Risk factors associated with increased mortality of farmed Pacific oysters in Ireland during 2011

The Pacific oyster, Crassostrea gigas, plays a significant role in the aquaculture industry in Ireland. Episodes of increased mortality in C. gigas have been described in many countries, and in Ireland since 2008. The cause of mortality events in C. gigas spat and larvae is suspected to be multifactorial, with ostreid herpesvirus 1 (OsHV-1, in particular OsHV-1 μvar) considered a necessary, but not sufficient, cause. The objectives of the current study were to describe mortality events that occurred in C. gigas in Ireland during the summer of 2011 and to identify any associated environmental, husbandry and oyster endogenous factors. A prospective cohort study was conducted during 2010–2012, involving 80 study batches, located at 24 sites within 17 bays. All 17 bays had previously tested positive for OsHV-1 μvar. All study farmers were initially surveyed to gather relevant data on each study batch, which was then tracked from placement in the bay to first grading. The outcome of interest was cumulative batch-level mortality (%). Environmental data at high and low mortality sites were compared, and a risk factor analysis, using a multiple linear regression mixed effects model, was conducted. Cumulative batch mortality ranged from 2% to 100% (median = 16%, interquartile range: 10–34%). The final multivariable risk factor model indicated that batches imported from French hatcheries had significantly lower mortalities than non-French hatcheries; sites which tested negative for OsHV-1 μvar during the study had significantly lower mortalities than sites which tested positive and mortalities increased with temperature until a peak was reached. There were several differences between the seed stocks from French and non-French hatcheries, including prior OsHV-1 μvar exposure and ploidy. A range of risk factors relating to farm management were also considered, but were not found significant. The relative importance of prior OsHV-1 μvar infection and ploidy will become clearer with ongoing selection towards OsHV-1 μvar resistant oysters. Work is currently underway in Ireland to investigate these factors further, by tracking seed from various hatchery sources which were put to sea in 2012 under similar husbandry and environmental conditions.     

Experimental infection of Pacific oyster Crassostrea gigas spat by ostreid herpesvirus 1: demonstration of oyster spat susceptibility

ABSTRACT: In 2008 and 2009, acute mortalities occurred in France among Pacific cupped oyster, Crassostrea gigas, spat. Different hypothesis including the implication of environmental factors, toxic algae and/or pathogens have been explored. Diagnostic tests indicated that OsHV-1 including a particular genotype, termed OsHV-1 μVar, was detected in most of samples and especially in moribund oysters with the highlighting of virus particles looking like herpes viruses by TEM examination. In this study, an experimental protocol to reproduce OsHV-1 infection in laboratory conditions was developed. This protocol was based on the intramuscular injection of filtered (0.22 μm) tissue homogenates prepared from naturally OsHV-1 infected spat collected on French coasts during mortality outbreaks in 2008. Results of the experimental trials showed that mortalities were induced after injection. Moreover, filtered tissue homogenates induced mortalities whereas the same tissue homogenates exposed to an ultraviolet (UV) treatment did not induce any mortality suggesting that oyster spat mortalities require the presence of a UV sensitive agent. Furthermore, analysis of injected oyster spat revealed the detection of high amounts of OsHV-1 DNA by real-time quantitative PCR. Finally, TEM analysis demonstrated the presence of herpes virus particles. The developed protocol allowed to maintain sources of infective virus which can be useful for the development of further studies concerning the transmission and the development of OsHV-1 infection.     

Ostreid herpesvirus 1 detection and relationship with Crassostrea gigas spat mortality in France between 1998 and 2006

ABSTRACT: Since its molecular characterisation, Ostreid herpesvirus 1 (OsHV-1) has been regularly detected in Crassostrea gigas in France. Although its pathogenicity was demonstrated on larval stages, its involvement during mortality outbreaks at the juvenile stage was highly suspected but not evidenced. To investigate mortality outbreaks, the French National Network for Surveillance and Monitoring of Mollusc Health (REPAMO) carried out two surveys in juvenile C. gigas. The first survey lasted from 1998 to 2006 and was an epidemiological inquiry occurring when oyster farmers reported mortality outbreaks. The second survey, a longitudinal one, was set up in 1998 to complete the network observations on OsHV-1. Data analysis showed a specific pattern of mortality outbreaks associated with OsHV-1 detection. Ostreid herpesvirus 1 detection mainly appeared during the summer, suggesting the influence of the seawater temperature on its occurrence. It mostly presented a patchy distribution in the field in contrast to the nursery. Significant relationship between OsHV-1 detection and spat mortality was found, preferentially in sheltered and closed environments. The longitudinal survey confirmed most of the network observations. Although subsequent works particularly epidemiological surveys would be useful to confirm the causal link between the detection of OsHV-1 and the mortality outbreaks in juvenile C. gigas, the role of OsHV-1 in oyster mortality is progressing.     

Isolation of microsatellite loci and reliable genotyping using noninvasive samples of a critically endangered primate,Trachypithecus leucocephalus

Genetic information can be critical in identifying conservation priorities and developing conservation strategies. There is an urgent need for noninvasive genetic tools to study the wild populations of Asian colobine monkeys. The majority of these species are threatened with habitat destruction, population reduction and even extinction, but generally lack information on their genetic diversity and population structure. Genetic sampling and tissue collection have been scarce in these species owing to strict regulations on manipulation of endangered species, and the difficulties and risks associated with capturing these arboreal and fast‐moving monkeys in the challenging environments that they inhabit. These difficulties have hindered the development of molecular genetic markers, which are usually derived from tissues or blood. In this study, we present a method for de novo microsatellite isolation and genotyping using DNA from noninvasive origins of a critically endangered Asian colobine, the white‐headed langur (Trachypithecus leucocephalus). Genomic DNA isolated from hair was shown to be sufficient for microsatellite enrichment and isolation, with similar isolation efficiencies as from tissue DNA. We identified and characterized 20 polymorphic microsatellite loci, and evaluated their amplification success and genotyping reliability with 86 field‐collected fecal samples. These results show that this panel of loci can produce reliable genotypes from fecal samples, and represent a useful tool for noninvasive investigation of genetic structure, individual identification and kinship assessment in this highly endangered species. Our approach can be applied to conservation genetic studies of other wild species that lack sequence information and tissue samples.     

Investigation of mortality in Pacific oysters associated with Ostreid herpesvirus-1 μVar in the Republic of Ireland in 2009

High levels of mortality in Pacific oysters Crassostrea gigas in the Republic of Ireland were recorded during the summer of 2009. The new variant of Ostreid herpes 1 (OsHV-1 μVar) which first emerged in France in 2008 was identified from affected stocks. Retrospective data was collected from 70 oyster farmers through an interviewer-administered questionnaire to investigate the distribution and determinants of the mortality. Based on farmer recall, data were recorded at the batch level for cumulative mortality during 2009, start dates and duration of the mortality event and the age of animals affected. Observable mortalities were recorded in 109 out of 346 batches at 47 sites; 104 of the 109 batches were located in bays where OsHV-1 μVar had been detected. The records from bays where OsHV-1 μVar had been detected were analysed to characterize the pattern of mortality and potential risk factors. Batch mortality averaged 37% (18-65% quartiles) but showed a bimodal distribution (half the batches had mortality less than 45%). Mortalities started at the end of May and continued until early August, peaking in early July. On average oysters died over a period of 18 days. Mortality varied considerably both between and within bays. Mortality started in recently introduced batches and occurred later in the summer in established oysters, which is consistent with the introduction of an infectious agent. Mortality was significantly lower in adults compared with other age groups, which supports observations from France. Three variables were significantly (P<0.05) associated, in both bivariate screening and a logistic regression, with high batch-level mortality (>40%): oysters (i) introduced as juveniles, (ii) during or since the winter of 2008/9 and (iii) which spent less than 8h out of water (in a tidal cycle) (compared with oysters introduced as adults before the winter of 2008/9 and spending more than 8h out of water). Twenty-one percent of triploid batches experienced "high" (>40%) mortality compared with 10% for diploid batches which was significant (P<0.05) in the initial bivariate screening but not in the final logistic regression model. Future studies should develop improved methods to assess oyster mortality and follow stocks over time to better determine the influence of management and environmental factors on mortality.     

Identification of the variant C of β-lactoglobulin in sheep using a polymerase chain reaction-restriction fragment length polymorphism method

β-Lactoglobulin (β-LG) is the major whey protein in the milk of ruminants and is able to bind and transport small hydrophobic molecules. However, its biological role is mainly unknown (G odovac -Z immermann 1988). Previously three genetic variants have been found in sheep: A, B and C. The genetic variants A and B differ at amino acid position 20, where variant A has a His and variant B has a Thr (K olde and B raunitzer 1983). The variant C is a subtype of variant A with a single amino acid exchange of Arg→Glu at position 148 (E rhardt et al. 1989). The genotype β-LG BB was found to be associated with higher milk yield, whereas genotypes AA and AB had a higher milk protein and casein content as well as yielding more curd (G arzon and M artinez 1992). No data is available concerning the relationship between the β-LG C allele and production traits or milk properties. Since DNA-based genotyping has already been performed for alleles A and B (S chlee et al. 1993), the aim of the present study was to develop a DNA-based method for identifying the β-LG C variant. However polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) typing has been established (P rinzenberg and E rhardt 1999) recently, and this study shows an alternative method to detect β-LG C.     

Precision Medicine in Cats: Novel Niemann‐Pick Type C1 Diagnosed by Whole‐Genome Sequencing

State‐of‐the‐art health care includes genome sequencing of the patient to identify genetic variants that contribute to either the cause of their malady or variants that can be targeted to improve treatment. The goal was to introduce state‐of‐the‐art health care to cats using genomics and a precision medicine approach. To test the feasibility of a precision medicine approach in domestic cats, a single cat that presented to the University of Missouri, Veterinary Health Center with an undiagnosed neurologic disease was whole‐genome sequenced. The DNA variants from the cat were compared to the DNA variant database produced by the 99 Lives Cat Genome Sequencing Consortium. Approximately 25× genomic coverage was produced for the cat. A predicted p.H441P missense mutation was identified in NPC1, the gene causing Niemann‐Pick type C1 on cat chromosome D3.47456793 caused by an adenine‐to‐cytosine transversion, c.1322A>C. The cat was homozygous for the variant. The variant was not identified in any other 73 domestic and 9 wild felids in the sequence database or 190 additionally genotyped cats of various breeds. The successful effort suggested precision medicine is feasible for cats and other undiagnosed cats may benefit from a genomic analysis approach. The 99 Lives DNA variant database was sufficient but would benefit from additional cat sequences. Other cats with the mutation may be identified and could be introduced as a new biomedical model for NPC1. A genetic test could eliminate the disease variant from the population.     

Representational differential analysis detects amplification of satellite sequences in postweaning multisystemic wasting syndrome of pigs.

Representational difference analysis (RDA) was used as a molecular approach to identify unique sequences associated with postweaning multisystemic wasting syndrome (PMWS) in pigs. Three rounds of subtractive hybridization and amplification between driver DNA extracted from normal pigs and tester DNA from PMWS-affected animals were performed. The final product corresponding to sequences associated with PMWS in pigs was analyzed using agarose gel electrophoresis, and 9 fragments were visualized after staining with ethidium bromide. Eight recombinants were successively cloned and sequenced, and the results were then compared with existing databases. Most of the PMWS clones isolated were satellite sequences from pig centrometric regions and 1 was a microsatellite sequence. One clone represented a microsatellite sequence, and 2 clones showed no homology with any gene found in the databases. The sequence comparison data did not reveal any homology with an infectious agent such as a virus or a bacterium. In the present experimental setting, it was concluded that PMWS in pigs triggers molecular changes such as an amplification of genomic regions containing repeated sequences.     

Molecular analysis of bovine leukemia virus in early epidemic phase in Japan using archived formalin fixed paraffin embedded histopathological specimens

Bovine leukemia virus (BLV) is an important pathogen associated with enzootic bovine leukosis. In this study, we performed PCR and sequencing analysis to characterize BLVgp51 sequences from formalin-fixed paraffin-embedded (FFPE) specimens made from 1974 to 2000 and successfully obtained BLV proviral genome sequences from 94% of the analyzed samples. Furthermore, from these samples, we reconstructed eight full-length and nearly full-length BLVgp51 sequences. These sequences were classified as BLV genotype 1, implying that genotype1 has already been circulating in Japan since the 1970s. In our results, the proviral DNA was detected in the 1970s, 1980s, and 1990s in the same manner, indicating that the detection of BLV proviral genome depends on storage conditions rather than storage period. The sequences obtained in this study provide direct insights into BLV sequences before 2000, which serves as a good calibrator for inferring ancient BLV diversity.     

Mitochondrial D‐loop analysis reveals low diversity in Mangalica pigs and their relationship to historical specimens

The genetic relationship between 195 Mangalica and 79 non‐Mangalica pigs was studied using mitochondrial D‐loop SNP genotyping. Altogether, 35 polymorphic sites and 27 haplotypes were identified. Of the haplotypes, eight and 16 are Mangalica and non‐Mangalica specific, respectively, while three contain both Mangalica and non‐Mangalica individuals. Genetic distance values and phylogenetic analysis indicate that Mangalica individuals are very closely related, and five haplotypes represent approximately 92% of the Mangalica pigs involved in the study, thus determining the major maternal lineages. In contrast to previous microsatellite studies, individuals of Mangalica could not be distinguished as three separate breeds using mtDNA genotyping. Comparing modern and archaeological mtDNA sequences revealed that present day Mangalica is related to pigs that lived in the Carpathian basin where postulated ancestors of Mangalica also lived. This is the first DNA‐based genetic evidence to support the described breeding history of Mangalica.     

Genetic characterization of chicken anemia virus from commercial broiler chickens in Alabama.

Chicken anemia virus (CAV) isolates show extremely limited genetic variability worldwide. We determined the nucleotide sequence of an 823-nucleotide portion of the 2.3-kb CAV genome found in 10 liver and/or spleen specimens of Alabama 29-to-49-day-old commercial broiler chickens exhibiting lymphocyte depletion of the thymus submitted to the state diagnostic laboratory because of problems unrelated to anemia. We determined the nucleotide sequence directly from DNA isolated from tissues, without isolation of virus in culture. This procedure enabled us to characterize CAV that might not have replicated in culture and avoided the potential for changes during passage. Results confirmed the limited genetic variability of CAV. All sequences were identical in 93% of nucleotide positions. The sequences encoded only two distinct VP1 hypervariable regions, and both had been found previously in other CAV isolates. A novel amino acid, glutamine, was found at VP1 position 22 in half the sequences, replacing the histidine residue encoded by most previously characterized CAV genomes. We were able to distinguish among CAV genomes with different codons at VP1 amino acid 22 and different hypervariable regions by restriction endonuclease analysis of polymerase chain reaction products.     

Applying molecular genetic tools to tiger conservation

The utility of molecular genetic approaches in conservation of endangered taxa is now commonly recognized. Over the past decade, conservation genetic analyses based on mitochondrial DNA sequencing and microsatellite genotyping have provided powerful tools to resolve taxonomy uncertainty of tiger subspecies, to define conservation units, to reconstruct phylogeography and demographic history, to examine the genetic ancestry of extinct subspecies, to assess population genetic status non-invasively, and to verify genetic background of captive tigers worldwide. The genetic status of tiger subspecies and populations and implications for developing strategies for the survival of this charismatic species both in situ and ex situ are discussed.     

Microsatellite genotyping reveals diversity within populations of Sodalis glossinidius, the secondary symbiont of tsetse flies

The aim of this study was to develop a PCR-based microsatellite genotyping method for identifying genetic diversity in Sodalis glossinidius, a symbiont associated with tsetse fly infection by trypanosomes causing human and animal trypanosomiasis. Allelic polymorphism at three loci, investigated on 40 fly gut extracts, evidenced eight alleles and the existence of five genotypes. This novel approach was shown to be efficient and suitable for routine large-scale genotyping of S. glossinidius present in the biologically complex tsetse fly extracts; it could favor progress in the fields of diagnosis, epidemiology, population genetics, and fly/symbiont/trypanosome interactions.     

Viral-derived DNA invasion and individual variation in an Indonesian population of large flying fox Pteropus vampyrus

Here, we performed next-generation sequencing (NGS) on six large flying foxes (Pteropus vampyrus) collected in Indonesia. Seventy-five virus species in the liver tissue of each specimen were listed. Viral homologous sequences in the bat genome were identified from the listed viruses. This finding provides collateral evidence of viral endogenization into the host genome. We found that two of the six specimens bore partial sequences that were homologous to the plant pathogens Geminiviridae and Luteoviridae. These sequences were absent in the P. vampyrus chromosomal sequences. Hence, plant viral homologous sequences were localized to the hepatocytes as extrachromosomal DNA fragments. Therefore, this suggests that the bat is a potential carrier or vector of plant viruses. The present investigation on wild animals offered novel perspectives on viral invasion, variation, and host interaction.     

微卫星标记及其在家畜遗传育种中的应用

本文阐述了微卫星的结构、遗传特性以及微卫星标记在家畜个体亲缘关系鉴定、构建基因图谱、标记辅助选择、评估遗传多样性等四方面的作用。认为微卫星标记是动物遗传育种的一种非常理想的分子遗传标记,但由于已知的微卫星位点较少,加之分析成本较高,微卫星扩增产物在检测过程中常出现各种误判因素等原因而存在应用缺陷,为了更好地利用微卫星,使其成为一种简捷、灵敏、低廉的重要遗传分析方法,必须对其进行更深入地研究。     

Identification of the putative ancestral allele of bovine single‐nucleotide polymorphisms

Identifying the action of natural selection from patterns of standing genetic variation has long been of interest to the population genetic community. Thanks to the availability of large single‐nucleotide polymorphism (SNP) data sets for many species and of high‐throughput SNP genotyping methods, whole‐genomic surveys to detect selective sweeps are now possible. Knowing the ancestral allele increases the power to detect selection. We present here a comparative genomic approach to determine the putative ancestral allele of bovine SNPs deposited in public databases. We analysed 19 551 488 SNPs and identified the putative ancestral allele for 14 339 107 SNPs. Our predicted ancestral alleles were in agreement with ancestral alleles detected by genotyping outgroup species for 97% SNPs from the BovineSNP50 BeadChip. This comparison indicates that our comparative genomic‐based approach to identify putative ancestral alleles is reliable.     

微卫星DNA的研究进展及其在寄生虫学的应用

微卫星DNA是广泛存在于原核生物和真核生物基因组中的短串联重复序列,具有快速突变性、多态性信息丰富、易于检测等特点.在动植物遗传育种、遗传图谱的构建、群体遗传学研究、肿瘤学、亲子鉴定、濒危野生动物保护及法医鉴定等方面的研究中被广泛应用.论文综述了微卫星DNA技术的研究进展及其在寄生虫学中的应用.     

双峰驼遗传多样性研究进展

综述了国内外近40年双峰驼在染色体核型、血液蛋白多态、基因组DNA和线粒体DNA遗传多样性上的研究进展.染色体核型、血液蛋白多态分析表明,双峰驼的遗传多样性有限,微卫星和线粒体DNA的RFLP分析表明双峰驼具有较丰富的遗传多样性,基于线粒体DNA全序列的骆驼科动物遗传进化分析有助于理解骆驼科动物进化历史.由于研究滞后,目前还未有关于双峰驼群体线粒体DNA序列的遗传多样性分析.     

First evidence of porcine circovirus type 2 (PCV-2) infection of pigs in the Czech Republic by semi-nested PCR

Three oligonucleotide primers for semi-nested polymerase chain reaction (PCR) were designed according to already published sequences of porcine circovirus types 1 (PCV-1) and 2 (PCV-2) isolates. These primers were used to detect PCV-2 DNA. A positive amplification reaction was visualized from a DNA suspension containing as few as 10 copies of virus DNA. In total. 77 samples of inguinal lymph nodes and nasal swabs from pigs in the Czech Republic were used to detect the virus. Thirty-seven of them were positive for PCV-2 DNA. In order to confirm specificity of the PCR reaction, seven DNA fragments were sequenced. Czech PCV sequences were found to have a 92-97% homology with other known PCV-2 strains and only 80-83% homology with PCV-1 strains.