Genomic characterization of equine influenza A subtype H3N8 viruses by long read sequencing and functional analyses of the PB1-F2 virulence factor of A/equine/Paris/1/2018.

Equine influenza virus (EIV) remains a threat to horses, despite the availability of vaccines. Strategies to monitor the virus and prevent potential vaccine failure revolve around serological assays, RT-qPCR amplification, and sequencing the viral hemagglutinin (HA) and neuraminidase (NA) genes. These approaches overlook the contribution of other viral proteins in driving virulence. This study assesses the potential of long-read nanopore sequencing for fast and precise sequencing of circulating equine influenza viruses. Therefore, two French Florida Clade 1 strains, including the one circulating in winter 2018-2019 exhibiting more pronounced pathogenicity than usual, as well as the two currently OIE-recommended vaccine strains, were sequenced. Our results demonstrated the reliability of this sequencing method in generating accurate sequences. Sequence analysis of HA revealed a subtle antigenic drift in the French EIV strains, with specific substitutions, such as T163I in A/equine/Paris/1/2018 and the N188T mutation in post-2015 strains; both substitutions were in antigenic site B. Antigenic site E exhibited modifications in post-2018 strains, with the N63D substitution. Segment 2 sequencing also revealed that the A/equine/Paris/1/2018 strain encodes a longer variant of the PB1-F2 protein when compared to other Florida clade 1 strains (90 amino acids long versus 81 amino acids long). Further biological and biochemistry assays demonstrated that this PB1-F2 variant has enhanced abilities to abolish the mitochondrial membrane potential ΔΨm and permeabilize synthetic membranes. Altogether, our results highlight the interest in rapidly characterizing the complete genome of circulating strains with next-generation sequencing technologies to adapt vaccines and identify specific virulence markers of EIV.

Full-length coding sequence analysis of genome segments A and B of infectious bursal disease virus in Thailand: identification of Chinese-like and recombinant virus in the field.

RESEARCH HIGHLIGHTS: For the first time, this work demonstrated a recombinant IBDV strain in Thailand.Two genogroups of IBDV were found in Thailand: including HLJ-504-like and recombinant virus.Analysis of the full coding sequence is essential for monitoring emerging variant IBDV.

A calf with hind limb paralysis and dysstasia and a genome sequence analysis of an isolated Clostridium perfringens toxinotype E strain.

Clostridium perfringens toxinotype E infections are rare in calves, and the development of intestinal lesions were commonly observed. In 2012, a 6-day-old calf in Japan exhibited swelling with emphysema on the right gluteal region, sudden paralysis of the hind limb and dysstasia. A pathological examination revealed myositis of the gluteal muscle and neuritis of the ischiatic nerve. C. perfringens type E strain CP118 was isolated from the affected muscle. However, the intestinal symptoms and lesions that commonly develop in type E infections in calves were not detected in the present case. Genome analyses revealed that CP118 possessed 16 virulence-related genes, including enterotoxin, and was closely related to other type E and F strains. Particularly, CP118 was more closely related to type E strains from humans, including a food poisoning case, than calf isolates, suggesting its potential to cause food poisoning in humans and, thus, its importance as a potential risk to public health. Since CP118 did not possess the reported toxin genes associated with neuropathy, pyogenic inflammation caused by CP118 and/or other bacteria may have damaged the ischiatic nerve, resulting in neuropathy. Alternatively, unidentified CP118 toxins may have caused the neuropathy. This is the first study to report C. perfringens type E infection with peripheral neuropathy. The distribution of all the reported virulence-related genes in the C. perfringens population as well as the details of this rare case will provide further insights into C. perfringens type E infections.

Detection and sequence analysis of Canine morbillivirus in multiple species of the Mustelidae family.

BACKGROUND: Canine morbillivirus (canine distemper virus, CDV) is a member of the Paramyxoviridae family. Canine distemper is a serious viral disease that affects many mammalian species, including members of the Mustelidae family. These animals have an elusive nature, which makes related virological studies extremely challenging. There is a significant knowledge gap about the evolution of their viruses and about the possible effects of these viruses to the population dynamics of the host animals. Spleen and lung tissue samples of 170 road-killed mustelids belonging to six species were collected between 1997 and 2022 throughout Hungary and tested for CDV with real-time RT-PCR. RESULTS: Three species were positive for viral RNA, 2 out of 64 Steppe polecats (Mustela eversmanii), 1 out of 36 European polecats (Mustela putorius) and 2 out of 36 stone martens (Martes foina); all 18 pine martens (Martes martes), 10 least weasels (Mustela nivalis) and 6 stoats (Mustela erminea) tested negative. The complete CDV genome was sequenced in five samples using pan-genotype CDV-specific, amplicon-based Nanopore sequencing. Based on the phylogenetic analysis, all five viral sequences were grouped to the Europe/South America 1 lineage and the distribution of one sequence among trees indicated recombination of the Hemagglutinin gene. We verified the recombination with SimPlot analysis. CONCLUSIONS: This paper provides the first CDV genome sequences from Steppe polecats and additional complete genomes from European polecats and stone martens. The infected specimens of various species originated from distinct parts of the country over a long time, indicating a wide circulation of CDV among mustelids throughout Hungary. Considering the high virulence of CDV and the presence of the virus in these animals, we highlight the importance of conservation efforts for wild mustelids. In addition, we emphasize the importance of full genomic data acquisition and analysis to better understand the evolution of the virus. Since CDV is prone to recombination, specific genomic segment analyses may provide less representative evolutionary traits than using complete genome sequences.

Multi-locus sequence analysis reveals great genetic diversity among Mycoplasma capricolum subsp. capripneumoniae strains in Asia.

Multi-Locus Sequence Analysis (MLSA) of Mycoplasma capricolum subsp. capripneumoniae (Mccp) strains from Asia revealed unforeseen diversity and a central position for genotyping groups representing strains from Central/East Asia, suggesting a possible origin of contagious caprine pleuropneumonia in this continent. A better assessment of the emergence, diversity and distribution of Mccp in Asia and Africa calls for renewed efforts to dramatically enlarge the sample of strains. Availability and affordability in the field, added to superior typeability (directly from poor samples) and high stability, discriminatory power and concordance with epidemiological and phylogenetic analyses, make MLSA an excellent tool for such investigations.

Whole genome sequence analyses-based assessment of virulence potential and antimicrobial susceptibilities and resistance of Enterococcus faecium strains isolated from commercial swine and cattle probiotic products.

Enterococcus faecium is one of the more commonly used bacterial species as a probiotic in animals. The organism, a common inhabitant of the gut of animals and humans, is a major nosocomial pathogen responsible for a variety infections in humans and sporadic infections in animals. In swine and cattle, E. faecium-based probiotic products are used for growth promotion and gut functional and health benefits. The objective of this study was to utilize whole genome sequence-based analysis to assess virulence potential, detect antimicrobial resistance genes, and analyze phylogenetic relationships of E. faecium strains from commercial swine and cattle probiotics. Genomic DNA extracted from E. faecium strains, isolated from commercial probiotic products of swine (n = 9) and cattle (n = 13), were sequenced in an Illumina MiSeq platform and analyzed. Seven of the nine swine strains and seven of the 13 cattle strains were identified as Enterococcus lactis, and not as E. faecium. None of the 22 probiotic strains carried major virulence genes required to initiate infections, but many carried genes involved in adhesion to host cells, which may benefit the probiotic strains to colonize and persist in the gut. Strains also carried genes encoding resistance to a few medically important antibiotics, which included aminoglycosides [aac(6')-Ii, aph(3')-III, ant(6)-Ia], macrolide, lincosamide and streptogramin B (msrC), tetracyclines [tet(L) and tet(M)], and phenicols [cat-(pc194)]. The comparison of the genotypic to phentypic AMR data showed presence of both related and unrelated genes in the probiotic strains. Swine and cattle probiotic E. faecium strains belonged to diverse sequence types. Phylogenetic analysis of the probiotic strains, and strains of human (n = 29), swine (n = 4), and cattle (n = 4) origin, downloaded from GenBank, indicated close clustering of strains belonging to the same species and source, but a few swine and cattle probiotic strains clustered closely with other cattle and human fecal strains. In conclusion, the absence of major virulence genes characteristic of the clinical E. faecium strains suggests that these probiotic strains are unlikely to initiate opportunistic infection. However, the carriage of AMR genes to medically important antibiotics and close clustering of the probiotic strains with other human and cattle fecal strains suggests that probiotic strains may pose risk to serve as a source of transmitting AMR genes to other gut bacteria.

Probiotics, also called direct-fed microbials, are widely used in swine and cattle production systems, as an alternative for antibiotics. The benefits of feeding probiotic products include growth promotion and gut functional benefits. One of the more common bacterial species used in swine and cattle commercial probiotic products is Enterococcus faecium. The species is also a member of the normal flora of hindgut of humans and animals. In recent years, the species has emerged as a major hospital-acquired infection in humans, mainly because of the propensity to become resistant to antibiotics. In the United States, the species is considered as generally recognized as safe. In this study, the virulence and antimicrobial resistance genes profiles of 9 and 13 E. faecium strains isolated from commercial swine and cattle probiotics, respectively, were assessed by sequencing the whole genome DNA. The analysis indicated that 14 of 22 strains were Enterococcus lactis, and not E. faecium. The absence of major virulence genes characteristic of the clinical E. faecium strains suggests that the strains are unlikely to initiate opportunistic infection. However, the carriage of genes that confer resistance to medically important antibiotics suggests that probiotic strains may pose risk as a source of antimicrobial resistance genes to other bacteria.

The Sequence Analysis of Mitochondrial DNA Revealed Some Major Centers of Horse Domestications: The Archaeologist's Cut.

The question about the time and the place of horse domestication, a process which had a profound impact on the progress of mankind, is disputable. According to the most widely accepted hypothesis, the earliest domestication of the horse happened in the western parts of the Eurasian steppes, between the Northern Black Sea region and present-day Kazakhstan and Turkmenistan. It seems that it occurred not earlier than the first half and most probably during the middle (even the last third) of the fourth millennium BC (from ∼ 5.5 kya). The next steps of large-scale horse breeding occurred almost simultaneously in Eurasia and North Africa due to the development of the social structure of human communities. On the other hand, the morphological differences between wild and domestic animals are rather vague and the genetic introgression between them is speculative. In this review, we have tried to gather all available scientific data on the existing possible hypotheses for the earliest domestication of the horse, as well as to highlight some data on the most plausible ones. This is due to the frequency of some significant data on the frequency of strictly defined mitotypes in different historical periods of human civilizations existing in the same periods.

Sequence analysis, intra-genotyping variation, and phylogenetic study of nad1 gene in Echinococcus granulosus sensu lato genotypes from intermediate hosts in southwestern Iran.

Cystic echinococcosis (CE) is caused by the larval stage of Echinococcus granulosus sensu lato (s. l.). The disease is cosmopolitan, and Iran is a highly endemic area for CE. This parasite exhibits high genetic diversity, which can be related to its life cycle, transmission, and pathogenesis. This study was aimed at determining the phylogenetic relationship and intra-genotyping variation of E. granulosus s.l. in a vast area in the southwest of Iran (SWI). Eighty hydatid cyst isolates of intermediate hosts (i.e., cattle, sheep, goat, buffalo, camel, and human) were collected. The sequence analysis of the nad1 gene exhibited the three genotypes of G1 (n = 70, 87.5%), G3 (n = 8, 10%), and G6/G7 (n = 2, 2.5%). Also, 16, 2, and 1 unique haplotypes were identified for the G1, G3, and G6/G7 genotypes, respectively. According to the phylogenetic tree topology, the nad1 gene similarities were found for some G1 isolates in some vast areas, and the G1 genotype showed a heterogeneous population worldwide. The only SWI G6/G7 haplotype was at a distant position in E. canadensis clade, indicating the notable difference of this haplotype from other isolates from Iran and other countries. The presence of the G6/G7 genotype in the SWI may be due to the transmission of the genotype from other regions or the role of camel/wild boar or other possible hosts in the expansion of this genotype in SWI. The results of the present study can be used in CE control programs, molecular epidemiology, and phylogenetic studies in Iran and other countries for future goals.

Whole-genome sequence analysis of Shiga toxin-producing Escherichia coli O157 strains isolated from wild deer and boar in Japan.

The prevalence of Shiga toxin-producing Escherichia coli O157 (STEC O157) strains in wild deer and boar in Japan was investigated. STEC O157 strains were isolated from 1.9% (9/474) of the wild deer and 0.7% (3/426) of the wild boar examined. Pulsed-field gel electrophoresis (PFGE) analysis classified the wild deer and boar strains into five and three PFGE patterns, respectively. The PFGE pattern of one wild boar strain was similar to that of a cattle strain that had been isolated from a farm in the same area the wild boar was caught, suggesting that a STEC O157 strain may have been transmitted between wild boar and cattle. Clade analysis indicated that, although most of the strains were classified in clade 12, two strains were classified in clade 7. Whole-genome sequence (WGS) analysis indicated that all the strains carried mdfA, a drug resistance gene for macrolide antibiotics, and also pathogenicity-related genes similar to those in the Sakai strain. In conclusion, our study emphasized the importance of food hygiene in processing meat from Japanese wild animals for human consumption.

Sequence analysis of the mitochondrial D-loop region throws a new light on the origin of Hungarian Nonius, Danubian Horse and Serbian Nonius.

The objective of our study was to investigate the genetic structure of yet uninvestigated populations of three closely related horse breeds - the Danubian Horse, the Hungarian Nonius and the Serbian Nonius - in order to clarify their origin and genetic diversity. A 640-bp-long fragment of the mtDNA D-loop region was amplified and sequenced. The results showed that the investigated breeds have different genetic profiles although they share some common characteristics. We identified nine of the 17 haplogroups described in modern horses. Most of the obtained sequences fall into the M, L, G, and O'P lineages, which is indicative of the genetic profile of the ancestral mares that had probably been used at the initial stages of the formation of the breeds. The population of the Danubian Horse is characterised by a high prevalence of the Anatolian specific haplogroup G (45%), followed by the Western Eurasian specific haplogroups L and M (both about 21%). In the Hungarian Nonius breed we found the highest frequency of the Western Eurasian haplogroup M (44%), followed by the Middle Eastern O'P (26%) and the Central Asian specific E (13%) and G (13%). The Serbian Nonius showed a distinct genetic profile, characterised by a high prevalence of the rare European haplogroup D (67%), followed by the Central Asian specific haplogroup G (17%). The high percentage of haplogroups shared especially between the Danubian and the Hungarian Nonius indicates the possibility of a common origin of the two breeds. In contrast, the Serbian Nonius showed a specific genetic profile, which can be explained by a different and independent origin.

Characterization of Campylobacter jejuni isolated from dogs and humans using flaA-SVR fragment sequencing in Ismailia, Egypt.

INTRODUCTION: Dogs are known as asymptomatic carriers forCampylobacter jejuni. The number of pet dogs is increasing in Egypt in the last decade. OBJECTIVE: This study aimed to investigate the frequency ofC. jejuni infection in dogs and humans, molecular typing of associated virulence genes, and flaA-SVR gene using sequencing. METHODOLOGY: 152 unpaired fecal swabs from dogs (n = 72) and humans (80) were examined for the presence of C. jejuni and Campylobacter 23S rRNA, and the pathogenicity genes including mapA genes, virB11, flaA, wlaN, iam, tetO, and aadA genes. Sequencing of the flaA- amplicon was also performed for the representative isolates. RESULTS: The isolation rate ofC. jejuni was 20.8 % and 31.2 %, respectively in dogs and humans, and all isolates were tested positive for 23S rRNA and mapA genes. C. jejuni harbor virB11 and wlaN (20 %, 0%), iam (10 %, 20 %), tetO and aadA1 (40 %, both), and flaA (40 %, 20 %) in human and dog strains, respectively. The flaA-SVR sequences revealed high identity between human and dog isolates (94.8 %), but revealed 18 substitutions in the amino acid sequence, and showed that the dog and human C. jejuni were close to strains isolated from human and poultry sources. CONCLUSION: this study demonstrated the comparative sequence analysis ofC. jejuni flaA-SVR fragment in dogs and some Egyptians, which indicated a high identity percentage between them. The results suggest that C. jejuni reservoirs dogs is an alarming public health concern and effective hygienic measures are necessary for house-holding pets to prevent C. jejuni zoonosis in Egypt's community.

The situation of echinococcosis in stray dogs in Turkey: the first finding of Echinococcus multilocularis and Echinococcus ortleppi.

Echinococcosis, caused by larval stage of the genus Echinococcus, is one of the most important zoonotic diseases worldwide. The purpose of this study was to determine the presence and prevalence of Echinococcus species in stray dogs of Erzurum, a highly endemic region for cystic echinococcosis (CE) and alveolar echinococcosis (AE) in Turkey. The study samples consisted of 446 stray dog faecal specimens collected from an animal shelter in Erzurum, Turkey, between October 2015 and February 2016. The faecal samples were collected from individual dogs for the isolation of taeniid eggs using the sequential sieving and flotation method (SSFM). Molecular analyses and sequencing revealed the prevalence of Echinococcus spp. as 14.13% (63/446) in faecal samples. The stray dogs harboured five different Echinococcus spp.: E. granulosus s.s. (G1/G3) (n = 41), E. equinus (G4) (n = 3), E. ortleppi (G5) (n = 1), E. canadensis (G6/G7) (n = 3) and E. multilocularis (n = 16). E. granulosus s.s. was the most abundant species. Surprisingly, the occurrence of E. multilocularis in dogs was revealed for the first time in Turkey. E. ortleppi was also reported for the first time in Turkey. These findings highlight a significant public health risk for human AE and CE, presenting useful baseline data on Echinococcus spp. infection in dogs for designing control strategies.

EGFR Overexpression and Sequence Analysis of KRAS, BRAF, and EGFR Mutation Hot Spots in Canine Intestinal Adenocarcinoma.

Epidermal growth factor receptor (EGFR) is overexpressed in many human colorectal cancers and anti-EGFR agents are employed as immunotherapies. However, KRAS, EGFR, and BRAF gene mutations can influence the activity of the anti-EGFR agents. We evaluated EGFR expression at protein and mRNA levels in canine intestinal adenocarcinomas using immunohistochemistry (IHC) and RNA in situ hybridization (RNA-ISH). We also investigated the mutation status of EGFR, KRAS, and BRAF to aid the development of anti-EGFR agents for canine intestinal adenocarcinoma. EGFR expression was highest in adenocarcinoma, followed by intramucosal neoplasia (adenoma and in situ carcinoma), and nonneoplastic canine intestinal tissue, at both protein (P = .000) and mRNA (P = .005) levels. The EGFR, KRAS, and BRAF genes showed wild-type sequences at the mutation hot spots in all 13 specimens. Thus, EGFR might serve as a promising diagnostic marker in canine intestinal adenocarcinoma, and further studies would be needed to develop EGFR-targeted anticancer therapies.

Development and Characterization of 12 Novel Polymorphic Microsatellite Loci for the Mammal Chewing Louse Geomydoecus aurei (Insecta: Phthiraptera) and a Comparison of Next-Generation Sequencing Approaches for Use in Parasitology.

Next-generation sequencing methodologies open the door for evolutionary studies of wildlife parasites. We used 2 next-generation sequencing approaches to discover microsatellite loci in the pocket gopher chewing louse Geomydoecus aurei for use in population genetic studies. In one approach, we sequenced a library enriched for microsatellite loci; in the other approach, we mined microsatellites from genomic sequences. Following microsatellite discovery, promising loci were tested for amplification and polymorphism in 390 louse individuals from 13 pocket gopher hosts. In total, 12 loci were selected for analysis (6 from each methodology), none of which exhibited evidence of null alleles or heterozygote deficiencies. These 12 loci showed adequate genetic diversity for population-level analyses, with 3-9 alleles per locus with an average HE per locus ranging from 0.32 to 0.70. Analysis of Molecular Variance (AMOVA) indicated that genetic variation among infrapopulations accounts for a low, but significant, percentage of the overall genetic variation, and individual louse infrapopulations showed FST values that were significantly different from zero in the majority of pairwise infrapopulation comparisons, despite all 13 infrapopulations being taken from the same locality. Therefore, these 12 polymorphic markers will be useful at the infrapopulation and population levels for future studies involving G. aurei. This study shows that next-generation sequencing methodologies can successfully be used to efficiently obtain data for a variety of evolutionary questions.

Confirmation and Sequence analysis of N gene of PPRV in South Xinjiang, China / Confirmação e análise de sequência do gene N de PPRV em Xinjiang do Sul, China

In China, Peste des petits ruminants (PPR) was officially first reported in 2007. From 2010 until the outbreak of 2013, PPRV infection was not reported. In November 2013, PPRV re-emerged in Xinjiang and rapidly spread to 22 P/A/M (provinces, autonomous regions and municipalities) of China. In the study, suspected PPRV-infected sheep in a breeding farm of South Xinjiang in 2014 were diagnosed and the characteristics of complete sequence of N protein gene of PPRV was analyzed. The sheep showed PPRV-infected signs, such as fever, orinasal secretions increase, dyspnea and diarrhea, with 60% of morbidity and 21.1% of fatality rate. The macroscopic lesions after autopsy and histopathological changes were observed under light microscope including stomatitis, broncho-interstitial pneumonia, catarrhal hemorrhagic enteritis and intracytoplasmic eosinophilic inclusions in multinucleated giantcell in lung. The formalin-fixed mixed tissues samples were positive by nucleic acid extraction and RT-PCR detection. The nucleotide of N protein gene of China/XJNJ/2014 strain was extremely high homology with the China/XJYL/2013 strain, and the highest with PRADESH_95 strain from India in exotic strains. Phylogenetic analysis based on complete sequence of N protein gene of PPRV showed that the China/XJNJ/2014 strain, other strain of 2013-2014 in this study and Tibetan strains all belonged to lineage Ⅳ, but the PPRV strains of 2013-2014 in this study and Tibetan strains were in different sub-branches.(AU)

Na China, Peste des petits ruminants (PPR) foi relatado oficialmente em 2007. De 2010 até o surto de 2013, não houve relato de infecção por PPRV. Em Novembro de 2013, PPRV ressurgiu em Xinjiang e rapidamente se espalhou para 22 P/A/M (províncias, regiões autônomas e municípios) da China. No estudo, ovelhas com suspeita de infecção por PPRV em uma fazenda de reprodução no sul de Xinjiang form diagnosticadas em 2014 e as características da sequência completa da proteína N do gene do PPRV foi analisada. As ovelhas tinham sinais de infecção pelo PPRV, como febre, aumento de secreções oro-nasais, dispneia e diarreia, com 60% de morbidade e 21.1% de fatalidade. As lesões macroscópicas após mudanças histopatológicas foram observadas sob microscópio, incluindo estomatite, pneumonia bronco-intersticial, enterite hemorrágica catarral e inclusões eosinofílicas intracitoplasmáticas em células gigantes multinucleares no pulmão. As amostras de tecido fixadas em formalina testaram positivo para detecção de RT-PCR por extração de ácido nucleico. Os nucleotídeos da proteína N do gene da linhagem China/XJNJ/2014 apresentou extrema homologia com o China/XJYL/2013, e homologia ainda maior com a variedade PRADESH-95 da Índia. Análise filogenética baseada na sequencia completa da proteína N do gene de PPRV mostrou que as variedades China/XJNJ/2014, outra de 2013-2014 mostrada nesse estudo e as Tibetanas todas pertenciam à linhagem Ⅳ, mas as PPRV de 2013-2014 nesse estudo e as Tibetanas estavam em diferentes agrupamentos.(AU)

Glycoprotein-G-gene-based molecular and phylogenetic analysis of rabies viruses associated with a large outbreak of bovine rabies in southern Brazil

A large outbreak of hematophagous-bat-associated bovine rabies has been occurring in Rio Grande do Sul (RS), the southernmost Brazilian state, since 2011, with official estimates exceeding 50,000 cattle deaths. The present article describes a genetic characterization of rabies virus (RABV) recovered from 59 affected cattle and two sheep, from 56 herds in 16 municipalities (2012-2016). Molecular analysis was performed using the nucleotide (nt) and predicted amino acid (aa) sequences of RABV glycoprotein G (G). A high level of nt and aa sequence identity was observed among the examined G sequences, ranging from 98.4 to 100%, and from 97.3 to 100%, respectively. Likewise, high levels of nt and aa sequence identity were observed with bovine (nt, 99.8%; aa, 99.8%) and hematophagous bat (nt, 99.5%; aa, 99.4%) RABV sequences from GenBank, and lower levels were observed with carnivore RABV sequences (nt, 92.8%; aa, 88.1%). Some random mutations were observed in the analyzed sequences, and a few consistent mutations were observed in some sequences belonging to cluster 2, subcluster 2b. The clustering of the sequences was observed in a phylogenetic tree, where two distinct clusters were evident. Cluster 1 comprised RABV sequences covering the entire study period (2012 to 2016), but subclusters corresponding to different years could be identified, indicating virus evolution and/or introduction of new viruses into the population. In some cases, viruses from the same location obtained within a short period grouped into different subclusters, suggesting co-circulation of viruses of different origins. Subcluster segregation was also observed in sequences obtained in the same region during different periods, indicating the involvement of different viruses in the cases at different times. In summary, our results indicate that the outbreaks occurring in RS (2012 to 2016) probably involved RABV of different origins, in addition to a possible evolution of RABV isolates within this period. (AU) i

The Prevalence of Canine Oral Protozoa and Their Association with Periodontal Disease.

Periodontal disease is one of the most important health concerns for companion animals. Research into canine forms of periodontitis has focused on the identification and characterization of the bacterial communities present. However, other microorganisms are known to inhabit the oral cavity and could also influence the disease process. A novel, broad spectrum 18S PCR was developed and used, in conjunction with next-generation sequencing analyses to target the identification of protists. Trichomonas sp. and Entamoeba sp. were identified from 92 samples of canine plaque. The overall prevalence of trichomonads was 56.52% (52/92) and entamoebae was 4.34% (4/92). Next-generation sequencing of pooled healthy, gingivitis, early-stage periodontitis, and severe periodontitis samples revealed the proportion of trichomonad sequences to be 3.51% (health), 2.84% (gingivitis), 6.07% (early periodontitis), and 35.04% (severe periodontitis), respectively, and entamoebae to be 0.01% (health), 0.01% (gingivitis), 0.80% (early-stage periodontitis), and 7.91% (severe periodontitis) respectively. Both genera of protists were statistically associated with plaque from dogs with periodontal disease. These findings provide the first conclusive evidence for the presence of oral protozoa in dog plaque and suggest a possible role for protozoa in the periodontal disease process.

Reducing animal sequencing redundancy by preferentially selecting animals with low-frequency haplotypes.

Many studies leverage targeted whole-genome sequencing (WGS) experiments to identify rare and causal variants within populations. As a natural consequence of their experimental design, many of these surveys tend to sequence redundant haplotype segments due to their high frequency in the base population, and the variants discovered within sequencing data are difficult to phase. We propose a new algorithm, called inverse weight selection (IWS), that preferentially selects individuals based on the cumulative presence of rare frequency haplotypes to maximize the efficiency of WGS surveys. To test the efficacy of this method, we used genotype data from 112,113 registered Holstein bulls derived from the US national dairy database. We demonstrate that IWS is at least 6.8% more efficient than previously published methods in selecting the least number of individuals required to sequence all haplotype segments ≥4% frequency in the US Holstein population. We also suggest that future surveys focus on sequencing homozygous haplotype segments as a first pass to achieve a 50% reduction in cost with an added benefit of phasing variant calls efficiently. Together, this new selection algorithm and experimental design suggestion significantly reduce the overall cost of variant discovery through WGS experiments, making surveys for causal variants influencing disease and production even more efficient.

Astaxanthin Normalizes Epigenetic Modifications of Bovine Somatic Cell Cloned Embryos and Decreases the Generation of Lipid Peroxidation.

Astaxanthin is an extremely common antioxidant scavenging reactive oxygen species (ROS) and blocking lipid peroxidation. This study was conducted to investigate the effects of astaxanthin supplementation on oocyte maturation, and development of bovine somatic cell nuclear transfer (SCNT) embryos. Cumulus-oocyte complexes were cultured in maturation medium with astaxanthin (0, 0.5, 1.0, or 1.5 mg/l), respectively. We found that 0.5 mg/l astaxanthin supplementation significantly increased the proportion of oocyte maturation. Oocytes cultured in 0.5 mg/l astaxanthin supplementation were used to construct SCNT embryos and further cultured with 0, 0.5, 1.0 or 1.5 mg/l astaxanthin. The results showed that the supplementation of 0.5 mg/l astaxanthin significantly improved the proportions of cleavage and blastulation, as well as the total cell number in blastocysts compared with the control group, yet this influence was not concentration dependent. Chromosomal analyses revealed that more blastomeres showed a normal chromosomal complement in 0.5 mg/l astaxanthin treatment group, which was similar to that in IVF embryos. The methylation levels located on the exon 1 of the imprinted gene H19 and IGF2, pluripotent gene OCT4 were normalized, and global DNA methylation, H3K9 and H4K12 acetylation were also improved significantly, which was comparable to that in vitro fertilization (IVF) embryos. Moreover, we also found that astaxanthin supplementation significantly decreased the level of lipid peroxidation. Our findings showed that the supplementation of 0.5 mg/l astaxanthin to oocyte maturation medium and embryo culture medium improved oocyte maturation, SCNT embryo development, increased chromosomal stability and normalized the epigenetic modifications, as well as inhibited overproduction of lipid peroxidation.

Epidemiology and molecular characterisation of duck hepatitis A virus from different duck breeds in Egypt.

Duck hepatitis virus (DHV) is an acute highly contagious disease of ducklings caused by three distinct serotypes of duck hepatitis A virus (DHAV), a member of the RNA family Picornaviridae, where serotype 1 is the most widespread serotype worldwide. To date, little if any is known about the prevalence and genetic characterisation of DHAV outside Asia. The current study describes surveillance on DHV in 46 commercial duck farms in Egypt with a history of high mortality in young ducklings from 3 to 15 day-old from 2012 to 2014. Clinical samples were examined by generic RT-PCR assays followed by partial sequence analysis of the 5'UTR, VP1 and 3D genes of the vaccine strain and 15 field viruses. The overall positive rate was 37% (n=17/46). All duck breeds (Pekin, Muscovy, Mallard and Green Winged) were susceptible to the disease with mortality ranged from 15% to 96.7%. Sequence and phylogenetic analyses indicated that the Egyptian strains cluster in the DHAV serotype 1 with Asian viruses and distinguishable from the vaccine strains. So far, this is the first report on the genetic characterisation of DHAV in Egypt. This study may be useful to better understand the epidemiology and evolution of DHAV.