Identification of a new candidate locus for uric acid nephrolithiasis

Renal stone formation is a common multifactorial disorder, of unknown etiology, with an established genetic contribution. Lifetime risk for nephrolithiasis is approximately 10% in Western populations, and uric acid stones account for 5%-10% of all stones, depending on climatic, dietary, and ethnic differences. We studied a small, isolated founder population in Sardinia, characterized by an increased prevalence of uric acid stones, and performed a genomewide search in a deep-rooted pedigree comprising many members who formed uric acid renal stones. The pedigree was created by tracing common ancestors of affected individuals through a genealogical database based on archival records kept by the parish church since 1640. This genealogical information was used as the basis for the study strategy, involving screening for alleles shared among affected individuals, originating from common ancestors, and utilization of large pedigrees to obtain greater power for linkage detection. We performed multistep linkage and allele-sharing analyses. In the initial stage, 382 markers were typed in 14 closely related affected subjects; interesting regions were subsequently investigated in the whole sample. We identified two chromosomal regions that may harbor loci with susceptibility genes for uric acid stones. The strongest evidence was observed on 10q21-q22, where a LOD score of 3.07 was obtained for D10S1652 under an affected-only dominant model, and a LOD score of 3.90 was obtained using a dominant pseudomarker assignment. The localization was supported also by multipoint allele-sharing statistics and by haplotype analysis of familial cases and of unrelated affected subjects collected from the isolate. In the second region on 20q13.1-13.3, multipoint nonparametric scores yielded suggestive evidence in a approximately 20-cM region, and further analysis is needed to confirm and fine-map this putative locus. Replication studies are required to investigate the involvement of these regions in the genetic contribution to uric acid stone formation.     

Seroprevalence of <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato and <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> in Danish horses

Background

Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum are able to infect horses. However, the extend to which Danish horses are infected and seroconvert due to these two bacteria is unknown. The aim of the present study was to evaluate the seroprevalence of B. burgdorferi sensu lato and A. phagocytophilum in Danish horses.

Methods

A total of 390 blood samples collected from all major regions of Denmark and with a geographical distribution corresponding to the density of the Danish horse population were analyzed. All samples were examined for the presence of antibodies against B. burgdorferi sensu lato and A. phagocytophilum by the use of the SNAP^®4DX ^® ELISA test.

Results

Overall, 29.0% of the horses were seropositive for B. burgdorferi sensu lato whereas 22.3% were seropositive for A. phagocytophilum.

Conclusions

Antibodies against B burgdorferi sensu lato and A. phagocytophilum are commonly found among Danish horses thus showing that Danish horses are frequently infected by these organisms.

     

Estimates of DNA and protein sequence divergence: an examination of some assumptions

Some of the assumptions underlying estimates of DNA and protein sequence divergence are examined. A solution for the variance of these estimates that allows for different mutation rates and different population sizes in each species and for an arbitrary structure in the initial population is obtained. It is shown that these conditions do not strongly affect estimates of divergence. In general, they cause the variance of divergence to be smaller than a binomial variance. Thus, the binomial variance that is usually assumed for these estimates is safely conservative. It is shown that variability in the mutation rate among sites can have an effect as large as or larger than variability in the mutation rate among bases. Variability in the mutation rate among bases and among sites causes the number of substitutions between two sequences to be underestimated. Protein and DNA sequences from several species are collected to estimate the variability in mutation rates among sites. When many homologous sequences are known, standard methods to estimate this variability can be used. The estimates of this variability show that this factor is important when considering the spectrum of spontaneous mutations and is strongly reflected in the divergence of sequences. Smaller variability is found for the third position of codons than for the first and second codon positions. This may be because of less selective constraints on this position or because the third position has been saturated with mutations for the sequences examined.      

Not all isolates are equal: linkage disequilibrium analysis on Xq13.3 reveals different patterns in Sardinian sub-populations

Recent studies indicate that, whereas the Sardinian population as a whole is comparable to outbred populations for linkage disequilibrium (LD) mapping of common variants, LD in Sardinian sub-isolates is more extended, making these populations particularly suitable for this approach. To evaluate the extent of LD between microsatellite markers, we compared different sub-populations within Sardinia selected on the basis of their geographical position and isolation: two small isolated villages (Talana, Urzulei), two larger but remote areas (Ogliastra, Nuoro province) and a cohort of samples representing the wider Sardinian population. LD analysis was carried out by using six microsatellite markers that are located on Xq13.3 and that have been extensively studied in different populations. We found different extents and patterns of LD in the sub-population samples depending on their degree of isolation and demographic history. All LD measurements and haplotype analyses indicate that there is a decreasing trend from Talana (the most inbred population, LD up to 9.5-11.5 Mb) to the more outbred Sardinian population (LD only for intervals <2 Mb). In one village (Talana), five haplotype classes accounting for 80% of the entire sample perfectly matched five Ogliastra clusters, supporting the origin of the village from the Ogliastra genetic pool. In contrast, the other village (Urzulei) showed a different pattern of haplotypes with a closer relationship to the Nuoro region sub-population. LD analyses therefore show that even neighbouring isolate villages may differ in their genetic background. Here, we highlight the importance of selecting appropriate populations and/or sub-populations for the analysis of complex traits. Isolated sub-populations showing different extents of LD can provide a powerful method for mapping complex traits by LD scanning at relatively low marker density.     

Sheep with scrapie and mastitis transmit infectious prions through the milk

Prions are misfolded proteins that are infectious and naturally transmitted, causing a fatal neurological disease in humans and animals. Prion shedding routes have been shown to be modified by inflammation in excretory organs, such as the kidney. Here, we show that sheep with scrapie and lentiviral mastitis secrete prions into the milk and infect nearly 90% of naïve suckling lambs. Thus, lentiviruses may enhance prion transmission, conceivably sustaining prion infections in flocks for generations. This study also indicates a risk of prion spread to sheep and potentially to other animals through dietary exposure to pooled sheep milk or milk products.Prion diseases have emerged globally as a significant threat to human and animal health. Recently, human-to-human spread of prions is believed to have occurred through blood transfusions (9, 12, 16), underscoring the importance of understanding possible transmission routes. PrP^Sc, a misfolded, aggregated form of the normal prion protein, PrP^C, commonly accumulates in the follicles of lymphoid tissues, prior to entering the central nervous system (2, 11, 14). Inflammation can cause lymphoid follicles to form in other organs, such as liver and kidney, which leads to prion invasion of organs that are not typically prion permissive (1). In mice, prion infection in the inflamed kidney has the untoward consequence of prion excretion in urine (13). This finding, together with our report of sheep with PrP^Sc in the inflamed mammary gland (8), has raised concerns of prion secretion into milk.Here, we investigated whether PrP^Sc in the inflamed mammary gland leads to prion secretion in milk and infection of naïve lambs through suckling. Prion infectivity has been detected in the milk of sheep expressing a prion gene (Prnp) coding for VRQ/VRQ or VRQ/ARQ at polymorphic codons 136, 154, and 171 (3, 4). However, whether (i) sheep-to-lamb transmission of prions in milk leads to clinical prion disease or (ii) sheep with the common ARQ/ARQ Prnp genotype can infect lambs through milk is unknown. We induced a chronic lentiviral mastitis and inoculated ARQ/ARQ Sarda breed sheep with infectious prions. After 14 months, we bred the sheep and collected the milk. To avoid cross-contamination of newborn lambs, we fed the milk to imported known-naïve lambs and then monitored the lambs for signs of prion infection (Fig. ​(Fig.1A1A).Open in a separate windowFIG. 1.Sheep infected with prions and maedi-visna virus (MVV) develop lymphofollicular mastitis with PrP^Sc. (A) Experimental scheme. Sheep were inoculated with culture medium or MVV and were then orally exposed to scrapie prions and bred. Milk was collected near the time point that neurologic signs of scrapie developed and was fed to naïve lambs. The ratio of lambs with detectable PrP^Sc to lambs fed the indicated milk is shown for each experiment. (B) PrP immunohistochemistry assay of brain and tonsil from milk source sheep shows staining for PrP^Sc in the brainstem, particularly in the vagal nucleus (indicated by asterisks) and in the tonsillar follicles of scrapie-infected sheep (arrows). (C) Mammary gland (MG) of milk source sheep shows lymphoid follicles (arrowheads) with associated PrP^Sc (arrows) adjacent to milk ducts (md) in the MVV-inoculated sheep, whereas the medium-inoculated sheep had a histologically normal MG with no detectable PrP^Sc. Insets show a high magnification of follicles containing PrP^Sc. Scale bar = 100 μm; scale bar in inset = 25 μm. (D) Western blot analysis shows PrP^Sc detection in MG of sheep inoculated with MVV/scrapie agents but not in sheep inoculated with scrapie prions only. The sheep identification number is indicated for each lane. PK, proteinase K digested; pos. br, positive brain control; neg. br, negative brain control.To induce a chronic lymphofollicular mastitis, we exposed 7- to 10-day-old lambs (groups of 10) by intratracheal and intravenous routes to a common sheep lentivirus known as maedi-visna virus (MVV) or to cell culture medium only. To do this, lambs were inoculated with 3.5 ml intravenously and 0.5 ml intratracheally of MVV in culture supernatant containing 1.5 × 10⁶ tissue culture infectious doses/ml of the “rapid/high” MVV strain 85/34 (5, 15). Twenty days later, all lambs were orally inoculated with 25 ml of 10% scrapie-infected brain homogenate from a pool of naturally infected Sarda sheep.We sequenced the entire Prnp gene and found that all lambs expressed the ARQ/ARQ Prnp genotype, indicating that the sheep should be susceptible to scrapie. As negative controls, 2 lambs of Prnp genotype ARR/ARR and ARQ/ARQ were mock inoculated with cell culture medium and healthy brain homogenate. All lambs originated from scrapie-free flocks that had been monitored for clinical scrapie cases for at least 3 years.All inoculated sheep were naturally bred to rams at 15 months postinoculation (p.i.) and produced lambs at 20 months p.i. Sheep developed early signs of scrapie just after the lambs were born. Milk from each sheep was manually collected and frozen daily.Eight of 10 MVV-and-scrapie (denoted MVV/scrapie)-inoculated sheep and 9 of 10 scrapie-inoculated sheep showed clinical signs of scrapie, with mean incubation periods of 22 ± 1.4 and 23 ± 1.5 months postinoculation, respectively, and were euthanized. There was no significant difference in incubation period between the groups (Student''s t test, P = 0.5), indicating that inflammation associated with the MVV infection does not accelerate prion disease. This finding is consistent with the results of previous studies that showed that chronic pancreatitis or nephritis did not affect the scrapie incubation period (1). Scrapie infection was confirmed postmortem by the detection of PrP^Sc in brain and lymphoid tissues by Western blot and immunohistochemistry assays (Fig. ​(Fig.1B).1B). Interestingly, scrapie did not develop in 3 sheep with a Prnp gene encoding a rare polymorphism at codon 176 (K), consistent with recent reports describing scrapie resistance for this genotype (10).Antibodies to MVV were detected in serum of all the MVV-inoculated sheep by indirect enzyme-linked immunosorbent assay (ELISA) (Elitest kit; Hyphen BioMed). Five of 8 MVV/scrapie-infected sheep (63%) showed a lymphofollicular mastitis (Fig. ​(Fig.1C),1C), and 3 had a diffuse interacinar lymphoid infiltrate. Of the 5 sheep with lymphofollicular mastitis, 4 had PrP^Sc deposits detectable by immunohistochemistry and Western blot assays (Fig. 1C and D), whereas no sheep with diffuse lymphoid infiltrates had detectable PrP^Sc. Surprisingly, 2 of 9 sheep inoculated only with scrapie also had lymphofollicular mastitis and anti-MVV antibodies, one of which had visible PrP^Sc deposits. MVV is a common pathogen in Europe, and it is possible that these sheep were infected from the dam. The remaining 7 scrapie-inoculated sheep had histologically normal mammary glands (Fig. ​(Fig.1C)1C) and no detectable PrP^Sc (Fig. ​(Fig.1D)1D) or anti-MVV antibodies.We selected the stored milk from the 4 MVV/scrapie-infected sheep with PrP^Sc in the mammary glands and from the 7 scrapie-infected sheep with histologically normal mammary glands. Milk samples from the early, middle, and late stages of lactation were pooled for each group. We imported naïve Cheviot lambs (n = 9) from flocks that originated from scrapie-free New Zealand and had been bred and housed under strict biosecurity containment in the United Kingdom to ensure that the lambs had not been exposed to scrapie. The Sarda lambs (n = 4) originated from a scrapie-free flock in Sardinia. We then fed pooled milk from MVV/scrapie-infected sheep to each of 8 naïve ARQ/ARQ lambs and from scrapie-infected sheep to 3 naïve ARQ/ARQ lambs ad libitum. Each lamb ingested a total volume of 1 to 2 liters over a total period of 3 days (Table ​(Table1).1). Two lambs were orally inoculated with brain homogenate pooled from the scrapie-infected milk donors as positive controls. Groups of lambs were housed in separate stalls and subjected to isolation conditions.

TABLE 1.

Genotype, breed, and PrP^Sc detection in lambs fed milk from MVV/scrapie- or scrapie-infected sheep

Molecular phylogeny of diplomonads and enteromonads based on SSU rRNA,alpha-tubulin and HSP90 genes: Implications for the evolutionary history of the double karyomastigont of diplomonads

Background  

Fornicata is a relatively recently established group of protists that includes the diplokaryotic diplomonads (which have two similar nuclei per cell), and the monokaryotic enteromonads, retortamonads and Carpediemonas, with the more typical one nucleus per cell. The monophyly of the group was confirmed by molecular phylogenetic studies, but neither the internal phylogeny nor its position on the eukaryotic tree has been clearly resolved.