Search

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., (© 2024. The Author(s).)

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.

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.

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., (© 2022. The Author(s).)

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., (© 2022. The Author(s).)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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.

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.

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 H E 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 F ST 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.

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., (© 2016 The Author(s) The Journal of Eukaryotic Microbiology published by Wiley Periodicals, Inc. on behalf of International Society of Protistologists.)

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., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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., (© 2015 Blackwell Verlag GmbH.)

Unlabelled: Canine mammary gland tumour (MGT) is the most common neoplasm in female dogs and has similar biological characteristics to human MGT. Spontaneous canine MGT is a more attractive clinical model in oncological research than that of the murine experimental model. Tumour-associated antigens (TAAs), which are produced in tumour cells, are applied as tumour markers, tumour vaccine antigens and molecular targets of therapeutic drugs. In this study, we have primarily identified 13 different TAAs of canine MGT by serological immunoscreening of cDNA expression library. The results of serological mini-arrays of identified antigens showed that CCDC41 antigen specially reacted with 35% of sera from MGT-dogs and did not react with control sera. We also found that HSPH1 mRNA expression levels increased significantly in MGT tissues. These findings will contribute to the development of diagnostic technologies and translational target therapies for dogs., Clinical Relevance: HSPH1, which is strongly expressed in the tumour tissue, will be a possible vaccine antigen of canine MGT., (© 2013 Blackwell Publishing Ltd.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

GnRH receptors play vital roles in mammalian reproduction via regulation of gonadotropin secretion, which is essential for gametogenesis and production of gonadal steroids. GnRH receptors for more than 20 mammalian species have been sequenced, including human, mouse, and dog. This study reports the molecular cloning and sequencing of GnRH receptor (GnRHR) cDNA from the pituitary gland of the domestic cat, an important species in biomedical research. Feline GnRHR cDNA is composed of 981 nucleotides and encodes a 327 amino acid protein. Unlike the majority of mammalian species sequenced so far, but similar to canine GnRHR, feline GnRHR protein lacks asparagine in position three of the extracellular domain of the protein. At the amino acid level, feline GnRHR exhibits 95.1% identity with canine, 93.8% with human, and 88.9% with mouse GnRHR. Comparative sequence analysis of GnRHRs for multiple mammalian species led to resequencing of canine GnRHR, which differed from that previously published by a single base change that translates to a different amino acid in position 193. This single base change was confirmed in dogs of multiple breeds. Reverse transcriptase PCR analysis of GnRHR messenger RNA in different tissues from four normal cats indicated the presence of amplicons of varying lengths, including full-length as well as shortened GnRHR amplicons, pointing to the existence of truncated GnRHR transcripts in the domestic cat. This study is the first insight into molecular composition and expression of feline GnRHR and promotes better understanding of receptor organization, and distribution in various tissues of this species., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Group A Rotaviruses (RVA) have been established as significant contributory agents of acute gastroenteritis in young children and many animal species. In 2008, we described the first RVA strain detected in a giraffe calf (RVA/Giraffe-wt/IRL/GirRV/2008/G10P[11]), presenting with acute diarrhoea. Molecular characterisation of the VP7 and VP4 genes revealed the bovine-like genotypes G10 and P[11], respectively. To further investigate the origin of this giraffe RVA strain, the 9 remaining gene segments were sequenced and analysed, revealing the following genotype constellation: G10-P[11]-I2-R2-C2-M2-A3-N2-T6-E2-H3. This genotype constellation is very similar to RVA strains isolated from cattle or other members of the artiodactyls. Phylogenetic analyses confirmed the close relationship between GirRV and RVA strains with a bovine-like genotype constellation detected from several host species, including humans. These results suggest that RVA strain GirRV was the result of an interspecies transmission from a bovine host to the giraffe calf. However, we cannot rule out completely that this bovine-like RVA genotype constellation may be enzootic in giraffes. Future RVA surveillance in giraffes may answer this intriguing question., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Echinococcus granulosus (EG) complex, the cause of cystic echinococcosis (CE), infects humans and several other animal species worldwide and hence the disease is of public health importance. Ten genetic variants, or genotypes designated as (G1-G10), are distributed worldwide based on genetic diversity. The objective of this study was to provide some sequence data and phylogeny of EG isolates recovered from the Sudanese one-humped camel (Camelus dromedaries). Fifty samples of hydatid cysts were collected from the one- humped camels (Camelus dromedaries) at Taboul slaughter house, central Sudan. DNAs were extracted from protoscolices and/or associated germinal layers of hydatid cysts using a commercial kit. The mitochondrial NADH dehydrogenase subunit 1 (NADH1) gene and the cytochrome C oxidase subunit 1 (cox1) gene were used as targets for polymerase chain reaction (PCR) amplification. The PCR products were purified and partial sequences were generated. Sequences were further examined by sequence analysis and subsequent phylogeny to compare these sequences to those from known strains of EG circulating globally., Results: The identity of the PCR products were confirmed as NADH1 and cox1 nucleotide sequences using the Basic Local Alignment Search Tool (BLAST) of NCBI (National Center for Biotechnology Information, Bethesda, MD). The phylogenetic analysis showed that 98% (n = 49) of the isolates clustered with Echinococcus canadensis genotype 6 (G6), whereas only one isolate (2%) clustered with Echinococcus ortleppi (G5)., Conclusions: This investigation expands on the existing sequence data generated from EG isolates recovered from camel in the Sudan. The circulation of the cattle genotype (G5) in the one-humped camel is reported here for the first time.

Edwardsiella tarda is an enteric fish pathogen that has caused significant economic losses in a range of fish species residing in diverse ecological conditions. Several molecular methods relying on DNA fingerprinting (RAPD, RFLP and ERIC-PCR) and the gyrB gene marker have been used to characterize E. tarda isolates. However, all had drawbacks in resolving power and reproducibility. The present study was aimed at developing a novel Multi-locus Sequence Analysis (MLSA) scheme for genetic characterization of E. tarda isolates originating from multiple sources. MLSA has been described as an effective molecular tool with superior discriminatory power and reproducibility for exploring intra-species genetic diversity of several bacterial species. Nucleotide sequence fragments of eight protein coding housekeeping genes (gyrB, mdh, adk, dnaK, phoR, metG, pyrG and aroE2) were obtained from 23 fish pathogenic E. tarda isolates of different geographical origins, one human isolate and 3 reference strains. The phylogenetic relationships between isolates in individual gene analyses were not consistent, although some common patterns were apparent. Phylogenetic analysis based on concatenated sequences of seven gene loci, however, buffered the conflicting phylogenetic signals and resolved isolates according to their geographical origin and/or fish host. The MLSA revealed two major genetically diverging clusters in E. tarda isolates examined, one cluster representing isolates from fish and the other representing (in the main) human isolates, with E. ictaluri cluster situated in between. The results suggest, therefore, that the fish pathogenic E. tarda isolates may have been previously misclassified and probably represent one or more as yet unrecognized taxa within the genus Edwardsiella. The MLSA described here was robust enough in discriminating E. tarda isolates not only with respect to their geographical origins but also within different hosts from the same geographical location, high-lighting its potential application in tracing the source of infection and understand the epidemiological relationships among isolates of environmental, fish, other domestic animals or human origins., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Angiotensinogen (AGT) is the precursor of the renin-angiotensin system and contributes to osmoregulation, acute-phase and immune responses. A full-length cDNA of the AGT (2004 bp with a 1389 bp coding region) was isolated from rock bream (Rb), Oplegnathus fasciatus. The encoded polypeptide of 463 amino acids had a predicted molecular mass of 51.6 kDa. RbAGT possessed a deduced signal peptide of 22 residues upstream of a putative angiotensin I sequence ((23)NRVYVHPFHL(32)). RbAGT possessed a specific domain profile and a signature motif which are characteristics of the serpin family. Sequence homology and phylogenetic analysis indicated that RbAGT was evolutionarily closest to AGT of Rhabdosargus sarba. The mRNA expression profile of RbAGT was determined by quantitative RT-PCR and it demonstrated a constitutive and tissue-specific expression with the highest transcript level in the liver. Significantly up-regulated RbAGT expression was elicited by systemic injection of a lipopolysaccharide, rock bream iridovirus (RBIV) and bacteria (Edwardsiella tarda and Streptococcus iniae), revealing its pathogen inducibility. RbAGT manifested a down-regulated response to systemic injury, contemporaneously with two other serpins, protease nexin-1 (PN-1), and heparin cofactor II (HCII). In addition, a synchronized expression pattern was elicited by RbAGT and RbTNF-α in response to injury, suggesting that TNF-α might be a potential modulator of AGT transcription., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Caseous lymphadenitis, caused by Corynebacterium pseudotuberculosis, has a high prevalence in many regions of the world, including Argentina and Brazil. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for the identification of this microorganism was designed based on the hypervariable region of the polymorphic RNA polymerase β-subunit gene (rpoB). All available CorynebacteriumrpoB sequences were analyzed by computer-assisted restriction analysis. The rpoB PCR-RFLP pattern predicted by using endonucleases MseI and StuI clearly differentiated C. pseudotuberculosis from sixty-one other Corynebacterium species. This method was successfully applied to identify twelve wild C. pseudotuberculosis ovine isolates and one caprine isolate. It was also used to differentiate C. pseudotuberculosis from Arcanobacterium pyogenes, an ovine pathogen with similar clinical characteristics. These results indicate that this new molecular method can be used for the reliable identification of the pathogen, essential for the timely detection of infected animals and for epidemiological studies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The incidence of canine mammary tumours (CMTs) differs significantly between breeds, strongly supporting an influence of genetic risk factors. We aimed at identifying germline genetic variations in mammary tumour-associated genes in dogs and survey whether these might alter the encoded proteins. We sequenced 11 genes (BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EGFR, ESR1, HER2, PTEN, STK11 and TP53) and screened for genetic variations. Sixty-four single nucleotide polymorphisms (SNPs) were identified. Nine of the coding SNPs were non-synonymous, of which four were located in gene regions conserved across four species. Three of the non-synonymous SNPs might be damaging according to PolyPhen predictions. One of the indels identified has previously been associated with CMTs. Because of the founder effects, genetic drift and inbreeding in many dog breeds the allele frequencies of the genes studied are likely to vary significantly between breeds and contribute to the considerable difference in genetic risk associated with cancer., (© 2011 Blackwell Publishing Ltd.)

Three Haliotrema spp. are reported from the Vietnamese grouper. Morphological and morphometric characters show minor deviations from original descriptions of H. cromileptis Young, 1968 and H. epinepheli Young, 1968 . The third encountered species ( Haliotrema sp.) appears to be new to science. Genetically, H. cromileptis clusters with Bravohollisia , Pseudohaliotrema , and Haliotrema . The group is well supported by partial large subunit rDNA (LSU), complete small subunit rDNA (SSU), and partial SSU + ITS1 rDNA analyses. Ingroup phylogenetic relationships are not well resolved. Haliotrema cromileptis , H. fleti Young, 1968 , and Pseudohaliotrema sphincteroporus Yamaguti, 1953 are closely related to a monophyletic group of 5 Haliotrema spp. characterized by bell- or horn-shaped bases of the male copulatory organ (MCO), which contains an accessory piece. Based on SSU rDNA, H. cromileptis is a sister species to P. sphincteroporus and, together, they form a clade to 3 other Haliotrema spp. characterized by a bell-shape based MCO with an accessory piece. Data analysis conducted on partial SSU + ITS1 rDNA confirms the close phylogenetic relationship of H. cromileptis , H. fleti , H. chenhsintaoi Zhang, 2001 (possessing a horn-shaped base of the MCO), and Bravohollisia rosetta Lin, 1995. However, because major differences in diagnostic characters exist, this genetic relationship needs further elucidation.

1. Spot 14 alpha acts as a transcription factor involved in the regulation of adipogenic enzymes via three thyroid response elements in its promoter region. The objective of the current research was to clone and sequence the Spot 14 alpha gene in geese. 2. We cloned the cDNA sequence of goose Spot 14 alpha. The gene was predicted to encode a peptide of 128 amino acids, which has sequence identities of 87% cDNA and 84% amino acids, with the duck counterparts. High percentages of G and C nucleotides were found in exon and 3' untranslated region of the goose Spot 14 alpha cDNA. 3. A novel frameshift mutation that leads to a damaged leucine zipper motif was observed at nucleotide position 399-400. This can influence the homodimerisation of Spot 14 alpha, probably resulting in dysfunction in the Spot 14 family in vivo. 4. Phylogenetic analysis revealed that goose and duck Spot 14 alpha form a monophyletic group. The Spot 14 alpha mRNA was highly expressed in the liver and adipose tissue of geese. The mRNA concentration and polymorphism of Spot 14 alpha in the lipogenic tissues of geese were related to the fatness trait.

This report describes the first recognised case of Angiostrongylus vasorum infection in a native Scottish dog and its diagnostic confirmation using polymerase chain reaction. The use of this technique to definitively diagnose canine angiostrongylosis confirms the presence of this epidemiologically significant case. The implications of the changing distribution of this parasite are discussed.

P-glycoprotein (P-gp) is an important drug transporter, which is expressed in a variety of cells, such as the intestinal enterocytes, the hepatocytes, the renal tubular cells and the intestinal and peripheral blood lymphocytes. We have studied the localization and the gene and protein expression of P-gp in these cells in horse. In addition we have compared the protein sequence of P-gp in horse with the protein sequences of P-gp in several other species. Real time RT-PCR and Western blot showed gene and protein expression of horse P-gp in all parts of the intestines, but there was no strict correlation between these parameters. Immunohistochemistry showed localization of P-gp in the apical cell membranes of the enterocytes and, in addition, staining was observed in the intestinal intraepithelial and lamina propria lymphocytes. Peripheral blood lymphocytes also stained for P-gp, and gene and protein expression of P-gp were observed in these cells. There was a high gene and protein expression of P-gp in the liver, with P-gp-immunoreactivity in the bile canalicular membranes of the hepatocytes. Gene and protein expression of P-gp were found in the kidney with localization of the protein in different parts of the nephrons. Protein sequence alignment showed that horse P-gp has two amino acid insertions at the N-terminal region of the protein, which are not present in several other species examined. One of these is a 99 amino acid long sequence inserted at amino acid positions 23-121 from the N-terminal. The other is a six amino acid long sequence present at the amino acid positions 140-145 from the N-terminal. The results of the present study indicate that P-gp has an important function for oral bioavailability, distribution and excretion of substrate compounds in horse.

Expression of prophenoloxidase (proPO) cDNA was determined from haemocytes of the giant freshwater prawn Macrobrachium rosenbergii by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA using oligonucleotide primers based on the proPO sequence of tiger shrimp Penaeus monodon, freshwater crayfish Pacifastacus leniusculus, green tiger shrimp Penaeus semisulcatus, kuruma shrimp Marsupenaeus japonicus, and white shrimp Litopenaeus vannamei. The proPO of M. rosenbergii was constitutively expressed. The 2,547-bp cDNA contained an open reading frame (ORF) of 2,013 bp, a 96-bp 5'-untranslated region, and a 438-bp 3'-untranslated region containing the poly A tail. The molecular mass of the deduced amino acid (aa) sequence (671 aa) was 76.7 kDa with an estimated pI of 7.05. It contained putative copper-binding sites, a complement-like motif (GCGWPRHM), a proteolytic activation site, and a conserved C-terminal region common to all known proPOs. However, no signal peptide sequence was detected in giant freshwater prawn proPO. Comparison of amino acid sequences showed that prawn proPO is similar to the proPO of penaeid, crayfish and lobster. Prawn proPO was only synthesised in haemocytes. The proPO transcript was significantly increased in the A stage and achieved the highest level in the B stage, and then declined sharply in the C stage and reached the lowest level in the D(2)/D(3) stage.

Infectious hematopoietic necrosis virus (IHNV), an aquatic rhabdovirus, causes a highly lethal disease of salmonid fish in North America. To evaluate the genetic diversity of IHNV from throughout the Columbia River basin, excluding the Hagerman Valley, Idaho, the sequences of a 303 nt region of the glycoprotein gene (mid-G) of 120 virus isolates were determined. Sequence comparisons revealed 30 different sequence types, with a maximum nucleotide diversity of 7.3% (22 mismatches) and an intrapopulational nucleotide diversity of 0.018. This indicates that the genetic diversity of IHNV within the Columbia River basin is 3-fold higher than in Alaska, but 2-fold lower than in the Hagerman Valley, Idaho. Phylogenetic analyses separated the Columbia River basin IHNV isolates into 2 major clades, designated U and M. The 2 clades geographically overlapped within the lower Columbia River basin and in the lower Snake River and tributaries, while the upper Columbia River basin had only U clade and the upper Snake River basin had only M clade virus types. These results suggest that there are co-circulating lineages of IHNV present within specific areas of the Columbia River basin. The epidemiological significance of these findings provided insight into viral traffic patterns exhibited by IHNV in the Columbia River basin, with specific relevance to how the Columbia River basin IHNV types were related to those in the Hagerman Valley. These analyses indicate that there have likely been 2 historical events in which Hagerman Valley IHNV types were introduced and became established in the lower Columbia River basin. However, the data also clearly indicates that the Hagerman Valley is not a continuous source of waterborne virus infecting salmonid stocks downstream.

Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus which infects salmon and trout and may cause disease with up to 90% mortality. In the Hagerman Valley of Idaho, IHNV is endemic or epidemic among numerous fish farms and resource mitigation hatcheries. A previous study characterizing the genetic diversity among 84 IHNV isolates at 4 virus-endemic rainbow trout farms indicated that multiple lineages of relatively high diversity co-circulated at these facilities (Troyer et al. 2000 J Gen Virol. 81:2823-2832). We tested the hypothesis that high IHNV genetic diversity and co-circulating lineages are present in aquaculture facilities throughout this region. In this study, 73 virus isolates from 14 rainbow trout farms and 3 state hatcheries in the Hagerman Valley, isolated between 1978 and 1999, were genetically characterized by sequence analysis of a 303 nucleotide region of the glycoprotein gene. Phylogenetic and epidemiological analyses showed that multiple IHNV lineages co-circulate in a complex pattern throughout private trout farms and state hatcheries in the valley. IHNV maintained within the valley appears to have evolved significantly over the 22 yr study period.

The evolutionary success of arthropods has been attributed partly to the diversity of their limb morphologies. Large morphological diversity and increased specialization are observed in water flea (Cladocera) limbs, but it is unclear whether the increased limb specialization in different cladoceran orders is the result of shared ancestry or parallel evolution. We inferred a robust among-order cladoceran phylogeny using small-subunit and large-subunit rRNA nuclear gene sequences, signature sequence regions, novel stem-loops and secondary structure morphometrics to assess the phylogenetic distribution of limb specialization. The sequence-based and structural rRNA morphometric phylogenies were congruent and suggested monophyly of orders with raptorial limbs, but paraphyly of orders with reduced numbers of specialized limbs. These results highlight the utility of complex molecular structural characters in resolving ancient rapid radiations.

The spike glycoprotein of infectious bronchitis virus (IBV), a coronavirus, is translated as a precursor protein (So), then cleaved into two subunits (S1 and S2) by host cell serine proteases. In this study, we compared the cleavage recognition site of 55 IBV isolates to determine if the cleavage recognition site sequence, which consists of five basic amino acid residues, correlates with host cell range, serotype, geographic origin, and pathogenicity as it does in orthomyxoviruses and paramyxoviruses. The most common cleavage recognition site observed (33 of 55 viruses) was Arg-Arg-Ser-Arg-Arg, representing at least 11 different serotypes. Thus, cleavage recognition site does not appear to correlate with serotype. We also determined that cleavage recognition site sequence does not correlate with pathogenicity because attenuated and pathogenic isolates (different passages of the same virus) contain identical cleavage recognition site sequences. In addition, nephropathogenic strains had the same cleavage recognition site sequence as many nonnephropathogenic isolates. Cleavage recognition site sequence does correlate with viruses in different geographic regions, which may be an important characteristic to examine in epidemiologic studies. An IBV monoclonal antibody neutralization-resistant mutant (NR 18) had an unusual substitution of Ile for Arg at the fourth position, giving the sequence Arg-Arg-Ser-Ile-Arg, which likely prevents cleavage and, thus, destroys the conformationally dependent monoclonal antibody binding epitope. Six residues on the amino-terminal side of the cleavage recognition site are conserved in 31% of the isolates and consist of only one or two basic amino acids. Thus, the number of basic residues around the cleavage recognition site does not appear to correlate with increased cleavability, host cell range, and increased virulence as it does with envelope glycoproteins in orthomyxoviruses and paramyxoviruses.

Objective: To determine stability of the restriction fragment length polymorphism (RFLP) pattern of a porcine reproductive and respiratory syndrome vaccine virus and patterns of other viral strains as they replicate in pigs., Sample Population: Field samples of porcine reproductive and respiratory syndrome virus (PRRSV) and samples from 2 weaned pigs, 2 nursery-age pigs, and 5 gilts experimentally infected with PRRSV., Procedure: PRRSV was isolated from field samples, experimentally infected pigs, or pigs that were in contact with experimentally infected pigs. For each virus, RNA was isolated from infected cells, and RFLP patterns were determined., Results: 61% of field samples had 2-5-2 RFLP patterns characteristic of the vaccine virus, 32% had field virus RFLP patterns, and 7% had intermediate RFLP patterns that indicated a virus with a close relationship to the vaccine virus. Viruses isolated from experimentally infected pigs had no change in RFLP patterns after up to 13 weeks of in vivo replication and transmission to contact pigs., Conclusions and Clinical Relevance: RFLP patterns distinguish the vaccine and field strains of PRRSV; however, as the vaccine virus spreads among a swine population, the RFLP pattern can change to a related intermediate pattern. A glycine at residue 151 of open reading frame 5 is another marker for the vaccine virus; this glycine is rapidly lost and eventually replaced with arginine as the vaccine virus replicates in pigs.

A polymerase chain reaction (PCR) assay was developed for the detection of avian leukosis virus strain J (ALV-J) in chickens. Primers were based in the E element and the 3' terminus of the long terminal repeat of proviral ALV-J. PCR products were amplified from genomic DNA extracted from chicken embryo fibroblasts (CEF) infected with either strain HPRS-103, the prototype of ALV-J, or field isolates of ALV-J obtained from broiler breeder flocks in the United States that exhibited myeloid leukosis. The newly developed PCR detected ALV-J in DNA prepared from CEF inoculated with ALV-J but not from CEF inoculated with subgroup A, B, C, D, or E. The PCR also detected ALV-J in DNA prepared from blood, combs, and toes obtained from chickens experimentally infected with ALV-J and in DNA obtained from peripheral blood monocytes from naturally infected broiler breeder chickens. The PCR described here offers a specific and sensitive alternative to conventional virus isolation tests for ALV-J.