Prevalence of Bartonella infection in wild African lions (Panthera leo) and cheetahs (Acinonyx jubatus).

Bartonella species are emerging pathogens that have been isolated worldwide from humans and other mammals. Our objective was to estimate the prevalence of Bartonella infection in free-ranging African lions (Panthera leo) and cheetahs (Acinonyx jubatus). Blood and/or serum samples were collected from a convenience sample of 113 lions and 74 cheetahs captured in Africa between 1982 and 2002. Whole blood samples available from 58 of the lions and 17 of the cheetahs were cultured for evidence of Bartonella spp., and whole blood from 54 of the 58 lions and 73 of the 74 cheetahs tested for the presence of Bartonella DNA by TaqMan PCR. Serum samples from the 113 lions and 74 cheetahs were tested for the presence of antibodies against Bartonella henselae using an immunofluorescence assay. Three (5.2%) of the 58 lions and one (5.9%) of the 17 cheetahs were bacteremic. Two lions were infected with B. henselae, based on PCR/RFLP of the citrate synthase gene. The third lion and the cheetah were infected with previously unidentified Bartonella strains. Twenty-three percent of the 73 cheetahs and 3.7% of the 54 lions tested by TaqMan PCR were positive for Bartonella spp. B. henselae antibody prevalence was 17% (19/113) for the lions and 31% (23/74) for the cheetahs. The prevalence of seropositivity, bacteremia, and positive TaqMan PCR was not significantly different between sexes and age categories (juvenile versus adult) for both lions and cheetahs. Domestic cats are thus no longer the only known carriers of Bartonella spp. in Africa. Translocation of B. henselae seronegative and TaqMan PCR negative wild felids might be effective in limiting the spread of Bartonella infection.

First findings of Cryptosporidium and Giardia in California sea lions (Zalophus californianus).

We report the detection and identification of Cryptosporidium and Giardia from 1 of 3 species of pinnipeds. Fecal samples were collected from Pacific harbor seal (Phoca vitulina richardsi), northern elephant seal (Mirounga angustirostris), and California sea lion (Zalophus californianus) in the northern California coastal area. By means of fluorescently labeled monoclonal antibodies, Cryptosporidium oocysts were detected in 3 samples from California sea lions, 1 of which also contained Giardia cysts. Oocysts of Cryptosporidium and cysts of Giardia were morphologically indistinguishable from oocysts of C. parvum and cysts of G. duodenalis from other animal origins. Oocysts and cysts were then purified using immunomagnetic separation techniques and identified by polymerase chain reaction (PCR), from which species-specific products were obtained. Sequence analysis revealed that the 452-bp and 358-bp PCR products of Cryptosporidium isolated from California sea lion had identities of 98% with sequences of their template fragments of C. parvum obtained from infected calves. Based on morphological, immunological, and genetic characterization, the isolates were identified as C. parvum and G. duodenalis, respectively. The findings suggested that California sea lions could serve as reservoirs in the environmental transmission of Cryptosporidium and Giardia.

Determination of hydramethylnon residues in grass by liquid chromatography with confirmation by liquid chromatography/mass spectrometry.

An improved method for determination of hydramethylnon residues in pasture grass is described. The method uses (1) the hydrochloride salt of hydramethylnon to improve its water solubility and (2) an acid-methanol precipitation to remove chlorophylls while leaving the analyte in solution. The liquid chromatographic method has a validated sensitivity of 0.05 ppm with controls showing < 0.004 ppm. The overall average recovery from 0.05 to 0.50 ppm was 98%, with a standard deviation of 11%. Samples showing a positive response (0.05 ppm or higher) and requiring mass spectrometric confirmation are directly amenable to liquid chromatography/mass spectrometry without additional sample preparation.

A new fumonisin from solid cultures of Fusarium moniliforme.

A new fumonisin has been isolated from Fusarium moniliforme isolate MRC826 grown on corn. It was shown by NMR and mass spectrometry to be an isomer of fumonisin B2 that has free hydroxyl groups at C-3 and C-10 instead of the normal C-3 and C-5. This new fumonisin was detected in cultures of most isolates of F. moniliforme that were examined and was usually present at concentrations similar to those of fumonisin B2. Two isolates of F. moniliforme that produce significantly higher levels of this new isomer were identified.

A kinetic method for measuring functional delivery of amphotericin B by drug delivery systems.

The human toxicity of amphotericin B can be considerably reduced by associating the drug with liposomes of varying lipid compositions. Some lipid compositions are much more effective than others. We show that a simple kinetic fluorescence assay using pyranine as an indirect probe of amphotericin-induced K+ currents may be used to study different liposomal drug delivery systems in vitro. We find that lipid mixtures composed of DMPC/DMPG/amphotericin at a 7:3:1 mole ratio show very slow functional delivery with a preference for ergosterol over cholesterol-containing membrane vesicles. On the other hand, amphotericin delivered from egg phosphatidylcholine liposomes lead to 100-fold increases in K+ leakage at one-fifth the amphotericin concentration of the 7:3:1 system. The egg phosphatidylcholine system as well as micellar amphotericin also show a slight selectivity towards cholesterol-containing vesicles over ergosterol. These results are consistent with previous clinical and in vitro cellular studies and this technique may prove valuable in screening of other delivery systems.

Potassium-selective amphotericin B channels are predominant in vesicles regardless of sidedness.

Amphotericin B (AmB) is a membrane-active antibiotic which has been shown to increase ion and small molecule permeability in a variety of model and biological membrane systems. A major mechanistic model, based on BLM systems, proposes that amphotericin forms barrellike pores with cholesterol which are cation selective when added to one side of the membrane and anion selective when added to both sides. We have tested this hypothesis on small and reverse-phase large unilamellar vesicles (SUV and REV) with and without cholesterol. The method used to measure K+, Cl-, and net ion currents is based on ion/H+ exchange detected by the entrapped pH probe pyranine. We find that AmB forms channels which have net selectivity for K+ over Cl- regardless of sidedness or sterol content in SUV. REV with 10% cholesterol also show net K+ selectivity with double-sided addition. Differences are noted between cholesterol- and non-sterol-containing vesicles consistent with at least two separate modes of action: (1) cholesterol-containing SUV form some larger diameter pores which allow the passage of larger ions especially when added to both sides; (2) SUV without sterol form pores which are still K+ over Cl- selective, but larger ions do not pass. The latter mode of action precludes a sterol/pore type of model but not necessarily a barrellike model consisting only of amphotericin molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Amphotericin B and Nystatin show different activities on sterol-free vesicles.

It has generally been assumed that the polyene antibiotics Nystatin and Amphotericin B cause membrane damage by the same mechanism. However, using kinetic fluorescence methods we have found that AmB and Nystatin have very different activities on sterol-free dioleoyl phosphatidylcholine and egg phosphatidylcholine small unilamellar vesicles. At very low AmB concentrations (less than 1/1000 lipids in egg phosphatidylcholine) significant K+ permeability enhancement is observed. However, even at very high Nystatin to lipid ratios (1/100) very little K+ current is induced, particularly in dioleoyl phosphatidylcholine vesicles. The novel technique described here uses a K+/H+ exchange mechanism to detect minute transmembrane K+ currents by monitoring internal membrane vesicle pH changes with pyranine.

Interrelationship of dietary silver with copper in the chick.

Adding 900 p.p.m. silver (as silver nitrate) to a practical diet for chicks significantly depressed growth, increased wet and dry heart weight to body weight ratios and markedly increased mortality during a four-week experimental period. Blood packed cell volume was not affected. Supplementing the diet containing silver with 50 p.p.m. copper prevented cardiac enlargement and mortality, but only partially corrected the growth depression. Glycogen content of the heart was not affected, but aortic elastin content was significantly reduced by silver and restored to normal by supplemental copper. Dietary silver significantly reduced the copper content of blood, spleen, brain, liver, but except for the brain, the level of copper in these tissues was restored to normal by dietary copper supplementation. No significant differences in copper content of kidney tissue were observed among the treatment. Copper content of the excreta was not significantly increased by adding dietary silver, but was greatly increased by adding 50 p.p.m copper to the diet containing silver.

Induced exudative diathesis in chicks by dietary silver.

Two experiments were conducted with chicks to compare the effectiveness of vitamin E,selenium and cystine in preventing the deleterious effects of dietary silver. Adding 900 p.p.m. silver to a diet marginal in vitamin E and selenium significantly depressed growth and caused a high mortality during the four-week experiment. Most of the mortality was due to exduative diathesis. Including either 1 p.p.m. selenium or 100 I.U. vitamin E per kg. in the diets with silver prevented the growth depression and mortality. Adding 0.15% cystine stimulated growth, but failed to rpevent mortality. In a second experiment, chicks were grown on the diet containing silver with and without cystine to 15 days of age, at which time approximately 50% of the chicks exhibited signs of exudative diathesis. At that time they were either continued on the same diet of fed diets supplemented with selenium or vitamin E. Both vitamin E and selenium reduced mortality during the following two-week period, but vitamin E. was more effective than selenium.