The locus for bovine dilated cardiomyopathy maps to chromosome 18

Bovine dilated cardiomyopathy (BDCMP) is a severe and terminal disease of the heart muscle observed in Holstein-Friesian cattle over the last 30 years. There is strong evidence for an autosomal recessive mode of inheritance for BDCMP. The objective of this study was to genetically map BDCMP, with the ultimate goal of identifying the causative mutation. A whole-genome scan using 199 microsatellite markers and one SNP revealed an assignment of BDCMP to BTA18. Fine-mapping on BTA18 refined the candidate region to the MSBDCMP06-BMS2785 interval. The interval containing the BDCMP locus was confirmed by multipoint linkage analysis using the software loki. The interval is about 6.7 Mb on the bovine genome sequence (Btau 3.1). The corresponding region of HSA19 is very gene-rich and contains roughly 200 genes. Although telomeric of the marker interval, TNNI3 is a possible positional and a functional candidate for BDCMP given its involvement in a human form of dilated cardiomyopathy. Sequence analysis of TNNI3 in cattle revealed no mutation in the coding sequence, but there was a G-to-A transition in intron 6 (AJ842179:c.378+315G>A). The analysis of this SNP using the study's BDCMP pedigree did not conclusively exclude TNNI3 as a candidate gene for BDCMP. Considering the high density of genes on the homologous region of HSA19, further refinement of the interval on BTA18 containing the BDCMP locus is needed.

The horse genome project - sequence based insights into male reproductive mechanisms

The growing knowledge on physiology, cell biology and biochemistry of the reproductive organs has provided many insights into molecular mechanisms that are required for successful reproduction. Research directed at the investigation of reproduction physiology in domestic animals was hampered in the past by a lack of species-specific genomic information. The genome sequences of dog, cattle and horse have become publicly available in 2005, 2006 and 2007 respectively. Although the gene content of mammalian genomes is generally very similar, genes involved in reproduction tend to be less conserved than the average mammalian gene. The availability of genome sequences provides a valuable resource to check whether any protein that may be known from human or mouse research is present in cattle and/or horse as well. Currently there are more than 200 genes known that are involved in the production of fertile sperm cells. Great progress has been made in the understanding of genetic aberrations that lead to male infertility. Additionally, the first genetic mechanisms are being discovered that contribute to the quantitative variation of fertility traits in fertile male animals. Here, I will review some selected aspects of genetic research in male fertility and offer some perspectives for the use of genomic sequence information.

Neuropathological and molecular comparison between clinical and asymptomatic bovine spongiform encephalopathy cases

Interest in the proper neuropathological and molecular characterization of bovine spongiform encephalopathy (BSE) has increased since asymptomatic and atypical cases were detected in the cattle population by active disease surveillance. In this respect we investigated a total of 95 confirmed BSE cases originating from different active and passive surveillance categories (clinical suspects, emergency-slaughter, fallen stock and routinely slaughter) in Switzerland for their neuropathological and molecular phenotype. We looked for measurable differences between these categories in lesion profile, severity of spongiform change, degree of astrocytosis as well as immunohistochemical and molecular patterns of the disease-associated isoform of the prion protein (PrPd) in the caudal brainstem. Our results indicate significantly higher intensities of spongiform change in clinically affected compared to asymptomatic BSE cases. Similar effects were in trend observed for the intensities of PrPd deposition and astrocytosis, whereas the frequencies of morphological PrPd types and the molecular patterns in Western immunoblot were not different. Importantly, none of the animals included in this study revealed features of atypical BSE. Taken together, this study suggests that both clinically affected as well as asymptomatic Swiss BSE cases in cattle share the neuropathological and molecular phenotype of classical BSE and that asymptomatic classical BSE cases are at a pre-clinical stage of the disease rather than representing a true sub-clinical form of BSE.

Induction of tachyzoite egress from cells infected with the protozoan Neospora caninum by nitro- and bromo-thiazolides, a class of broad-spectrum anti-parasitic drugs

Neospora caninum represents an important pathogen causing stillbirth and abortion in cattle and neuromuscular disease in dogs. Nitazoxanide (NTZ) and its deacetylated metabolite tizoxanide (TIZ) are nitro-thiazolyl-salicylamide drugs with a broad-spectrum anti-parasitic activity in vitro and in vivo. In order to generate compounds potentially applicable in food and breeding animals, the nitro group was removed, and the thiazole-moiety was modified by other functional groups. We had shown earlier that replacement of the nitro-group by a bromo-moiety did not notably affect in vitro efficacy of the drugs against N. caninum. In this study we report on the characterization of two bromo-derivatives, namely Rm4822 and its de-acetylated putative metabolite Rm4847 in relation to the nitro-compounds NTZ and TIZ. IC(50) values for proliferation inhibition were 4.23 and 4.14 microM for NTZ and TIZ, and 14.75 and 13.68 microM for Rm4822 and Rm4847, respectively. Complete inhibition (IC(99)) was achieved at 19.52 and 22.38 microM for NTZ and TIZ, and 18.21 and 17.66 microM for Rm4822 and Rm4847, respectively. However, in order to exert a true parasiticidal effect in vitro, continuous culture of infected fibroblasts in the presence of the bromo-thiazolide Rm4847 was required for a period of 3 days, while the nitro-compound TIZ required 5 days continuous drug exposure. Both thiazolides induced rapid egress of N. caninum tachyzoites from their host cells, and egress was inhibited by the cell membrane permeable Ca(2+)-chelator BAPTA-AM. Host cell entry by N. caninum tachyzoites was inhibited by Rm4847 but not by TIZ. Upon release from their host cells, TIZ-treated parasites remained associated with the fibroblast monolayer, re-invaded neighboring host cells and resumed proliferation in the absence of the drug. In contrast, Rm4847 inhibited host cell invasion and respective treated tachyzoites did not proliferate further. This demonstrated that bromo- and nitro-thiazolides exhibit differential effects against the intracellular protozoan N. caninum and bromo-thiazolides could represent a valuable alternative to the nitro-thiazolyl-salicylamide drugs.

Bacterial colitis increases susceptibility to oral prion disease

Dietary exposure to prion-contaminated materials has caused kuru and variant Creutzfeldt-Jakob disease in humans and transmissible spongiform encephalopathies (TSEs) in cattle, mink, and felines. The epidemiology of dietary prion infections suggests that host genetic modifiers and possibly exogenous cofactors may play a decisive role in determining disease susceptibility. However, few cofactors influencing susceptibility to prion infection have been identified. In the present study, we investigated whether colitis might represent one such cofactor. We report that moderate colitis caused by an attenuated Salmonella strain more than doubles the susceptibility of mice to oral prion infection and modestly accelerates the development of disease after prion challenge. The prion protein was up-regulated in intestines and mesenteric lymph nodes of mice with colitis, providing a possible mechanism for the effect of colitis on the pathogenesis of prion disease. Therefore, moderate intestinal inflammation at the time of prion exposure may constitute one of the elusive risk factors underlying the development of TSE.

The bovine dilated cardiomyopathy locus maps to a 1.0-Mb interval on chromosome 18

Cardiomyopathies are myocardial diseases that lead to cardiac dysfunction, heart failure, arrhythmia, and sudden death. In human medicine, cardiomyopathies frequently warrant heart transplantation in children and adults. Bovine dilated cardiomyopathy (BDCMP) is a heart muscle disorder that has been observed during the last 30 years in cattle of Holstein-Friesian origin. In Switzerland BDCMP affects Swiss Fleckvieh and Red Holstein breeds. BDCMP is characterized by a cardiac enlargement with ventricular remodeling and chamber dilatation. The common symptoms in affected animals are subacute subcutaneous edema, congestion of the jugular veins, and tachycardia with gallop rhythm. A cardiomegaly with dilatation and hypertrophy of all heart chambers, myocardial degeneration, and fibrosis are typical postmortem findings. It was shown that all BDCMP cases reported worldwide traced back to a red factor-carrying Holstein-Friesian bull, ABC Reflection Sovereign. An autosomal recessive mode of inheritance was proposed for BDCMP. Recently, the disease locus was mapped to a 6.7-Mb interval MSBDCMP06-BMS2785 on bovine Chr 18 (BTA18). In the present study the BDCMP locus was fine mapped by using a combined strategy of homozygosity mapping and association study. A BAC contig of 2.9 Mb encompassing the crucial interval was constructed to establish the correct marker order on BTA18. We show that the disease locus is located in a gene-rich interval of 1.0 Mb and is flanked by the microsatellite markers DIK3006 and MSBDCMP51.

Neospora caninum and bone marrow-derived dendritic cells: parasite survival, proliferation, and induction of cytokine expression

Dendritic cells (DCs) represent the first line defence of the innate immune system following infection with pathogens. We exploratively addressed invasion and survival ability of Neospora caninum, a parasite causing abortion in cattle, in mouse bone marrow DCs (BMDCs), and respective cytokine expression patterns. Immature BMDCs were exposed to viable (untreated) and nonviable parasites that had been inactivated by different means. Invasion and/or internalization, as well as intracellular survival and proliferation of tachyzoites were determined by NcGRA2-RT-PCR and transmission electron microscopy (TEM). Cytokine expression was evaluated by reverse transcription (RT)-PCR and cytokine ELISA. Transmission electron microscopy of DCs stimulated with untreated viable parasites revealed that N. caninum was able to invade and proliferate within BMDCs. This was confirmed by NcGRA2-RT-PCR. On the other hand, no viable parasite organisms were revealed by TEM when exposing BMDCs to inactivated parasites (nonviability demonstrated by NcGRA2-RT-PCR). Cytokine expression analysis (as assessed by both RT-PCR and ELISA) demonstrated that both viable and nonviable parasites stimulated mBMDCs to express IL-12p40, IL-10 and TNF-alpha, whereas IL-4 RNA expression was not detected. Thus, exposure of mBMDCs to both viable and nonviable parasites results in the expression of cytokines that are relevant for a mixed Th1/Th2 immune response.

DNA-based identification of novel bovine casein gene variants

In cattle, at least 39 variants of the 4 casein proteins (α(S1)-, β-, α(S2)- and κ-casein) have been described to date. Many of these variants are known to affect milk-production traits, cheese-processing properties, and the nutritive value of milk. They also provide valuable information for phylogenetic studies. So far, the majority of studies exploring the genetic variability of bovine caseins considered European taurine cattle breeds and were carried out at the protein level by electrophoretic techniques. This only allows the identification of variants that, due to amino acid exchanges, differ in their electric charge, molecular weight, or isoelectric point. In this study, the open reading frames of the casein genes CSN1S1, CSN2, CSN1S2, and CSN3 of 356 animals belonging to 14 taurine and 3 indicine cattle breeds were sequenced. With this approach, we identified 23 alleles, including 5 new DNA sequence variants, with a predicted effect on the protein sequence. The new variants were only found in indicine breeds and in one local Iranian breed, which has been phenotypically classified as a taurine breed. A multidimensional scaling approach based on available SNP chip data, however, revealed an admixture of taurine and indicine populations in this breed as well as in the local Iranian breed Golpayegani. Specific indicine casein alleles were also identified in a few European taurine breeds, indicating the introgression of indicine breeds into these populations. This study shows the existence of substantial undiscovered genetic variability of bovine casein loci, especially in indicine cattle breeds. The identification of new variants is a valuable tool for phylogenetic studies and investigations into the evolution of the milk protein genes.

Molecular epidemiology of Bacillus anthracis: determining the correct origin

We analyzed and compared strains of Bacillus anthracis isolated from husbandry and industrial anthrax cases in Switzerland between 1952 and 1981 with published data using multiple-locus variable-number tandem repeat analysis. Strains isolated from autochthonous cases of anthrax in cattle belong to genotype B2, together with strains from continental Europe, while human B. anthracis strains clustered with genotype A4. These strains could be traced back to outbreaks of human anthrax that occurred between 1978 and 1981 in a factory processing cashmere wool from the Indian subcontinent. We interpret the worldwide occurrence of B. anthracis strains of cluster A4 to be due to the extensive global trade of untreated cashmere wool during the last century.

Bovine mastitis: the diagnostic properties of a PCR-based assay to monitor the Staphylococcus aureus genotype B status of a herd, using bulk tank milk

Staphylococcus aureus genotype B (GTB) is a contagious mastitis pathogen in cattle, occurring in up to 87% of individuals. Because treatment is generally insufficient, culling is often required, leading to large economic loss in the Swiss dairy industry. As the detection of this pathogen in bulk tank milk (BTM) would greatly facilitate its control, a novel real-time quantitative PCR-based assay for BTM has previously been developed and is now being evaluated for its diagnostic properties at the herd level. Herds were initially classified as to their Staph. aureus GTB status by a reference method. Using BTM and herd pools of single-quarter and 4-quarter milk, the herds were then grouped by the novel assay, and the resulting classifications were compared. A total of 54 dairy herds were evaluated. Using the reference method, 21 herds were found to be GTB positive, whereas 33 were found to be negative. Considering the novel assay using both herd pools, all herds were grouped correctly, resulting in maximal diagnostic sensitivities (100%) and specificities (100%). For BTM samples, diagnostic sensitivities and specificities were 90 and 100%, respectively. Two herds were false negative in BTM, because cows with clinical signs of mastitis were not milked into the tank. Besides its excellent diagnostic properties, the assay is characterized by its low detection level, high efficiency, and its suitability for automation. Using the novel knowledge and assay, eradication of Staph. aureus GTB from a dairy herd may be considered as a realistic goal.

Phylogenetic positions of Clostridium chauvoei and Clostridium septicum based on 16S rRNA gene sequences

The sequences of the 16S rRNA genes (rrs genes) of Clostridium chauvoei, the causative agent of blackleg in cattle, and the phenotypically related organism Clostridium septicum were determined. After amplification of 1,507-bp PCR fragments from the corresponding rrs genes, the sequences were determined in a single round of sequencing by using conserved region primers. A sequence similarity analysis of the sequences revealed the close phylogenetic relationship of C. chauvoei and C. septicum in Clostridium cluster I (M. D. Collins, P. A. Lawson, A. Willems, J. J. Cordoba, J. Fernandez-Garayzabal, P. Garcia, J. Cai, H. Hippe, and J. A. E. Farrow, Int. J. Syst. Bacteriol. 44:812-826, 1994), which includes Clostridium carnis, Clostridium perfringens, Clostridium botulinum, and Clostridium tetani. We found that 99.3% of the nucleotides in the genes of C. chauvoei and C. septicum are identical.

Diseases of the Tendons and Tendon Sheaths

Contracted flexor tendon leading to flexural deformity is a common congenital defect in cattle. Arthrogryposis is a congenital syndrome of persistent joint contracture that occurs frequently in Europe as a consequence of Schmallenberg virus infection of the dam. Spastic paresis has a hereditary component, and affected cattle should not be used for breeding purposes. The most common tendon avulsion involves the deep digital flexor tendon. Tendon disruptions may be successfully managed by tenorrhaphy and external coaptation or by external coaptation alone. Medical management alone is unlikely to be effective for purulent tenosynovitis.

A 4 Mb high resolution BAC contig on bovine chromosome 1q12 and comparative analysis with human chromosome 21q22

The bovine RPCI-42 BAC library was screened to construct a sequence-ready ~4 Mb single contig of 92 BAC clones on BTA 1q12. The contig covers the region between the genes KRTAP8P1 and CLIC6. This genomic segment in cattle is of special interest as it contains the dominant gene responsible for the hornless or polled phenotype in cattle. The construction of the BAC contig was initiated by screening the bovine BAC library with heterologous cDNA probes derived from 12 human genes of the syntenic region on HSA 21q22. Contig building was facilitated by BAC end sequencing and chromosome walking. During the construction of the contig, 165 BAC end sequences and 109 single-copy STS markers were generated. For comparative mapping of 25 HSA 21q22 genes, genomic PCR primers were designed from bovine EST sequences and the gene-associated STSs mapped on the contig. Furthermore, bovine BAC end sequence comparisons against the human genome sequence revealed significant matches to HSA 21q22 and allowed the in silico mapping of two new genes in cattle. In total, 31 orthologues of human genes located on HSA 21q22 were directly mapped within the bovine BAC contig, of which 16 genes have been cloned and mapped for the first time in cattle. In contrast to the existing comparative bovine-human RH maps of this region, these results provide a better alignment and reveal a completely conserved gene order in this 4 Mb segment between cattle, human and mouse. The mapping of known polled linked BTA 1q12 microsatellite markers allowed the integration of the physical contig map with existing linkage maps of this region and also determined the exact order of these markers for the first time. Our physical map and transcript map may be useful for positional cloning of the putative polled gene in cattle.

Bovine Staphylococcus aureus: Subtyping, evolution, and zoonotic transfer.

Staphylococcus aureus is globally one of the most important pathogens causing contagious mastitis in cattle. Previous studies using ribosomal spacer (RS)-PCR, however, demonstrated in Swiss cows that Staph. aureus isolated from bovine intramammary infections are genetically heterogeneous, with Staph. aureus genotype B (GTB) and GTC being the most prominent genotypes. Furthermore, Staph. aureus GTB was found to be contagious, whereas Staph. aureus GTC and all the remaining genotypes were involved in individual cow disease. In addition to RS-PCR, other methods for subtyping Staph. aureus are known, including spa typing and multilocus sequence typing (MLST). They are based on sequencing the spa and various housekeeping genes, respectively. The aim of the present study was to compare the 3 analytic methods using 456 strains of Staph. aureus isolated from milk of bovine intramammary infections and bulk tanks obtained from 12 European countries. Furthermore, the phylogeny of animal Staph. aureus was inferred and the zoonotic transfer of Staph. aureus between cattle and humans was studied. The analyzed strains could be grouped into 6 genotypic clusters, with CLB, CLC, and CLR being the most prominent ones. Comparing the 3 subtyping methods, RS-PCR showed the highest resolution, followed by spa typing and MLST. We found associations among the methods but in many cases they were unsatisfactory except for CLB and CLC. Cluster CLB was positive for clonal complex (CC)8 in 99% of the cases and typically positive for t2953; it is the cattle-adapted form of CC8. Cluster CLC was always positive for t529 and typically positive for CC705. For CLR and the remaining subtypes, links among the 3 methods were generally poor. Bovine Staph. aureus is highly clonal and a few clones predominate. Animal Staph. aureus always evolve from human strains, such that every human strain may be the ancestor of a novel animal-adapted strain. The zoonotic transfer of IMI- and milk-associated strains of Staph. aureus between cattle and humans seems to be very limited and different hosts are not considered as a source for mutual, spontaneous infections. Spillover events, however, may happen.

Tritrichomonas--systematics of an enigmatic genus.

Tritrichomonas spp. are parasitic protozoans that proliferate on mucus membranes of the urogenital, gastro-intestinal or nasal tract. For instance, Tritrichomonas foetus is an important cause of reproductive failure in cattle. Some years ago, T. foetus was also identified as a causative agent of diarrhoea in cats. Previous studies on the morphological, physiological and molecular levels have raised doubts as to the phylogenetic relationship among some Tritrichomonas species, particularly in relation to T. foetus, Tritrichomonas suis, and Tritrichomonas mobilensis. With the advent of molecular genetic tools, it has become clear that these three tritrichomonad species are closely related or may even represent the same species. Indeed, since recently, T. suis and T. foetus are generally considered as one species, with T. mobilensis being a closely related sister taxon. To date, molecular studies have not yet been able to resolve the taxonomic (specific) status of T. foetus from cattle and cats. In the future, novel genomic approaches, particularly those involving next generation sequencing are poised to resolve the taxonomy of Tritrichomonas spp. Here, we review the literature on the current state of knowledge of the taxonomy of T. foetus, T. suis, and T. mobilensis with special reference to the relationship between T. foetus from cattle and cats.