropositive birds was 2.8% at Banks Island in 1994, but increased to 8.2% during 1995 and 1996 when an estimated 40,000-60,000 snow geese were infected. Approximately 50% of the infected birds died during the epizootic and a portion of the surviving birds may have become carriers of the disease. This pattern of prevalence indicated that enzootic levels of infection with P. multocida occurred at both breeding colonies. When no avian cholera epizootics occurred (Wrangel Island, Banks Island in 1994), female snow geese (4.7%) had higher antibody prevalence than males (2.0%).

A cryopreservation method for Pasteurella multocida from wetland samples.

A cryopreservation method and improved isolation techniques for detection of Pasteurella multocida from wetland samples were developed. Wetland water samples were collected in the field, diluted in dimethyl sulfoxide (DMSO, final concentration 10%), and frozen at -180 C in a liquid nitrogen vapor shipper. Frozen samples were transported to the laboratory where they were subsequently thawed and processed in Pasteurella multocida selective broth (PMSB) to isolate P. multocida. This method allowed for consistent isolation of 2 to 18 organisms/ml from water seeded with known concentrations of P. multocida. The method compared favorably with the standard mouse inoculation method and allowed for preservation of the samples until they could be processed in the laboratory.

Pasteurella multocida serotype 1 isolated from a lesser snow goose: evidence of a carrier state.

Pharyngeal swabs were collected from 298 lesser snow geese (Chen caerulescens caerulescens) at Banks Island (Northwest Territories. Canada) in the summer of 1994. Pasteurella multocida serotype 1 was isolated from an adult male bird and P. multocida serotype 3 was isolated from an adult female goose. Pathogenicity of the serotype 1 isolate was confirmed by inoculation in Pekin ducks (Anas platyrhynchos). The serotype 3 isolate was non-pathogenic in Pekin ducks. This is the first documented isolation of pathogenic P. multocida serotype 1 from apparently healthy wild snow geese.

Exposure of wild waterfowl to Mycoplasma anatis.

We developed an ELISA procedure to assess the presence of M. anatis-specific serum antibody in ducks. Sera from exposed and unexposed Pekin ducks (Anas platyrhynchos) were used to standardize the ELISA and to establish reference ranges to classify ELISA results as exposed or not exposed. We conducted serological surveys of female waterfowl in the central and eastern United States between 1988 and 1992 to assess the frequency of exposure in wild waterfowl. Adult breeding mallards (Anas platyrhynchos), wintering mallards, and black ducks (Anas rubripes) had high prevalences of exposure to M. anatis (25% to > 80%). In comparison, none of the breeding adult canvasbacks (Aythya valisineria) had serum antibody levels indicating exposure. Approximately 50% of the juvenile mallards and black ducks were exposed to M. anatis by 8 months of age, indicating high transmission rates among wild birds.

The occurrence of mycoplasmas in selected wild North American waterfowl.

We determined the prevalence of mycoplasma infection in breeding mallard (Anas platyrhynchos) and canvasback (Aythya valisineria) hens and their broods from the central United States (1988 to 1990); and wintering American black duck (Anas rubripes) and mallard hens from the eastern United States (1990 to 1993). Mycoplasmas were isolated by culturing tracheal swabs from 656 live birds and tissue samples from 112 dead waterfowl. Nine (18%) of 51 mycoplasma isolates were identified as Mycoplasma anatis; M. anatis was recovered from four mallards, a black duck, and a gadwall (Anas strepera) duckling. Nineteen (37%) of 51 mycoplasma isolates were identified as Mycoplasma cloacale; these isolates were obtained from mallard, canvasback, and black duck adults, and from a mallard duckling. Additional unspeciated mycoplasmas were isolated from mallards, black ducks, and one canvasback.

Effects of Mycoplasma anatis and cold stress on hatching success and growth of mallard ducklings.

We inoculated game-farm mallard (Anas platyrhynchos) eggs and 1-day-old birds with Mycoplasma anatis to determine its effect on hatching success and growth rates of ducklings. Inoculations of eggs reduced hatching success, hatchling size, and duckling growth rates, compared to controls. Intratracheal inoculations of 1-day-old birds did not affect growth rates. Hatchlings and 1-day-old ducklings grew much slower for the first 7 to 10 days when raised at 17 to 19 C, compared to controls raised at 30 to 35 C. The effect of cold stress on growth was greater than the effect of M. anatis infection; we found no synergistic effects between cold stress and M. anatis infection.

Mortality from duck plague virus in immunosuppressed adult mallard ducks.

Environmental contaminants contain chemicals that, if ingested, could affect the immunological status of wild birds, and in particular, their resistance to infectious disease. Immunosuppression caused by environmental contaminants, could have a major impact on waterfowl populations, resulting in increased susceptibility to contagious disease agents. Duck plague virus has caused repeated outbreaks in waterfowl resulting in mortality. In this study, several doses of cyclophosphamide (CY), a known immunosuppressant, were administered to adult mallards (Anas platyrhynchos) to determine if a resultant decrease in resistance to a normally sub-lethal strain of duck plague virus would occur, and induce mortality in these birds. Death occurred in birds given CY only, and in birds given virus and CY, but not in those given virus only. There was significantly greater mortality and more rapid deaths in the duck plague virus-infected groups than in groups receiving only the immunosuppressant. A positively correlated dose-response effect was observed with CY mortalities, irrespective of virus exposure. A fuel oil and a crude oil, common environmental contaminants with immunosuppressive capabilities, were tested to determine if they could produce an effect similar to that of CY. Following 28 days of oral oil administration, the birds were challenged with a sub-lethal dose of duck plague virus. No alteration in resistance to the virus (as measured by mortality) was observed, except in the positive CY control group.

Effects of sewage sludge on the immune defenses of mallards.

Sewage sludge contains numerous chemicals that, if ingested, could affect the immunological status of wild birds and, in particular, their resistance to infectious disease. Mallard ducks were fed a diet containing 0, 5, or 20% dried sewage sludge from either Milwaukee or Madison, Wisconsin, for 28 and 57 days, respectively. When subsequently challenged with Pasteurella multocida, the mortality in the sludge-treated groups was no greater than that in the untreated control groups. There was, however, significantly more cadmium (two- to three-fold higher concentration) retained in the livers of birds receiving 20% of either sludge in their feed, as compared to controls.