Spleen DNA Research Articles

High prevalence of Lynx rufus gammaherpesvirus 1 in wild Vermont bobcats.

Gammaherpesviruses (GHVs) are host specific DNA viruses that infect a large range of mammalian species. These viruses preferentially target host lymphocyte cell populations and infection may lead to morbidity or mortality in immunocompromised, co-infected, or non-adapted hosts. In this study, we tested for the presence of Lynx rufus gammaherpesvirus 1 (LruGHV1) in a northeastern United States population of wild bobcats (L. rufus). We estimated prevalence of infection and viral load in infected individuals using quantitative real-time PCR analysis of spleen DNA from 64 Vermont bobcats. We observed an overall prevalence of 64% using this methodology. Bobcat age was significantly positively associated with GHV infection status, and we noted a trend for higher viral loads in young animals, but prevalence and viral load were similar in male and female bobcats. A single LruGHV1 variant was identified from the sequencing of the viral glycoprotein B gene of Vermont bobcats. This gene sequence was 100% similar to that reported in Florida bobcats and slightly variant from other isolates identified in the Western USA. Our work suggests broad geographic distribution and high prevalence of LruGHV1 in bobcat populations across the United States with infection attributes that suggest horizontal transmission of the agent. Geographic differences in viral genotype may reflect historical migration and expansion events among bobcat populations.

Municipal leachates health risks: Chemical and cytotoxicity assessment from regulated and unregulated municipal dumpsites in Lebanon

The proper management of municipal waste is critical for resource recovery, sustainability and health. Lebanon main approach for managing its municipal waste consisted of landfill disposal with minimal recycling capacity. This approach contributed to exceeding the holding capacity of existing landfills leading eventually to their closures. The closure of a major landfill (Naameh landfill) servicing Beirut and Mount Lebanon areas led to municipal wastes piling in the streets and forests for more than a year in 2016. The main problem identified in the municipal wastes consisted of untreated leachates (from regulated and unregulated dumpsites) going straight into the Mediterranean Sea. Therefore leachate samples were collected and subjected to chemical characterization followed by biological assessment. The chemical characterization and profiling of the Lebanese leachates were compared to results reported in Lebanon, Europe and United States as well as to the toxicity reference values (TRV). The biological assessment was conducted in vitro using human derived immortalized cell cultures. This strategy revealed significant alarming cellular organelles and DNA damages using in vitro cytotoxicity assays (MTS and comet assay). The significant damages observed at the cellular level prompted further animal model investigations using BALB/c mice. The animal data pointed to significant upregulation of liver activity enzymes coupled with significant damage expression in liver spleen and bone marrow DNA. The presented research clearly indicated that there is an urgent need for development of national waste strategies for proper treatment and disposal of municipal waste leachates in Lebanon.

Application of a specific quantitative real-time PCR (qPCR) to identify Leishmania infantum DNA in spleen, skin and hair samples of wild Leporidae

The aim of this study was to compare a quantitative real-time PCR (qPCR) validated for the detection of Leishmania infantum in dogs with a nested PCR but in wild Leporidae. Additionally, L. infantum results from indirect immunofluorescent antibody test (IFAT) and in vitro culture were also compared with qPCR. Different samples (spleen, skin and hair) recovered from 224 European rabbits and 70 Iberian hares from two green areas of Madrid Council were analysed for the detection of L. infantum. The presence of Leishmania kDNA was detected by qPCR in 58 out of 221 (26.24%), 162 out of 203 (79.8%) and 22 out of 33 (66.67%) analysed rabbits on spleen, skin and hair samples, respectively; and in 7 out of 69 (10.14%), 39 out of 70 (55.71%) and 17 out of 32 (53.13%) test hares on spleen, skin and hair samples, respectively. The qPCR in all test samples resulted to be more sensitive than nested PCR, with a limit of detection of 1.43 fg/reaction (0.039 parasites) for L. infantum genomic DNA. Additionally, the percentage of positive animals detected by qPCR in at least two out of three samples (n=221 rabbits and 70 hares) tested was higher than those detected by IFAT (n=190 rabbits and 61 hares) and isolation (n=75 rabbits and 20 hares). The highest level of agreement was obtained by nested PCR on spleen/skin (89%/83%) samples and qPCR on spleen samples (81%), followed by IFAT (48%) and qPCR on skin (32%) samples. Our results demonstrate this qPCR is a suitable method for detecting L. infantum DNA in different samples suggesting hair could be considered an adequate sample for direct, reliable and non-invasive diagnosis of L. infantum in wild animals.

ENDEMIC INFECTION OF STRANDED SOUTHERN SEA OTTERS (ENHYDRA LUTRIS NEREIS) WITH NOVEL PARVOVIRUS, POLYOMAVIRUS, AND ADENOVIRUS.

Over the past century, the southern sea otter (SSO; Enhydra lutris nereis) population has been slowly recovering from near extinction due to overharvest. The SSO is a threatened subspecies under federal law and a fully protected species under California law, US. Through a multiagency collaborative program, stranded animals are rehabilitated and released, while deceased animals are necropsied and tissues are cryopreserved to facilitate scientific study. Here, we processed archival tissues to enrich particle-associated viral nucleic acids, which we randomly amplified and deeply sequenced to identify viral genomes through sequence similarities. Anelloviruses and endogenous retroviral sequences made up over 50% of observed viral sequences. Polyomavirus, parvovirus, and adenovirus sequences made up most of the remaining reads. We characterized and phylogenetically analyzed the full genome of sea otter polyomavirus 1 and the complete coding sequence of sea otter parvovirus 1 and found that the closest known viruses infect primates and domestic pigs ( Sus scrofa domesticus), respectively. We tested archived tissues from 69 stranded SSO necropsied over 14 yr (2000-13) by PCR. Polyomavirus, parvovirus, and adenovirus infections were detected in 51, 61, and 29% of examined animals, respectively, with no significant increase in frequency over time, suggesting endemic infection. We found that 80% of tested SSO were infected with at least one of the three DNA viruses, whose tissue distribution we determined in 261 tissue samples. Parvovirus DNA was most frequently detected in mesenteric lymph node, polyomavirus DNA in spleen, and adenovirus DNA in multiple tissues (spleen, retropharyngeal and mesenteric lymph node, lung, and liver). This study describes the virome in tissues of a threatened species and shows that stranded SSO are frequently infected with multiple viruses, warranting future research to investigate associations between these infections and observed lesions.

HAdV-2-suppressed growth of SV40 T antigen-transformed mouse mammary epithelial cell-induced tumours in SCID mice

Human adenovirus (HAdV) vectors are promising tools for cancer therapy, but the shortage of efficient animal models for productive HAdV infections has restricted the evaluation of systemic effects to mainly immunodeficient mice. Previously, we reported a highly efficient replication of HAdV-2 in a non-tumorigenic mouse mammary epithelial cell line, NMuMG. Here we show that HAdV-2 gene expression and progeny formation in NMuMG cells transformed with the SV40 T antigen (NMuMG-T cells) were as efficient as in the parental NMuMG cells. Injection of HAdV-2 into tumours established by NMuMG-T in SCID mice caused reduced tumour growth and signs of intratumoural lesions. HAdV-2 replicated within the NMuMG-T-established tumours, but not in interspersed host-derived tissues within the tumours. The specific infection of NMuMG-T-derived tumours was verified by the lack of viral DNA in kidney, lung or spleen although low levels of viral DNA was occasionally found in liver.

Abstract 225: Assessing and enriching human tumor cell content in patient-derived cancer xenografts and co-cultures

Abstract Purpose: To develop a robust real-time PCR-based assay with improved accuracy for quantifying the relative fractions of human and mouse DNA in heterogeneous samples such as patient-derived cancer xenografts or cultures. Background: Patient derived mouse xenografts (PDXs), or irradiated mouse fibroblast conditionally reprogrammed cancer cultures (CRCs) provide valuable resources for studying cancers, but as mouse tissue in heterogeneous samples can significantly confound downstream functional and molecular assays (such as next-generation sequencing) it is important to determine the fraction of mouse DNA in mixed samples. Methods: Using an approach different from a previously reported PCR assay (Alcoser SY et al BMC Biotech 2011, 11:124), we established a Taqman real-time PCR assay employing mouse- and human-specific primers that amplified the same chromosomal region of PTGER2 on human chromosome 14q22.1 and mouse chromosome 14 22.68 cM. We also developed a method to enrich human tumor cells from PDXs using enzymatic digestion with Liberase (Roche), followed by differential centrifugation and adherence. Results: Utilizing serial dilutions of heterogeneous DNA mixtures of mouse spleen DNA and normal human white blood cell DNA at varied DNA ratios we demonstrated the assay to be linear (Pearson r = 0.9984, human DNA; r = 0.9998, mouse DNA; p<0.0001) and sensitive (>6.4 pg DNA/reaction). The assay was evaluated in 66 samples of human tumors (lung, ovary, neuroblastoma, colon, pancreas, stomach and melanoma) including 25 PDXs before and after tumor cell enrichment, 12 non-enriched PDXs, 2 CRC-derived xenografts and 2 CRCs. Of the PDXs 28 were subcutaneous tumors and 9 were malignant ascites. A wide range of human:mouse DNA ratios were found from zero to 99% among all samples, with two PDXs and 2 CRC-derived xenografts lacking detectable human DNA. The highest human cell fractions were in the malignant ascites samples. Of the enriched samples, 18 of 25 (72%) showed tumor cell enrichment of 2 to 71-fold as compared with the value of matching non-enriched PDX. Conclusions: We developed a robust real-time PCR assay for measuring the relative fraction of species-specific DNA in human:mouse heterogenous samples. The human:mouse DNA ratios in PDX samples varied widely and the human tumor cell populations in PDXs can be effectively enriched in vitro. Assessing human tumor cell content with this assay will facilitate molecular studies with PDXs and mouse-human cocultivated cells. Citation Format: Yu-An Zhang, Victor Stastny, Mahboubeh Papari-Zareei, Heather Davidson, Boning Gao, Brenda Timmons, Jingsheng Yan, C Patrick Reynolds, John D. Minna, Adi F. Gazdar. Assessing and enriching human tumor cell content in patient-derived cancer xenografts and co-cultures. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 225. doi:10.1158/1538-7445.AM2015-225

Protective Effect of Lycium ruthenicum Murr. Against Radiation Injury in Mice.

The protective effect of Lycium ruthenicum Murr. against radiation injury was examined in mice. Kunming mice were randomly divided into a control group, model group, positive drug group and L. ruthenicum high dose (8 g/kg), L. ruthenicum middle dose (4 g/kg), L. ruthenicum low dose (2 g/kg) treatment groups, for which doses were administered the third day, seventh day and 14th day after irradiation. L. ruthenicum extract was administered orally to the mice in the three treatment groups and normal saline was administered orally to the mice in the control group and model group for 14 days. The positive group was treated with amifostine (WR-2721) at 30 min before irradiation. Except for the control group, the groups of mice received a 5 Gy quantity of X-radiation evenly over their whole body at one time. Body weight, hemogram, thymus and spleen index, DNA, caspase-3, caspase-6, and P53 contents were observed at the third day, seventh day, and 14th day after irradiation. L. ruthenicum could significantly increase the total red blood cell count, hemoglobin count and DNA contents (p < 0.05). The spleen index recovered significantly by the third day and 14th day after irradiation (p < 0.05). L. ruthenicum low dose group showed a significant reduction in caspase-3 and caspase-6 of serum in mice at the third day, seventh day, and 14th day after irradiation and L. ruthenicum middle dose group experienced a reduction in caspase-6 of serum in mice by the seventh day after irradiation. L. ruthenicum could decrease the expression of P53. The results showed that L. ruthenicum had protective effects against radiation injury in mice.

English

Three groups (10 in each) (6 to 8 weeks old) of experimental mice were orally infected each with 500 (group A) 1000 (group B) and 2000 (group C) infective larvae of Ancylostoma caninum . Group (D) was kept as uninfected control for comparison. Two mice from each of the experimental groups were sacrificed on day 1, 4, 9, 16 and 30 after infection. Two mice from control group were also sacrificed on the same designated days. Spleen was separated and the content of DNA and RNA was estimated following standard methods. DNA and RNA levels in spleen of experimental groups (A, B and C) are lower and higher than control group during 1 to 30 days of infection respectively. The synthesis of DNA and RNA was influenced by three varied doses of infection; the occurrence of abnormal physiological changes in spleen indicates the influence of infection in the lymphoid organ.

Protection of DNA From Ionizing Radiation-Induced Lesions by Asiaticoside.

This study aims to investigate whether asiaticoside, a triterpene glycoside, can afford protection to DNA from alterations induced by gamma radiation under in vitro, ex vivo, and in vivo conditions. In vitro studies were done on plasmid pBR322 DNA, ex vivo studies were done on cellular DNA of human peripheral blood leukocytes, and in vivo investigations were conducted on cellular DNA of spleen and bone marrow cells of mice exposed to whole-body gamma radiation. The supercoiled form of the plasmid pBR322 DNA upon exposure to the radiation was converted into relaxed open circular form due to induction of strand breaks. Presence of asiaticoside along with the DNA during irradiation prevented the relaxation of the supercoiled form to the open circular form. When human peripheral blood leukocytes were exposed to gamma radiation, the cellular DNA suffered strand breaks as evidenced by the increased comet parameters in an alkaline comet assay. Asiaticoside, when present along with blood during irradiation ex vivo, prevented the strand breaks and the comet parameters were closer to that of the controls. Whole-body exposure of mice to gamma radiation resulted in a significant increase in comet parameters of DNA of bone marrow and spleen cells of mice as a result of radiation-induced strand breaks in DNA. Administration of asiaticoside prior to whole-body radiation exposure of the mice prevented this increase in radiation-induced increase in comet parameters, which could be the result of protection to DNA under in vivo conditions of radiation exposure. Thus, it can be concluded from the results that asiaticoside can offer protection to DNA from radiation-induced alterations under in vitro, ex vivo, and in vivo conditions.

Detection of Rickettsia felis in Wild Mammals from Three Municipalities in Yucatan, Mexico.

The aim of this study was to provide information of the occurrence of Rickettsia felis in wild mammals from three municipalities in Yucatan, Mexico. The reactivity of rodent serum to Rickettsia antigens was detected in 80.9% (17 of 21) samples using immunofluorescence assay. Polymerase chain reaction identified rickettsial DNA in spleens of 43.5% (10 of 23) rodents and 57.1% (4 of 7) opossums. The identification of the rickettsial DNA was confirmed as R. felis by restriction fragment length polymorphism and DNA sequencing. This study comprises the first report of R. felis detection in wild mammals in Yucatan.

Detection and characterization of zoonotic pathogens of free-ranging non-human primates from Zambia.

BackgroundWildlife may harbor infectious pathogens that are of zoonotic concern acting as a reservoir of diseases transmissible to humans and domestic animals. This is due to human-wildlife conflicts that have become more frequent and severe over recent decades, competition for the available natural habitats and resources leading to increased human encroachment on previously wild and uninhabited areas.MethodsA total of 88 spleen DNA samples from baboons and vervet monkeys from Zambia were tested for zoonotic pathogens using genus or species-specific PCR. The amplified products were then subjected to sequencing analysis.ResultsWe detected three different pathogenic agents, including Anaplasma phagocytophilum in 12 samples (13.6%), Rickettsia spp. in 35 samples (39.8%) and Babesia spp. in 2 samples (2.3%).ConclusionThe continuously increasing contacts between humans and primate populations raise concerns about transmission of pathogens between these groups. Therefore, increased medical and public awareness and public health surveillance support will be required to detect and control infections caused by these agents at the interface between humans and wildlife.

DNA damage in internal organs after cutaneous exposure to sulphur mustard

Sulphur mustard (SM) is a chemical warfare agent that attacks mainly skin, eye and lungs. Due to its lipophilic properties, SM is also able to diffuse through the skin and reach internal organs. DNA represents one of the most critical molecular targets of this powerful alkylating agent which modifies DNA structure by forming monoadducts and biadducts. These DNA lesions are involved in the acute toxicity of SM as well as its long-term carcinogenicity. In the present work we studied the formation and persistence of guanine and adenine monoadducts and guanine biadducts in the DNA of brain, lungs, kidneys, spleen, and liver of SKH-1 mice cutaneously exposed to 2, 6 and 60mg/kg of SM. SM-DNA adducts were detected in all studied organs, except in liver at the two lowest doses. Brain and lungs were the organs with the highest level of SM-DNA adducts, followed by kidney, spleen and liver. Monitoring the level of adducts for three weeks after cutaneous exposure showed that the lifetime of adducts were not the same in all organs, lungs being the organ with the longest persistence. Diffusion from skin to internal organs was much more efficient at the highest compared to the lowest dose investigated as the result of the loss of the skin barrier function. These data provide novel information on the distribution of SM in tissues following cutaneous exposures and indicate that brain is an important target.

Molecular evidence of Anaplasma phagocytophilum in wild boar (Sus scrofa) in Belgium.

BackgroundAnaplasma phagocytophilum is a tick-borne pathogen of veterinary and human importance. Both ticks as vectors and vertebrates as reservoir hosts are essential for the cycle maintenance of this bacterium. Currently, the whole range of animal species reservoirs for A. phagocytophilum in natural environment is still unknown. Therefore, the aim of this study was to estimate the prevalence of infection with A. phagocytophilum in the wild boar population in southern Belgium.ResultsIn the frame of a targeted surveillance program, 513 wild boars were sampled during the hunting season 2011. A nested 16S rRNA PCR was used to screen the presence of A. phagocytophilum DNA in spleen of boars. Within 513 samples, 5 (0,97%) were tested PCR positive and identification was confirmed by sequencing.ConclusionsThis study gives the first insight of presence of A. phagocytophilum in wild boars in southern Belgium.

Detection ofFrancisella tularensisin Voles in Finland

Francisella tularensis is a highly virulent intracellular bacterium causing the zoonotic disease tularemia. It recurrently causes human and animal outbreaks in northern Europe, including Finland. Although F. tularensis infects several mammal species, only rodents and lagomorphs seem to have importance in its ecology. Peak densities of rodent populations may trigger tularemia outbreaks in humans; however, it is still unclear to which extent rodents or other small mammals maintain F. tularensis in nature. The main objective of this study was to obtain information about the occurrence of F. tularensis in small mammals in Finland. We snap-trapped 547 wild small mammals representing 11 species at 14 locations around Finland during 6 years and screened them for the presence of F. tularensis DNA using PCR analysis. High copy number of F. tularensis-specific DNA was detected in tissue samples of five field voles (Microtus agrestis) originating from one location and 2 years. According to DNA sequences of the bacterial 23S ribosomal RNA gene amplified from F. tularensis-infected voles, the infecting agent belongs to the subspecies holarctica. To find out the optimal tissue for tularemia screening in voles, we compared the amounts of F. tularensis DNA in lungs, liver, spleen, and kidney of the infected animals. F. tularensis DNA was detectable in high levels in all four organs except for one animal, whose kidney was F. tularensis DNA-negative. Thus, at least liver, lung, and spleen seem suitable for F. tularensis screening in voles. Thus, liver, lung, and spleen all seem suitable for F. tularensis screening in voles. In conclusion, field voles can be heavily infected with F. tularensis subsp. holarctica and thus potentially serve as the source of infection in humans and other mammals.

Detection and characterization of zoonotic pathogens of free-ranging non-human primates from Zambia

Wildlife may harbor infectious pathogens that are of zoonotic concern acting as a reservoir of diseases transmissible to humans and domestic animals. This is due to human-wildlife conflicts that have become more frequent and severe over recent decades, competition for the available natural habitats and resources leading to increased human encroachment on previously wild and uninhabited areas. A total of 88 spleen DNA samples from baboons and vervet monkeys from Zambia were tested for zoonotic pathogens using genus or species-specific PCR. The amplified products were then subjected to sequencing analysis. We detected three different pathogenic agents, including Anaplasma phagocytophilum in 12 samples (13.6%), Rickettsia spp. in 35 samples (39.8%) and Babesia spp. in 2 samples (2.3%). The continuously increasing contacts between humans and primate populations raise concerns about transmission of pathogens between these groups. Therefore, increased medical and public awareness and public health surveillance support will be required to detect and control infections caused by these agents at the interface between humans and wildlife.

Genomic DNA characterization of pork spleen by Raman spectroscopy

In this paper, the study of Raman signal enhancement due to interaction between ZnO rods and pork spleen DNA is reported. ZnO microstructures were synthesized by the Sol-Gel method and afterward combined with porcine spleen DNA extracted in the previous stages, following standardized cell lysis, deproteinization, and precipitation processes. Raman spectroscopy was used for the characterization of structures of ZnO and ZnO-DNA complex, and the results show the respective bands of ZnO wurtzite hexagonal phase for modes E2 (M), A1(TO), E2(High), E1(LO), and 2LO. Due to the SERS effect in the spectral range from 200 to 1800 cm,−1 Raman bands caused by vibrations of the deoxyribose C-O-C binding were also observed, producing deformation of the ring as shown in the 559 cm−1 peak. The broad band at 782 cm−1, together with the complex vibration of the string 5'-COPO-C3′, is over a wide band of thymine (790 cm−1) or cytosine (780 cm−1). A prominent band near 1098 cm−1 assigned to symmetric stretching vibration phosphodioxy group (PO2−) DNA backbone is most favoured in intensity by the addition of ZnO particles originated by the SERS effect. This effect suggests a possible mechanism for enhancing the Raman signal due to the electromagnetic interaction between a DNA molecule and the flat surface of the ZnO rod.

Molecular and Histopathological Evidence of Mycobacteriosis in Paradise Fish Macropodus opercularis Imported into Korea

We report on mycobacteriosis in an imported tropical ornamental fish Macropodus opercularis commonly known as the paradise fish. Mass mortality occurred in paradise fish imported to Korea from Southeast Asia in 2008. The affected fish did not show any outward clinical signs, but enlargement of the spleen, kidneys, and liver was observed on dissection. Histopathological examination revealed numerous granulomas in the spleen, and acid-fast bacilli were observed in the centers of the granulomas. About 65% of spleen DNA samples were PCR positive using mycobacteria-specific primers targeting the 16S rRNA and hsp65 genes. The nucleotide identities of the 16S rRNA and hsp65 genes with those of Mycobacterium marinum were 99.5% and 99.4%, respectively. Although the bacterium was not cultured, the molecular diagnosis and histopathological findings were consistent with mycobacteriosis in paradise fish.

EVALUATION OF PARSLEY EFFECT AGAINST GENTAMICIN GENOTOXICITY IN RATS USING RAPD ANALYSIS

The present study was conducted to determine the protective effect of parsley (leaves extract, fresh leaves, seeds and oil) on rat injected intraperitoneally with gentamicin at a dose of 10 mg/kg b.wt. The toxic effects of gentamicin were assessed in term of the changes in relative organs weights in addition the genetic variation in DNA-RAPD profiles in both liver and spleen. Results showed that no significant differences were found between the negative control and all of tested groups for relative liver weights, while the group fed on parsley oil recorded the highest value of relative spleen weight which did not differ than negative control group. For RAPD profiles, the differences in RAPD patterns refer to band intensity, loss of normal bands and appearance of new bands as compared with the negative control group. All these differences were well represented in the patterns produced in gentamicin group as well as all rat groups fed on parsley. Changes in bands intensity which were major event increased by gentamicin effect, were decreased in all groups fed on parsley forms except the group fed on seeds (for liver DNA) and the group fed on parsley leaves extract (for spleen DNA) were increased. Parsley leaves and oil (for liver) and parsley oil (for spleen) exhibited the best effect in reducing the increase in band intensity. All forms of parsley caused a decrease in the genomic template stability (%) values compared to gentamicin group. The major decrease was for leaves extract with regard to liver DNA and for seeds with regard to spleen DNA. Results of this study demonstrated the genotoxic effects of gentamicin on rat's liver and spleen. In addition, the results revealed that parsley oil may be the effective form in reducing gentamicin genotoxic effect followed by parsley fresh leaves.

A real-time polymerase chain reaction assay for identification and quantification ofFlavobacterium psychrophilumand application to disease resistance studies in selectively bred rainbow troutOncorhynchus mykiss

Rapid detection and quantification of Flavobacterium psychrophilum, the causative agent of bacterial cold water disease (BCWD) in rainbow trout, are crucial to disease surveillance and encompass an essential component of BCWD research. Real-time, or quantitative polymerase chain reaction (qPCR) assays that have previously targeted the 16S rRNA gene of F.psychrophilum are complicated by polymorphisms and off-target amplification. Insignia primer and probe development software were used to identify a conserved single-copy signature sequence in the F.psychrophilum genome that codes for a hypothetical protein with unknown function. Primer and probes were used in a TaqMan qPCR assay that amplified 210 F.psychrophilum isolates with a lower limit of linear detection at 3.1 genome equivalents per reaction, with no amplification of 23 nontarget bacterial isolates. The assay was not inhibited by host spleen DNA or spleen homogenate. Methods were successfully applied to detect F.psychrophilum in rainbow trout from naturally occurring BCWD outbreaks and to quantify bacterial loads in experimentally infected rainbow trout. This assay will be applied to future studies to characterize disease pathogenesis in fish selectively bred for BCWD resistance.

A new LC-MS/MS method for the quantification of endogenous and vinyl chloride-induced 7-(2-Oxoethyl)guanine in sprague-dawley rats.

Vinyl chloride (VC) is an industrial chemical that is known to be carcinogenic to animals and humans. VC primarily induces hepatic angiosarcomas following high exposures (≥50 ppm). VC is also found in Superfund sites at ppb concentrations as a result of microbial metabolism of trichloroethylene and perchloroethylene. Here, we report a new sensitive LC-MS/MS method to analyze the major DNA adduct formed by VC, 7-(2-oxoethylguanine) (7-OEG). We used this method to analyze tissue DNA from both adult and weanling rats exposed to 1100 ppm [(13)C(2)]-VC for 5 days. After neutral thermal hydrolysis, 7-OEG was derivatized with O-t-butyl hydroxylamine to an oxime adduct, followed by LC-MS/MS analysis. The limit of detection was 1 fmol, and the limit of quantitation was 1.5 fmol on the column. The use of stable isotope VC allowed us to demonstrate for the first time that endogenous 7-OEG was present in tissue DNA. We hypothesized that endogenous 7-OEG was formed from lipid peroxidation and demonstrated the formation of [(13)C(2)]-7-OEG from the reaction of calf thymus DNA with [(13)C(18)]-ethyl linoleate (EtLa) under peroxidizing conditions. The concentrations of endogenous 7-OEG in liver, lung, kidney, spleen, testis, and brain DNA from adult and weanling rats typically ranged from 1.0 to 10.0 adducts per 10(6) guanine. The exogenous 7-OEG in liver DNA from adult rats exposed to 1100 ppm [(13)C(2)]-VC for 5 days was 104.0 ± 23.0 adducts per 10(6) guanine (n = 4), while concentrations in other tissues ranged from 1.0 to 39.0 adducts per 10(6) guanine (n = 4). Although endogenous concentrations of 7-OEG in tissues in weanling rats were similar to those of adult rats, exogenous [(13)C(2)]-7-OEG concentrations were higher in weanlings, averaging 300 adducts per 10(6) guanine in liver. Studies on the persistence of [(13)C(2)]-7-OEG in adult rats sacrificed 2, 4, and 8 weeks postexposure to [(13)C(2)]-VC demonstrated a half-life of 7-OEG of 4 days in both liver and lung.