Giraffe lineages are shaped by major ancient admixture events.

Strong genetic structure has prompted discussion regarding giraffe taxonomy,1,2,3 including a suggestion to split the giraffe into four species: Northern (Giraffa c. camelopardalis), Reticulated (G. c. reticulata), Masai (G. c. tippelskirchi), and Southern giraffes (G. c. giraffa).4,5,6 However, their evolutionary history is not yet fully resolved, as previous studies used a simple bifurcating model and did not explore the presence or extent of gene flow between lineages. We therefore inferred a model that incorporates various evolutionary processes to assess the drivers of contemporary giraffe diversity. We analyzed whole-genome sequencing data from 90 wild giraffes from 29 localities across their current distribution. The most basal divergence was dated to 280 kya. Genetic differentiation, FST, among major lineages ranged between 0.28 and 0.62, and we found significant levels of ancient gene flow between them. In particular, several analyses suggested that the Reticulated lineage evolved through admixture, with almost equal contribution from the Northern lineage and an ancestral lineage related to Masai and Southern giraffes. These new results highlight a scenario of strong differentiation despite gene flow, providing further context for the interpretation of giraffe diversity and the process of speciation in general. They also illustrate that conservation measures need to target various lineages and sublineages and that separate management strategies are needed to conserve giraffe diversity effectively. Given local extinctions and recent dramatic declines in many giraffe populations, this improved understanding of giraffe evolutionary history is relevant for conservation interventions, including reintroductions and reinforcements of existing populations.

Detection of Bartonella schoenbuchensis (sub)species DNA in different louse fly species in Saxony, Germany: The proof of multiple PCR analysis necessity in case of ruminant-associated bartonellae determination.

BACKGROUND: Hippoboscid flies are bloodsucking arthropods that can transmit pathogenic microorganisms and are therefore potential vectors for pathogens such as Bartonella spp. These Gram-negative bacteria can cause mild-to-severe clinical signs in humans and animals; therefore, monitoring Bartonella spp. prevalence in louse fly populations appears to be a useful prerequisite for zoonotic risk assessment. METHODS: Using convenience sampling, we collected 103 adult louse flies from four ked species (Lipoptena cervi, n = 22; Lipoptena fortisetosa, n = 61; Melophagus ovinus, n = 12; Hippobosca equina, n = 8) and the pupae of M. ovinus (n = 10) in the federal state of Saxony, Germany. All the samples were screened by polymerase chain reaction (PCR) for Bartonella spp. DNA, targeting the citrate synthase gene (gltA). Subsequently, PCRs targeting five more genes (16S, ftsZ, nuoG, ribC and rpoB) were performed for representatives of revealed gltA genotypes, and all the PCR products were sequenced to identify the Bartonella (sub)species accurately. RESULTS AND CONCLUSIONS: The overall detection rates for Bartonella spp. were 100.0%, 59.1%, 24.6% and 75.0% in M. ovinus, L. cervi, L. fortisetosa and H. equina, respectively. All the identified bartonellae belong to the Bartonella schoenbuchensis complex. Our data support the proposed reclassification of the (sub)species status of this group, and thus we conclude that several genotypes of B. schoenbuchensis were detected, including Bartonella schoenbuchensis subsp. melophagi and Bartonella schoenbuchensis subsp. schoenbuchensis, both of which have previously validated zoonotic potential. The extensive PCR analysis revealed the necessity of multiple PCR approach for proper identification of the ruminant-associated bartonellae.

Characterisation of IS1311 in Mycobacterium avium subspecies paratuberculosis genomes: Typing, continental clustering, microbial evolution and host adaptation.

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a global burden for livestock producers and has an association with Crohn's disease in humans. Within MAP there are two major lineages, S/Type I/TypeIII and C/Type II, that vary in phenotype including culturability, host preference and virulence. These lineages have been identified using the IS1311 element, which contains a conserved, single nucleotide polymorphism. IS1311 and the closely related IS1245 element belong to the IS256 family of insertion sequences, are dispersed throughout M. avium taxa but remain poorly characterised. To investigate the distribution and diversity of IS1311 in MAP, 805 MAP genomes were collated from public databases. IS1245 was absent, while IS1311 sequence, copy number and insertion loci were conserved between MAP S lineages and varied within the MAP C lineage. One locus was specific to the S strains, which contained nine IS1311 copies. In contrast, C strains contained either seven or eight IS1311 loci. Most insertion loci were associated with the boundaries of homologous regions that had undergone genome rearrangement between the MAP lineages, suggesting that this sequence may be a driver of recombination. Phylogenomic geographic clustering of MAP subtypes was demonstrated for the first time, at continental scale, and indicated that there may have been recent MAP transmission between Europe and North America, in contrast to Australia where importation of live ruminants is generally prohibited. This investigation confirmed the utility of IS1311 typing in epidemiological studies and resolved anomalies in past studies. The results shed light on potential mechanisms of niche/host adaptation, virulence of MAP and global transmission dynamics.

Functional convergence in gastric lysozymes of foregut-fermenting rodents, ruminants, and primates is not attributed to convergent molecular evolution.

Convergent evolution is a widespread phenomenon. While there are many examples of convergent evolution at the phenotypic scale, convergence at the molecular level has been more difficult to identify. A classic example of convergent evolution across scales is that of the digestive lysozyme found in ruminants and Colobine monkeys. These herbivorous species rely on foregut fermentation, which has evolved to function more optimally under acidic conditions. Here, we explored if rodents with similar dietary strategies and digestive morphologies have convergently evolved a lysozyme with digestive functions. At the phenotypic level, we find that rodents with bilocular stomach morphologies exhibited a lysozyme that maintained higher relative activities at low pH values, similar to the lysozymes of ruminants and Colobine monkeys. Additionally, the lysozyme of Peromyscus leucopus shared a similar predicted protonation state as that observed in previously identified digestive lysozymes. However, we found limited evidence of positive selection acting on the lysozyme gene in foregut-fermenting species and did not identify patterns of convergent molecular evolution in this gene. This study emphasizes that phenotypic convergence need not be the result of convergent genetic modifications, and we encourage further exploration into the mechanisms regulating convergence across biological scales.

Copy number and selection of MHC genes in ruminants are related to habitat, average life span and diet.

The ruminants, as the main group of livestock, have been extensively studied in terms of their physiology, endocrinology, biochemistry, genetics, and nutrition. Despite the wide geographic distribution and habitat diversity of animals in this group, their ecology and evolution remain poorly understood. In this study, we analyzed the gene copy number, selection, and ecological and evolutionary processes that have affected the evolution of major histocompatibility complex (MHC) genes across ruminant lineages based on available genomic data. The 51 species analyzed represented all six families of ruminants. Our finding indicated that the architecture of the MHC region is conserved in ruminants, but with variable copy numbers of MHC-I, MHC-IIA, and MHC-IIB genes. No lineage-specific gene duplication was observed in the MHC genes. The phylogenetic generalized least squares regression (PGLS) model revealed association between ecological and biological factors (habitat and lifespan) and gene duplication in DQA and DQB, but not in DRB. The selection pressure of DQA and DQB were related with lifespan, diet, and the ratio of genetic repeat elements. These results suggest that the MHC evolution in ruminants, including copy number and selection, has been influenced by genetic repeat elements, pathogen exposure risk, and intrinsic cost of possessing multiple MHC genes.

Bacterial community and diversity in the rumen of 11 Mongolian cattle as revealed by 16S rRNA amplicon sequencing.

Through microorganism in the rumen of ruminant, plant fiber can be converted to edible food such as meat and milk. Ruminants had a rich and complex microbial community within the rumen, and the bacteria comprised the dominant proportion of the ruminal microbes. High-throughput sequencing offered a viable solution for the study of rumen microbes. In this study, rumen fluid samples were taken from 11 cattle from Inner Mongolian, the DNA of 11 rumen fluid samples were extracted and bacterial amplicons of the V4 regions of 16S rRNA were subjected to Illumina sequencing. More than 90,000 raw reads and 60,000 effect Tags per sample were obtained. 28,122 operational taxonomic units (OTUs) were observed from 11 samples, in average 2557 ± 361 OTUs for each sample. Bacteroidetes (44.41 ± 7.31%), Firmicutes (29.07 ± 3.78%), and Proteobacteria (7.18 ± 5.63%) were the dominant phyla among the bacteria of rumen, accounting for 82%. At the genus level, the highest relative abundance was Prevotella. Their functions were predicted using the Kyoto Encyclopedia of Genes and Genomes (KEGG). The results showed that they included metabolism, genetic information processing, environmental information processing and cellular processes. It explored the bacterial community diversity and composition of the rumen of Mongolian cattle. On the whole, our research showed that there was a high diversity as well as rich bacterial flora function of rumen bacteria in Mongolian cattle. Meanwhile, these findings provided information for further studies on the relationship between the community, diversity, functions of rumen bacteria and the nutritional physiological functions of the host.

Ruminal microbiota-host crosstalks promote ruminal epithelial development in neonatal lambs with alfalfa hay introduction.

Ruminal microbiota is gradually established after birth, while microbiota maturation could be highly diverse because of varied solid dietary accessibility. However, how the ruminal microbiota accreted from postnatal hay diets alters rumen epithelial development, and how this affects animal health remains largely unknown. Here, neonatal lambs were introduced to starchy corn-soybean starter or corn-soybean starter + alfalfa hay (AH) to investigate the influences of early life ruminal microbiome on rumen epithelial development using integrated 16s rRNA sequencing-metagenome-transcriptome approaches. The results showed that AH introduction elevated average daily weight gain, rumen weight and volume, rumen epithelial papillae length, and rumen muscle layer thickness. Meanwhile, the relative abundance of fibrolytic bacteria (Christensenellaceae R-7 group, Prevotellaceae UCG-001, and Succinivibrio), acetate producer (Acetitomaculum and Mitsuokella), and propionate producer Succiniclasticum was increased in the rumen content by AH supplementation (P < 0.05). Moreover, AH introduction decreased the relative abundance of total CAZymes, CBM, and GH and increased the abundance of KO genes related to volatile fatty acid (VFA) generation in the rumen content. AH lambs had a higher relative abundance of Succiniclasticum, Megasphaera, Succinivibrio, and Suttonella (P < 0.05), while a lower relative abundance of Cloacibacillus, Desulfovibrio, Dialister, Intestinimonas, Parabacteroides, and Pseudoscardovia (P < 0.05) in the rumen epithelial samples. Furthermore, these alterations in ruminal microbial structure and function resulted in ruminal epithelial cell proliferation and development pathways activation. In summary, AH introduction benefited ruminal fiber degradation and VFA generation bacteria colonization and promoted ruminal epithelial development. These findings provide new insights into ruminal microbial-host interactions in the early life.IMPORTANCEWhile it is established that a fiber-rich diet promotes rumen development in lambs, further research is needed to investigate the precise response of rumen microbiota and epithelium to high-quality alfalfa hay. Here, we observed that the inclusion of alfalfa hay led to a discernible alteration in the developmental trajectory of the rumen. Notably, there was a favorable shift in the rumen's volume, morphology, and the development of rumen papillae. Furthermore, ruminal microbial structure and function resulted in ruminal epithelial cell proliferation and development pathways activation, collectively provide compelling evidence supporting the capacity of alfalfa hay to enhance rumen development and health through ruminal micrbiota-host crosstalks. Our findings elucidate the functional response of the rumen to alfalfa hay introduction, providing new insights into strategies for promoting healthy development of the rumen in young ruminants.

Comparison of 2 PCR assays on environmental samples cultured for Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of paratuberculosis, a chronic, contagious, and incurable enteric disease of ruminants. An in-house IS900 PCR assay validated for MAP detection in sheep has been shown to have a higher sensitivity than a commercial PCR and fecal culture. We have now compared the performance of this in-house IS900 PCR assay with a commercial ISMap02 PCR assay for the detection of MAP DNA in bovine dairy farm environmental samples. We purposefully selected 30 culture-positive, 62 culture-negative, and 62 non-interpretable environmental samples. We applied the IS900 PCR assay directly to the frozen inoculum of these samples. Inocula were incubated in an automated system, and growth was confirmed by an acid-fast bacilli stain and the IS900 PCR assay. Among culture-positive samples before incubation, the IS900 PCR assay yielded significantly more positive results than the ISMap02 PCR assay; however, among culture-negative samples, the IS900 PCR assay yielded positive results both before and after incubation. The ISMap02 PCR assay did not flag positively among the culture-negative samples either before or after incubation. The IS900 PCR assay is a sensitive method that can be used to detect MAP DNA in environmental samples before incubation. The ISMap02 PCR assay is a specific method used to detect MAP DNA in environmental samples both before and after incubation.

Population genomics of the muskox' resilience in the near absence of genetic variation.

Genomic studies of species threatened by extinction are providing crucial information about evolutionary mechanisms and genetic consequences of population declines and bottlenecks. However, to understand how species avoid the extinction vortex, insights can be drawn by studying species that thrive despite past declines. Here, we studied the population genomics of the muskox (Ovibos moschatus), an Ice Age relict that was at the brink of extinction for thousands of years at the end of the Pleistocene yet appears to be thriving today. We analysed 108 whole genomes, including present-day individuals representing the current native range of both muskox subspecies, the white-faced and the barren-ground muskox (O. moschatus wardi and O. moschatus moschatus) and a ~21,000-year-old ancient individual from Siberia. We found that the muskox' demographic history was profoundly shaped by past climate changes and post-glacial re-colonizations. In particular, the white-faced muskox has the lowest genome-wide heterozygosity recorded in an ungulate. Yet, there is no evidence of inbreeding depression in native muskox populations. We hypothesize that this can be explained by the effect of long-term gradual population declines that allowed for purging of strongly deleterious mutations. This study provides insights into how species with a history of population bottlenecks, small population sizes and low genetic diversity survive against all odds.

Isolation and functional analysis of acid-producing bacteria from bovine rumen.

Ruminants such as cattle rely mainly on microbes in the rumen to digest cellulose and hemicellulose from forage, and the digestion products are mainly absorbed and utilized by the host in the form of short chain fatty acids (SCFAs). This study aimed to isolate acid-producing strains from the cattle rumen and investigate their functions. A total of 980 strains of acid-producing bacteria were isolated from cattle rumen contents using a medium supplemented with bromocresol green. Combined with the test of acid production ability and 16S rRNA amplicon sequencing technology, five strains were selected based on their ability to produce relatively high levels of acid, including Bacillus pumillus, Enterococcus hirae, Enterococcus faecium, and Bacillus subtilis. Sheep were treated by gavage with a mixed bacterial suspension. The results showed that mixed bacteria significantly increased the body weight gain and feed conversion rate of sheep. To investigate the function of acid-producing bacteria in sheep, we used 16S rDNA sequencing technology to analyze the rumen microbes of sheep. We found that mixed bacteria changed the composition and abundance of sheep rumen bacteria. Among them, the abundance of Bacteroidota, Actinobacteriota, Acidobacteriota, and Proteobacteria was significantly increased, and the abundance of Firmicutes was significantly decreased, indicating that the changes in gut microbiota changed the function of the sheep rumen. The acid-producing bacteria isolated in this study can effectively promote the growth of ruminants, such as cattle and sheep, and can be used as additives to improve breeding efficiency, which lays a foundation for subsequent research on probiotics.

Single-cell transcriptome and metagenome profiling reveals the genetic basis of rumen functions and convergent developmental patterns in ruminants.

The rumen undergoes developmental changes during maturation. To characterize this understudied dynamic process, we profiled single-cell transcriptomes of about 308,000 cells from the rumen tissues of sheep and goats at 17 time points. We built comprehensive transcriptome and metagenome atlases from early embryonic to rumination stages, and recapitulated histomorphometric and transcriptional features of the rumen, revealing key transitional signatures associated with the development of ruminal cells, microbiota, and core transcriptional regulatory networks. In addition, we identified and validated potential cross-talk between host cells and microbiomes and revealed their roles in modulating the spatiotemporal expression of key genes in ruminal cells. Cross-species analyses revealed convergent developmental patterns of cellular heterogeneity, gene expression, and cell-cell and microbiome-cell interactions. Finally, we uncovered how the interactions can act upon the symbiotic rumen system to modify the processes of fermentation, fiber digestion, and immune defense. These results significantly enhance understanding of the genetic basis of the unique roles of rumen.

A Chinese indicine pangenome reveals a wealth of novel structural variants introgressed from other Bos species.

Chinese indicine cattle harbor a much higher genetic diversity compared with other domestic cattle, but their genome architecture remains uninvestigated. Using PacBio HiFi sequencing data from 10 Chinese indicine cattle across southern China, we assembled 20 high-quality partially phased genomes and integrated them into a multiassembly graph containing 148.5 Mb (5.6%) of novel sequence. We identified 156,009 high-confidence nonredundant structural variants (SVs) and 206 SV hotspots spanning ∼195 Mb of gene-rich sequence. We detected 34,249 archaic introgressed fragments in Chinese indicine cattle covering 1.93 Gb (73.3%) of the genome. We inferred an average of 3.8%, 3.2%, 1.4%, and 0.5% of introgressed sequence originating, respectively, from banteng-like, kouprey-like, gayal-like, and gaur-like Bos species, as well as 0.6% of unknown origin. Introgression from multiple donors might have contributed to the genetic diversity of Chinese indicine cattle. Altogether, this study highlights the contribution of interspecies introgression to the genomic architecture of an important livestock population and shows how exotic genomic elements can contribute to the genetic variation available for selection.

Differential diagnosis of theileriosis through blood smear examination and polymerase chain reaction in small ruminants from Pakistan.

Background: Ovine and caprine theileriosis is a tick-borne hemoprotozoan disease, caused by Theileria spp., responsible for heavy economic losses in terms of high mortality and morbidity rates. Diagnosis of ovine theileriosis is primarily based on clinical symptoms, microscopic screening of stained blood smears, and lymph node biopsy smears, but the limitations of these detection methods against Theileria spp. infection limits their specificity. Aim: To overcome these limitations, the current study reports the differential diagnosis of theileriosis through a blood smear examination and polymerase chain reaction (PCR) in small ruminants from Pakistan. Methods: The study was conducted on 1,200 apparently healthy small ruminants (737 sheep and 463 goats). First, blood smears were screened for the presence of Theileria piroplasms in red blood cells. Second, PCR amplification based on 18S rRNA gene was performed by using primers specific to Theileria spp. Results: Out of the 1,200 samples of examined blood smears, 100 animals (8.33%) were found positive for Theileria species, which showed intra-erythrocytic bodies in the form of dot and comma shapes. Amplification of the isolated DNA from randomly collected blood samples of 737 sheep and 463 goats showed that an amplicon size of 1,098 bp was positive for Theileria spp. In total, 315 out of the 1,200 small ruminants examined in this study were found positive for Theileria spp. DNA through PCR amplification. Notably, out of the 885 blood samples negative by PCR amplification, only 15 blood samples were found positive by the blood smear test. Conversely, 230 blood samples that tested negative in the smear technique produced a specific band through PCR amplification. Overall, the sensitivity and specificity rates were 26.98% and 98.31% for the blood smear method and 73.01% and 100% for the PCR assay, respectively. Conclusion: Our finding suggests that PCR is the gold standard method compared to the conventional method of smear examination for the diagnosis of ovine and caprine theileriosis in Pakistan.

A novel multiplex qPCR­HRM assay for the simultaneous detection of four abortive zoonotic agents in cattle, sheep, and goats.

Abortifacient pathogens induce substantial economic losses in the livestock industry worldwide, and many of these pathogens are zoonotic, impacting human health. As Brucella spp., Coxiella burnetii, Leptospira spp., and Listeria monocytogenes cause abortion, rapid differential molecular diagnostic tests are needed to facilitate early and accurate detection of abortion to establish effective control measures. However, the available molecular methods are laborious, time-consuming, or costly. Therefore, we developed and validated a novel multiplex real-time polymerase chain reaction (qPCR) method based on high-resolution melting (HRM) curve analysis to simultaneously detect and differentiate four zoonotic abortifacient agents in cattle, goats, and sheep. Our HRM assay generated four well-separated melting peaks allowing the differentiation between the four zoonotic abortifacients. Out of 216 DNA samples tested, Brucella spp. was detected in 45 samples, Coxiella burnetii in 57 samples, Leptospira spp. in 12 samples, and Listeria monocytogenes in 19 samples, co-infection with Brucella spp. and Coxiella burnetii in 41 samples, and 42 samples were negative. This assay demonstrated good analytical sensitivity, specificity, and reproducibility. This is a valuable rapid, cost-saving, and reliable diagnostic tool for detecting individual and co-infections for zoonotic abortifacient agents in ruminants.

Comparative studies of X chromosomes in Cervidae family.

The family Cervidae is the second most diverse in the infraorder Pecora and is characterized by variability in the diploid chromosome numbers among species. X chromosomes in Cervidae evolved through complex chromosomal rearrangements of conserved segments within the chromosome, changes in centromere position, heterochromatic variation, and X-autosomal translocations. The family Cervidae consists of two subfamilies: Cervinae and Capreolinae. Here we build a detailed X chromosome map with 29 cattle bacterial artificial chromosomes of representatives of both subfamilies: reindeer (Rangifer tarandus), gray brocket deer (Mazama gouazoubira), Chinese water deer (Hydropotes inermis) (Capreolinae); black muntjac (Muntiacus crinifrons), tufted deer (Elaphodus cephalophus), sika deer (Cervus nippon) and red deer (Cervus elaphus) (Cervinae). To track chromosomal rearrangements during Cervidae evolution, we summarized new data, and compared them with available X chromosomal maps and chromosome level assemblies of other species. We demonstrate the types of rearrangements that may have underlined the variability of Cervidae X chromosomes. We detected two types of cervine X chromosome-acrocentric and submetacentric. The acrocentric type is found in three independent deer lineages (subfamily Cervinae and in two Capreolinae tribes-Odocoileini and Capreolini). We show that chromosomal rearrangements on the X-chromosome in Cervidae occur at a higher frequency than in the entire Ruminantia lineage: the rate of rearrangements is 2 per 10 million years.

Convergent molecular evolution of thermogenesis and circadian rhythm in Arctic ruminants.

The muskox and reindeer are the only ruminants that have evolved to survive in harsh Arctic environments. However, the genetic basis of this Arctic adaptation remains largely unclear. Here, we compared a de novo assembled muskox genome with reindeer and other ruminant genomes to identify convergent amino acid substitutions, rapidly evolving genes and positively selected genes among the two Arctic ruminants. We found these candidate genes were mainly involved in brown adipose tissue (BAT) thermogenesis and circadian rhythm. Furthermore, by integrating transcriptomic data from goat adipose tissues (white and brown), we demonstrated that muskox and reindeer may have evolved modulating mitochondrion, lipid metabolism and angiogenesis pathways to enhance BAT thermogenesis. In addition, results from co-immunoprecipitation experiments prove that convergent amino acid substitution of the angiogenesis-related gene hypoxia-inducible factor 2alpha (HIF2A), resulting in weakening of its interaction with prolyl hydroxylase domain-containing protein 2 (PHD2), may increase angiogenesis of BAT. Altogether, our work provides new insights into the molecular mechanisms involved in Arctic adaptation.

Industrial and Ruminant Trans-Fatty Acids-Enriched Diets Differentially Modulate the Microbiome and Fecal Metabolites in C57BL/6 Mice.

Industrially originated trans-fatty acids (I-tFAs), such as elaidic acid (EA), and ruminant trans-fatty acids (R-tFAs), such as trans-palmitoleic acid (TPA), may have opposite effects on metabolic health. The objective was to compare the effects of consuming 2-3% I-tFA or R-tFA on the gut microbiome and fecal metabolite profile in mice after 7 and 28 days. Forty C57BL/6 mice were assigned to one of the four prepared formulations: lecithin nanovesicles, lecithin nanovesicles with EA or TPA, or water. Fecal samples and animals' weights were collected on days 0, 7, and 28. Fecal samples were used to determine gut microbiome profiles by 16S rRNA sequencing and metabolite concentrations by GC/MS. At 28 days, TPA intake decreased the abundance of Staphylococcus sp55 but increased Staphylococcus sp119. EA intake also increased the abundance of Staphylococcus sp119 but decreased Ruminococcaceae UCG-014, Lachnospiraceae, and Clostridium sensu stricto 1 at 28 days. Fecal short-chain fatty acids were increased after TPA while decreased after EA after 7 and 28 days. This study shows that TPA and EA modify the abundance of specific microbial taxa and fecal metabolite profiles in distinct ways.

Possible biased virulence attenuation in the Senegal strain of Ehrlichia ruminantium by ntrX gene conversion from an inverted segmental duplication.

Ehrlichia ruminantium is a tick-borne intracellular pathogen of ruminants that causes heartwater, a disease present in Sub-saharan Africa, islands in the Indian Ocean and the Caribbean, inducing significant economic losses. At present, three avirulent strains of E. ruminantium (Gardel, Welgevonden and Senegal isolates) have been produced by a process of serial passaging in mammalian cells in vitro, but unfortunately their use as vaccines do not offer a large range of protection against other strains, possibly due to the genetic diversity present within the species. So far no genetic basis for virulence attenuation has been identified in any E. ruminantium strain that could offer targets to facilitate vaccine production. Virulence attenuated Senegal strains have been produced twice independently, and require many fewer passages to attenuate than the other strains. We compared the genomes of a virulent and attenuated Senegal strain and identified a likely attenuator gene, ntrX, a global transcription regulator and member of a two-component system that is linked to environmental sensing. This gene has an inverted partial duplicate close to the parental gene that shows evidence of gene conversion in different E. ruminantium strains. The pseudogenisation of the gene in the avirulent Senegal strain occurred by gene conversion from the duplicate to the parent, transferring a 4 bp deletion which is unique to the Senegal strain partial duplicate amongst the wild isolates. We confirmed that the ntrX gene is not expressed in the avirulent Senegal strain by RT-PCR. The inverted duplicate structure combined with the 4 bp deletion in the Senegal strain can explain both the attenuation and the faster speed of attenuation in the Senegal strain relative to other strains of E. ruminantium. Our results identify nrtX as a promising target for the generation of attenuated strains of E. ruminantium by random or directed mutagenesis that could be used for vaccine production.

Comparison of RT-qPCR and RT-ddPCR with Rift valley fever virus (RVFV) RNA.

Rift valley fever (RVF) is an important zoonotic disease caused by the Rift valley fever virus (RVFV) which can affect ruminants and humans. In this study, a comparison was done of the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription-droplet digital PCR (RT-ddPCR) assays with synthesized RVFV RNA, cultured viral RNA, and mock clinical RVFV RNA samples. The genomic segments (L, M, and S) of three RVFV strains (BIME01, Kenya56, and ZH548) were synthesized and used as templates for in vitro transcription (IVT). Both the RT-qPCR and RT-ddPCR assays for RVFV did not react with any of the negative reference viral genomes. Thus, both the RT-qPCR and RT-ddPCR assays are specific to RVFV. The comparison of both the RT-qPCR and RT-ddPCR assays with serially diluted templates showed that the LoD of both assays are similar, and a concordant of the results was observed. The LoD of both assays reached the practical measurable minimum concentration. Taken altogether, the sensitivity of the RT-qPCR and RT-ddPCR assays is similar, and the material measured by RT-ddPCR can be used as a reference material for RT-qPCR.