Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Comparative analysis of stress agents in a simplified in vitro system of Neospora caninum bradyzoite production

Neospora caninum is an apicomplexan parasite identified as a major cause of abortion in cattle and neurological disease in various animal species. It is closely related to Toxoplasma gondii, sharing the ability to persist indefinitely in latent stage within the host as a tissue cyst containing slow-dividing bradyzoites. In this study, we compared different stress methods to induce in vitro bradyzoite conversion, using MARC-145 cells infected with Nc-Liverpool isolate. The tachyzoite-to-bradyzoite conversion rate was monitored at days 3, 5, and 7 after stress in a double-immunofluorescence assay using a monoclonal antibody against the tachyzoite antigen SAG1 (alphaSAG1) and a rabbit serum directed to the intracytoplasmic bradyzoite antigen BAG1 (alphaBAG1). Seven days of treatment with 70 microM sodium nitroprusside offered the highest bradyzoite transformation rate and the best yield of total parasitophorous vacuoles observed. In the present work, we introduce an alternative, simplified, and more advantageous method for bradyzoite production of N. caninum, using a reliable cell culture system easy to handle and with promising capacity of parasite purification.     

Characterization of pathology and parasite load in outbred and inbred mouse models of chronic Neospora caninum infection

Here, we analyzed histological findings and parasite burden in chronic Neospora caninum infection in BALB/c and ICR mice and studied the correlation between lesion severity and parasite load in brain. To obtain a better understanding of the infection, we examined the influence of various host pathogen factors. Groups of outbred (ICR) and inbred (BALB/c) mice were inoculated using several NC-1 parasite doses (4 x 10(5), 10(6), and 5 x 10(6) tachyzoites), inoculation routes (intraperitoneal and subcutaneous), and 3 immunosuppressive treatments (methylprednisolone, cyclophosphamide, and vinblastine). Lesion severity was analyzed in the liver, lung, heart, and brain tissues, and parasite load was measured by real-time polymerase chain reaction in brain tissue. The results indicated more severe cerebral lesions and higher brain parasite burdens in inbred than in outbred mice. Hepatic tissue was the primary lesion site in immunosuppressed ICR mice. We also observed that increased inoculum size was reflected in greater lesion severity and a higher cerebral parasite load. No difference was observed with respect to inoculation route. The study also showed an association between brain parasite burden and severity of cerebral lesions in BALB/c mice.     

Grazing behaviour and performance of lactating suckler cows, ewes and goats on partially improved heathlands

The foraging behaviour (grazing time and diet composition), live-weight (LW) changes and parasitic infection (faecal egg counts of gastrointestinal nematodes) of 12 beef cows, 84 ewes and 84 goats suckling their offspring, managed in mixed grazing on heathlands with 24% of improved pasture of perennial ryegrass-white clover, were studied during the years 2003 and 2006. The results showed that goats tended to graze for a longer time, and utilised significantly more heathland vegetation than cattle and sheep, including in their diet in an average of 0.39 herbaceous plants, 0.11 gorse and 0.51 heather over the grazing season, comparing with respective mean values of 0.85, 0.02 and 0.13 in cattle and sheep. Dietary overlap was higher between cattle and sheep (0.76) than between sheep and goats (0.53) or between cattle and goats (0.47). Despite the high performance of autumn-calving cows in spring, sheep had the best LW changes per livestock unit (LU) during the whole grazing season. Goats' performance per LU was lower than in sheep but higher than in spring-calving cows. Regarding offspring production, lambs had higher LW gains per LU than calves and kids. Mean nematode eggs/g fresh faeces (epg) during the grazing season were higher in goats (91 epg) compared with sheep (34 epg) and cattle (14 epg), particularly from September to December. In conclusion, sheep performed best on these heathlands with improved pasture areas if the entire grazing season was considered, despite the good level of production in spring from autumn-calving cows. The results suggested the complementary use of goats in order to increase the effective utilisation of the available vegetation, achieving production levels similar to those from spring-calving cows. Therefore, mixed flocks of sheep and goats would be the most appropriate sustainable systems from the animal production and vegetation use points of view.     

A radioimmunoprecipitation method in the serodiagnosis of HIV infection: the development of the optimal conditions for its performance and the evaluation of the possibilities for its use

The optimum conditions for using the method of radioimmunoprecipitation (RIP) for the detection of human immunodeficiency virus (HIV) in serum samples have been established. Out of several available cell lines persistently infected with HIV, specially selected line 17 has been chosen. The characteristic feature of this is the high and stable (under the conditions of prolonged cultivation) accumulation of virus-specific proteins in infected cells. The optimum conditions for making the test and its evaluation have also been established. The data of literature on the advantages of the method of RIP over such traditional methods as the enzyme immunoassay and immunoblotting have been confirmed. Thus, the presence of specific antibodies in several serum samples registered as false negative has been established. The intertypical reactivity of two serotypes of the virus, HIV-1 and HIV-2, has been studied. Cross reactivity of antibodies with respect to the HIV gene gag, but not with respect to viral glycoproteids, has been established. Ideas on the expediency and prospects of using RIP for the serological control of HIV infection are presented.