Spleen DNA Research Articles

Rickettsia helvetica in C3H/HeN mice: A model for studying pathogen-host interactions

An infection with the tick-borne Rickettsia helvetica has been associated with a broad spectrum of clinical manifestations in humans, but patients are only seldomly reported. Understanding its disease etiology necessitates well-stablished infection models, improving to recognize and diagnose patients with R. helvetica infection and facilitating the development of effective control strategies. In this study, we used C3H/HeN mice as a model to establish R. helvetica infection, achieving a high infection prevalence (89–100 %). While the liver and the spleen DNA consistently tested positive for infection in all challenged mice, additional infected organs included the kidneys, heart, and the lungs. Notably, a low prevalence of infection was observed in I. ricinus nymphs fed on R. helvetica-challenged mice. In addition, larvae were refractory to infection, suggesting that ticks exhibit low susceptibility to the pathogen. To the best of our knowledge, this is the first study of an animal model for R. helvetica infection. It serves as a valuable tool for advancing research on the interactions among the bacterium and its vertebrate host.

Using clotted, pelleted blood samples for direct molecular detection of Bartonella spp. in small mammal wildlife surveillance studies

ObjectiveBartonella are emerging bacterial zoonotic pathogens. Utilization of clotted blood samples for surveillance of these bacteria in wildlife has begun to supersede the use of tissues; however, the efficacy of these samples has not been fully investigated. Our objective was to compare the efficacy of spleen and blood samples for DNA extraction and direct detection of Bartonella spp. via qPCR. In addition, we present a protocol for improved DNA extraction from clotted, pelleted (i.e., centrifuged) blood samples obtained from wild small mammals.ResultsDNA concentrations from kit-extracted blood clot samples were low and A260/A280 absorbance ratios indicated high impurity. Kit-based DNA extraction of spleen samples was efficient and produced ample DNA concentrations of good quality. We developed an in-house extraction method for the blood clots which resulted in apposite DNA quality when compared to spleen samples extracted via MagMAX DNA Ultra 2.0 kit. We detected Bartonella in 9/30 (30.0%) kit-extracted spleen DNA samples and 11/30 (36.7%) in-house-extracted blood clot samples using PCR. Our results suggest that kit-based methods may be less suitable for DNA extraction from blood clots, and that blood clot samples may be superior to tissues for Bartonella detection.

Molecular Survey of Piroplasmids and Hemosporidians in Vampire Bats, with Evidence of Distinct Piroplasmida Lineages Parasitizing Desmodus rotundus from the Brazilian Amazon

Although bats can serve as reservoirs for several viruses and bacteria, there is limited knowledge regarding the diversity of apicomplexan protozoan belonging to the Piroplasmida and Haemosporida orders within this group of mammals. The present study aimed to investigate the occurrence and phylogenetic assessment of piroplasmids and hemosporidians in spleen samples collected from 229 vampire bats (228 Desmodus rotundus and 1 Diaemus youngii) in the states of Pará, Roraima, Amapá, and Amazonas, northern Brazil. Out of 229 bat spleen samples, 43 (18.77%) tested positive in a nested PCR for piroplasmids based on the 18S rRNA gene. Thirteen sequences (ranging from 474 to 828 base pairs) of the partial 18S rRNA gene showed 91.04–100% identity to Theileria sp., Babesia sp., and Piroplasmida previously detected in deer, tapirs, opossums, and crab-eating raccoons. The phylogenetic analysis based on the near-complete 18S rRNA gene positioned the obtained sequences from three D. rotundus in distinct clades (Theileria sensu stricto, Tapirus terrestris, and “South America Marsupialia”). All bat spleen DNA samples tested negative in a nested PCR assay for hemosporidians based on the cytB gene. The present study reported, for the first time, the presence Babesia sp. and Theileria sp. DNA in D. rotundus. The distinct positioning of the 18S rRNA gene sequences within different clades demonstrates the occurrence of different piroplasmid species in vampire bats.

Effect of ionic strength on the interaction of AuNPs with calf spleen DNA

Gold nanoparticles (AuNPs) are well-known biomedical and biotechnological applications because of their interesting properties. They easily cross the cell membranes and interact with intracellular materials. This study was designed to investigate the interaction of calf spleen DNA with AuNPs at a molar ratio of 2:1 in an aqueous solution with different ionic strengths (10, 50, and 100%). AuNPs and AuNPs/DNA complex were characterized by different techniques such as UV/Vis spectrophotometry, transmission electron microscopy (TEM), dynamic light scattering (DLS), and Fourier transform IR spectrophotometry. The results revealed that the maximum absorption (λmax) of AuNPs synthesis was observed at 520 nm, and the average particle size was about 13 nm. In addition to a negative zeta potential (− 37 mV), the interaction of AuNPs with DNA was confirmed by melting point and TEM. The melting point that reflects the DNA became unstable in the presence of AuNPs, and the melting temperature decreased by about 3–5 °C with different ionic strength. Additionally, the TEM image of AuNPs/DNA complex obviously illustrated the location of AuNPs on the DNA groove. Finally, these results clearly indicate the attachment of AuNPs with DNA.

Recombinant Ehrlichia canis GP19 Protein as a Promising Vaccine Prototype Providing a Protective Immune Response in a Mouse Model

Simple summaryOne of the limitations of vaccine development against E. canis infection is the indefinite knowledge of the protective immunity in the host. In this study, recombinant protein GP19 was produced as a vaccine prototype, rGP19, for inducing protective immune responses in a mouse model against E. canis. Antibody responses against E. canis were evaluated and revealed that the immunized mice with rGP19 showed higher antibody levels than in adjuvant-immunized and naive mice, both pre- and post-challenging with E. canis. DNA from blood, liver, and spleen were extracted to determine ehrlichial loads. The rGP19-immunized mice showed significantly lower ehrlichial loads in blood, liver, and spleen DNA compared with adjuvant-immunized mice. This study also detected IFN-γ-producing CD4+ T cells in the rGP19-immunized mice and then were later infected with E. canis on day 14 of the post-infection period using flow cytometry. Additionally, Cytokine mRNA expression was investigated and revealed up-regulation of IFNG and IL1 mRNA expression in rGP19-immunized mice. The present study provides evidence of rGP19 that can eliminate E. canis by manipulating both humoral and cell-mediated immune responses in the laboratory animal model.The intracellular bacterium Ehrlichia canis is the causative pathogen of canine monocytic ehrlichiosis (CME) in dogs. Despite its veterinary and medical importance, there is currently no available vaccine against this pathogen. In this study, the recombinant GP19 (rGP19) was produced and used as a recombinant vaccine prototype in a mouse model against experimental E. canis infection. The efficacy of the rGP19 vaccine prototype in the part of stimulating B and T cell responses and conferring protection in mice later challenged with E. canis pathogen were evaluated. The rGP19-specific antibody response was evaluated by ELISA after E. canis challenge exposure (on days 0, 7, and 14 post-challenge), and demonstrated significantly higher mean antibody levels in rGP19-immunized mice compared with adjuvant-immunized and naive mice. Significantly lower ehrlichial loads in blood, liver, and spleen DNA samples were detected in the immunized mice with rGP19 by qPCR. The up-regulation of IFNG and IL1 mRNA expression were observed in mice immunized with rGP19. In addition, this study detected IFN-γ-producing memory CD4+ T cells in the rGP19-immunized mice and later infected with E. canis on day 14 post-infection period using flow cytometry. The present study provided a piece of evidence that rGP19 may eliminate E. canis by manipulating Th1 and B cell roles and demonstrated a promising strategy in vaccine development against E. canis infection in the definitive host for further study.

HIV-1 but not SIVmac239 induces higher interferon-α antiviral state in chronic infected northern pig-tailed macaques (Macaca leonina)

Studies have shown that interferon (IFN)-α has an inhibitory effect on human immunodeficiency virus type 1 (HIV-1) replication in the acute infection stage, but its role in chronic infection is still unclear. We previously established a nonpathogenic HIV-1 and pathogenic simian immunodeficiency virus (SIV) model in northern pig-tailed macaques (NPMs, Macaca leonina). In the current study, we detected viral RNA and DNA in various tissues (axillary lymph nodes (LNs), inguinal LNs, and spleen) in HIV-1NL4-3- and SIVmac239-infected NPM during the chronic stage of infection. Results indicated that the levels of viral DNA and RNA were higher in the tested tissues (LNs and spleen) of the SIVmac239-infected NPMs than in the HIV-1NL4-3 infected NPMs. Furthermore, IFN-α expression was higher in the HIV-infected tissues than in the SIV-infected controls. The HIV restriction factors induced by IFN-α (i.e., tetherin and MX2), as well as inflammatory factors IFN-γ, tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6), were analyzed using real-time polymerase chain reaction (PCR) and immunofluorescence staining assays. Results showed that their expression levels were much higher in the HIV-infected tissues than in the SIV-infected controls. These findings were confirmed by in vitro experiments on healthy NPM peripheral blood mononuclear cells infected with HIV-1NL4-3, which showed lower viral replication, higher IFN-α expression, and an antiviral status. This study demonstrated that HIV-1 infection, but not SIVmac239 infection, in NPMs caused higher expression of IFN-α and induced a higher antiviral status. This may be one of the reasons why HIV-1 cannot replicate at a high level or develop into AIDS in NPMs.

Pathology of Coinfection of Mycoplasma hyorhinis and Aerococcus urinae Causing FiBrinous Pericarditis in a Piglet - A Case Report

Fibrinous pericarditis due to co infection with Mycoplasma hyorhinis and Aerococcus urinae is rarely reported in pigs. In this case report, a one-month crossbreed male piglet with sudden death was diagnosed with fibrinous pericarditis based on necropsy, microbiological and PCR assay. The thoracic cavity was filled with large amounts of serosanguineous fluids mixed with fibrins and thickened pericardial sac adhered to the epicardium looked like bread-and-butter appearance. The cranioventral lobes of lungs showed consolidation with fibrinous exudate. Histopathology of epicardium showed fibrocellular exudates and lungs showed hyperplastic changes with congestion. The antigen of Mycoplasma hyorhinis was demonstrated in heart and lungs. Bacteriological analysis of the heart blood and spleen showed the pure growth of Aerococcus urinae. The genomic DNA of heart, lungs and spleen showed positive amplification of Mycoplasma hyorhinis targeting 16srRNA gene. The present case was diagnosed as fibrinous pericarditis in piglet due to M. hyorhinis with coinfection of Aerococcus urinae, which is often overlooked as a risk factor.

Tissue‐specific differences in detection of Yersinia ruckeri carrier status in rainbow trout (Oncorhynchus mykiss)

Effective monitoring for subclinical infections is a cornerstone of proactive disease management in aquaculture. Salmonid fish that survive enteric redmouth disease (ERM) can carry Yersinia ruckeri as a latent infection for several months, potentially facilitating cryptic spread between facilities that exchange fish. In this study, fingerling rainbow trout (Oncorhynchus mykiss) were infected by immersion and sampled for up to 14weeks post-infection. Yersinia ruckeri was cultured from the posterior kidney of more than 89% of fish up to 4weeks post-infection, but from 2% or fewer of fish sampled at later time points. In contrast, qPCR-based detection of the Y.ruckeri 16s rRNA gene in intestine and spleen extracts revealed a much higher rate of infection: at 14weeks post-infection Y.ruckeri was detected in nearly 50% of spleens and 15% of intestines. The difference between spleen and intestine is likely due at least in part to technical limitations of qPCR on intestinal DNA extracts; accordingly, we propose that qPCR of spleen DNA ought to be considered the preferred standard for detection of carriers of Y.ruckeri.

Clonal Evolution and Sub-Clonal Architecture In Paediatric T-Cell Acute Lymphoblastic Leukaemia (T-ALL)

IntroductionThe issue of clonal heterogeneity in leukaemia has diverse implications for disease monitoring, targeted therapy design and understanding the biology of disease relapse. In this study, we aimed to perform single cell analysis to improve understanding of clonal evolution in T-ALL. We used fluorescence in situ hybridisation (FISH) and phylogenetic analysis of diagnostic T-ALL samples to infer the order of acquisition of genetic events and define the early and likely founder events in this subtype of leukaemia. Screening of paired diagnostic and xenograft material from two STIL-TAL1 positive cases allowed analysis of clonal heterogeneity and evolution in leukaemia initiating cells. MethodsDNA from seven diagnostic T-ALL patients aged 1 – 25 years (five STIL-TAL1 fusion positive, one TLX1 translocation positive, one TLX3 translocation positive) was screened using the Affymetrix® SNP 6.0 platform (analysed using CNAG version 3.3.0 software) and standard Sanger Sequencing to detect genome wide copy number alterations (CNA), mutations in NOTCH1 (exons 26, 27, 34) FBXW7 (exons 9,10), PTEN (exon 7) and IL7R (exon 6) and to define the genomic DNA sequence of the STIL-TAL1 fusion in relevant cases. Genomic localisation of CNA was used to design patient-specific FISH probes for the STIL-TAL1 fusion and losses of CDKN2A, PTEN, LEF1, SYNCRIP (marker of 6q deletion), MYC (marker of chromosome 8 gain) and ETV6 (marker of 12p loss). TLX1 status was assessed using a commercial Cytocell FISH probe and TLX3 status using an in-house probe set. Interphase FISH scoring of one to two hundred methanol-acetic acid fixed nuclei per patient with four - five differentially labelled FISH probes allowed phylogenetic analysis and reconstruction of the evolution of the malignant clone. In two of the STIL-TAL1 cases, we completed xenograft transplants using NOD/ShiSCID/IL2Rgamma null (NOG) mice. Mutation screening, copy number analysis and FISH were performed on xenograft spleen DNA and methanol-acetic acid fixed spleen nuclei. ResultsConstruction of phylogenetic trees for each patient based on single cell four - five colour FISH analysis demonstrated the presence of two - four abnormal sub-clones per patient. Both linear clonal progression and branching sub-clonal patterns were observed. STIL-TAL1 fusion, TLX1 and TLX3 translocations were placed at the root of each phylogenetic tree as they were present in all cells with an abnormal FISH pattern signal; the majority (but not all) cells also demonstrated loss of CDKN2A. Copy number losses of PTEN and LEF1 as well as losses of 12p and 6q and gain of chromosome 8 occurred at a lower level than CDKN2A loss. Sequencing of the STIL-TAL1 fusion sequence in the two paired diagnosis/xenograft cases (A and B) demonstrated that the fusion sequence was conserved in the xenografts. Case A harboured a NOTCH1 PEST domain truncating mutation detected in the xenograft. Sequencing of the diagnostic sample was suggestive of the presence of the same mutation at low level. At diagnosis, case B was found to have a PTEN exon 7 truncating mutation which was not detectable by Sanger Sequencing in the xenograft. In case B, the dominant diagnostic clone assessed by FISH had the STIL-TAL1 fusion plus losses of CDKN2A, 4p and 6q, compared to the dominant clone in the paired xenograft, which harboured the STIL-TAL1 fusion and loss of CDKN2A but retained both copies of 4p and 6q. This dominant xenograft clone was present at low level in the diagnostic sample. ConclusionsThese results support the hypothesis that, in accordance with other types of leukaemia, intra or inter-chromosomal rearrangements (STIL-TAL1 fusion, TLX1 and TLX3 translocation) are early events. As the STIL-TAL1 fusion was present in all cells with an abnormal FISH signal pattern in all five cases and the fusion sequence was conserved in the xenograft transplants, this provides evidence to support the hypothesis that it is a founder event in the development of T-ALL. CDKN2A loss also occurred early in the evolution of the leukaemogenic clone, in line with its key role in T-ALL. Assessment of the order of acquisition of genetic events in T-ALL and their stability as evolution of the malignant clone unfolds are key factors to determine rational treatment targets for molecularly targeted therapies. Future work will incorporate mutations and gene fusions in to the single cell phylogenetic analysis using a multiplex Q-PCR approach. Disclosures:Ferrando:Pfizer: Research Funding; BMS: Research Funding.

Pterocarpus santalinus L. extract mitigates gamma radiation-inflicted derangements in BALB/c mice by Nrf2 upregulation

Plant-based natural extracts contain several nutrients and bioactive compounds, such as phenolics and flavonoids, that possess various health-promoting activities. This study investigated the effects of polyphenols from Pterocarpus santalinus hydroalcoholic extract (PSHE) against gamma radiation-induced derangements via the upregulation of Nrf2. Ultra High Performance Liquid Chromatography Coupled to High Resolution Mass Spectrometry (UHPLC-HRMS/MS) analysis was performed to identify the possible radioprotectors. In vivo and in vitro studies, namely Real-Time-PCR (RT-PCR) analysis, Reactive Oxygen Species (ROS) scavenging activity, lipid peroxidation and GSH levels, DNA damage and cell death studies, anti-inflammatory (Sandwich ELISA), immunomodulatory studies (antibody staining), and model free radical scavenging assays, were performed. Vanillic acid, protocatechuic acid, para-hydroxybenzoic acid, chlorogenic acid, TNF-α inhibitor (Eudesmin), isoflavone (Daidzein 7-o-glucoside), astragalin (Kaempferol 3-o-glycoside), and other polyphenols were identified in PSHE using UHPLC-HRMS/MS analysis. Prophylactic administration of PSHE (−1 h) rendered more than 33% survival in mice exposed to 8 Gy whole-body-irradiation with increased mice survival and recovery of bone marrow and spleen cellularity. Real-time RT-PCR analysis showed that PSHE treatment (50 µg/mL) upregulated Nrf2, HO-1, and GPX-1 in mice splenocytes. At 50 µg/mL, PSHE reduced ROSscavenging activity, mitochondrial and spleen membrane lipid peroxidation levels, DNA damage, and cell death, and increased GSH levels. At 10 µg/mL, PSHE treatment diminished the content of IL-6 and TNF-α. At 50 µg/mL, PSHE suppressed lymphocyte proliferation. These findings indicate that polyphenols of PSHE possess marked antioxidant, anti-inflammatory, and immunomodulatory capacities, which play important roles in the prevention of radiation damage.

Molecular detection of Anaplasma phagocytophilum in hair and spleen of cats revealed a possible underestimation of feline vector-borne pathogens

Feline Vector-Borne Diseases show increased global prevalence and some Anaplasma and Ehrlichia species may pose a risk to human health. The diagnosis of Anaplasma and Ehrlichia species infection in cats is achieved by the combined use of different methods as cytologic examination evidencing intracytoplasmic morulae, serologic tests and molecular assays. The peripheral whole blood is considered the sample of choice for Anaplasma and Ehrlichia species DNA detection in cats, but false negative results are reported leading to underestimation of infection prevalence. In order to have a more accurate assessment of the spread of feline vector-borne pathogens, the presence of Anaplasma spp. and Ehrlichia spp. DNA in 37 owner and shelter cats subjected to necropsy were prospectively investigated by testing in end-point PCR spleen, bone marrow, blood clot and hair samples. The bacteria identified were genetically characterised. Three shelter cats tested positive for A. phagocytophilum DNA in spleen (one cat) or in hair samples (two cats). None of the cats tested positive in bone marrow and blood samples. From the results obtained, it can be assumed that the use of spleen or hair samples could allow a more reliable detection of A. phagocytophilum DNA in cats with blood tested negative. In the phylogeny constructed with a fragment of the heat shock (groEL) gene nucleotide sequences, all the identified A. phagocytophilum clustered with bacteria infecting a wide range of hosts, including humans, showing a potential zoonotic role.

Molecular Evidence of Bartonella spp. in Rodents: A Study in Pianosa Island, Italy.

Simple SummaryInvasive rats and field mice were captured under the project RESTO CON LIFE “Island conservation in Tuscany, restoring habitat not only for birds”, aiming to improve habitats preservation on the Italian islands of Pianosa, Elba, Montecristo, and Giannutri. Bartonella henselae DNA was detected in one captured animal. This is the first report of the presence of B. henselae DNA in rodents in Italy. B. henselae is the causative agent of cat scratch disease, and the detection of this bacterium in rodents might have public health implications. Wild rodents are reservoirs of several Bartonella species that cause human bartonellosis. The aim of this study was to assess the presence of Bartonella spp. DNA in wild rodents in Pianosa island, Italy. Rats (Rattus spp.; n = 15) and field mice (Apodemus spp.; n = 16) were captured and spleen DNA tested for the presence of Bartonella spp. by means of an initial screening using a qPCR amplifying a short segment of the 16S-23S rRNA gene intergenic transcribed spacer region (ITS, ~200 bp) followed by conventional PCR amplification of a longer ITS fragment (~600 bp) and of a citrate synthase (gltA, ~340 bp) gene segment. A total of 25 spleen DNA samples obtained from 31 rodent carcasses (81%) yielded positive qPCR results. Bartonella genus was confirmed by amplicon sequencing. By conventional PCR, eight out of 25 samples (32%) yielded bands on gels consistent with ITS segment, and 6/25 (24%) yielded bands consistent with the gltA locus. Amplicon sequencing identified B. henselae and B. coopersplainsensis in 1/25 (4%), and 4/25 (16%) samples, respectively. Moreover, 5/25 (20%) of Bartonella spp. positive samples showed gltA sequences with about 97% identity to B. grahamii. These results provide support to recently published observations suggesting that B. henselae circulates in wild rodent populations.

The red fox (Vulpes vulpes), a possible reservoir of Babesia vulpes, B. canis and Hepatozoon canis and its association with the tick Dermacentor reticulatus occurrence

The red fox (Vulpes vulpes) is known to be a reservoir host of various vector-borne protozoan parasites. Yet, the impact of these parasitic infections on the health status of the red fox is unknown. In this study, we compared the prevalence of haemoprotozoa in juvenile and adult foxes from four regions in Poland. The abundance of questing adult Dermacentor reticulatus ticks was shown to differ between regions and include a tick-endemic and non-endemic region, as well as two zones in which D. reticulatus recently expanded: an eastern and a western zone. Spleen and kidney enlargement indices were compared between infected, co-infected and non-infected foxes to evaluate pathogenic output of parasitic infections.Blood samples were collected from 383 necropsied red foxes and 25 live-trapped red fox cubs. The weights of spleens, kidneys and bodies were recorded for each necropsied individual. Blood and spleen DNA samples were screened by PCR to detect 18S rRNA and cox1 gene fragments of Babesia spp., and 18S rRNA of Hepatozoon spp. Prevalence of infection and mean organ weight indices were compared in relation to fox age and sex, origin (zone) and infection status.Hepatozoon canis (174/381; 45.7%) and Babesia vulpes (117/381; 30.7%) were the dominant pathogens infecting adult foxes. Babesia canis DNA was detected in 9 (2.4%) individuals. Two fox cubs from Western Poland were infected with B. vulpes (2/14; 14%), while H. canis infection (16/25; 59%) was detected in cubs from all three regions where trapping was carried out. Infection with B. vulpes was associated with significantly higher weights of spleens and kidneys. Spleen weight/ body weight and mean kidney weight/ body weight indices were shown as relevant in evaluating changes occurring during infection with B. vulpes.Babesia and Hepatozoon spp. infections differed in red foxes from D. reticulatus-endemic, non-endemic and newly inhabited areas. The prevalence of B. canis in foxes from four regions of Poland reflects the prevalence recorded in questing adult D. reticulatus collected in these areas. This finding suggests a contribution of red foxes to the establishment of new foci of B. canis-infected D. reticulatus ticks in areas recently invaded by this tick species. Spleen and kidney enlargement was identified in foxes infected with B. vulpes, particularly in individuals co-infected with B. vulpes and H. canis.

Abstract A06: Impact of cigarette smoke exposure on acute myeloid leukemia progression

Abstract Cigarette smoke is known to cause lung cancer but is also associated with increased incidence of leukemia. Acute myeloid leukemia (AML) is the most lethal form of leukemia, and patients who are or were smokers have increased risk of developing AML and have worse prognosis. There is also an increased incidence of AML in children whose parents smoked either during or after the pregnancy. It is currently unknown how cigarette smoke exposure (CSE) may be impacting leukemic progression or treatment efficacy in AML. Cigarette smoking increases levels of reactive oxygen species (ROS) in the tissue of smokers, including peripheral blood samples. Oxidative stress can alter epigenetic signaling, including DNA methylation. Oxidation of methylated DNA and oxidative lesions on DNA can alter normal DNA methylation patterns. Smokers have altered DNA methylation that can be retained decades after the cessation of the habit, and these changes can be found in the blood cells of smokers. Cigarette smoke condensate (CSC) treatment has also been seen to epigenetically prime nontransformed human bronchial cells for malignant transformation. These occurrences have been seen in the context of lung cancer and lung damage, but studies into other tissue damage have largely been overlooked. Thus, we hypothesize that CSE impacts leukemic progression and treatment in AML through these mechanisms. We have developed a model to study the impact of CSE on AML in mice. NOD-SCID mice are smoke exposed for two weeks prior to being engrafted with luciferase-labeled human AML cells. The CSE is performed in the SCIREQ Cigarette Smoking Robot, with 3R4F research cigarettes 5 days/week. We include experimental arms of smoking cessation (who stop once evidence of leukemic engraftment is detected) and continued smoking, to model patients who either stop or continue smoking upon diagnosis of AML. Leukemic burden is quantified through bioluminescent imaging throughout the experiment. From these models, we have observed that two weeks of CSE is sufficient to significantly increase (p-value<0.0001) early leukemic burden as compared to non-smoke exposed mice, in FLT3-ITD AML-bearing mice. Spleen samples revealed trends of increased levels of superoxides in the CSE mice. Currently, immunohistochemistry is being performed on collected organs to detect oxidative stress markers. Additionally, mouse spleen DNA underwent reduced representative bisulfide sequencing; the levels of DNA methylation in the mouse host cells and in the leukemia are compared between our CSE groups. This is the first description of CSE promoting leukemia growth in mouse model, which will enable the study of mechanistic aspects. This may help define if unique treatment strategies are needed for these patients. Citation Format: Yue Lu, Marcos Estecio, Marina Konopleva, Elias Jabbour, Joya Chandra, Mary Figueroa. Impact of cigarette smoke exposure on acute myeloid leukemia progression [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A06.

Quantitative polymerase chain reaction offers improved diagnostic and analytical sensitivity in identifying differential microsporidium (Loma morhua) infections among family lines of cultured Atlantic cod (Gadus morhua)

The failed attempt to establish Atlantic cod as an alternate aquaculture species in eastern Canada is due in part to hatchery outbreaks of the microsporidium Loma morhua during selective breeding programs. The sporadic nature of microsporidian epizootics during fish culture emphasizes the need for streamlined quantitative diagnostic approaches that help promote development of disease mitigation strategies. The current gold standard for assessing infection status relies on proficiency with parasitological confirmation of hypertrophied host cells (xenomas) that house infectious microsporidian spores. Parasite quantification is complicated with certain host organs showing better diagnostic utility, xenomas being heterogeneously distributed throughout these tissues, variable numbers of spores occurring within xenomas, and with pre-xenoma life-stages being difficult to identify using gold standard procedures. Herein, individuals from ten cod families with differential susceptibility to L. morhua were analyzed using a quantitative PCR (qPCR) assay, developed in accordance to the Minimum Information Required for Quantitative Experiments (MIQE) guidelines, to assess assay utility in quantifying infections relative to the gold standard procedure. Heterogeneous parasite distribution within spleens necessitated development of a tissue disruption protocol (FastPrep homogenizer; MP Biomedical) yielding a homogenous preparation for standardized DNA extractions. qPCR primers and a hydrolysis probe targeting a region of large subunit ribosomal DNA (rDNA) enable L. morhua to be distinguished from congeneric species infecting non-gadid hosts, but not those infecting gadid hosts. Parasite rDNA copies were normalized to Atlantic cod glyceraldehyde-3-phosphate dehydrogenase genomic DNA and to spleen weight. The qPCR assay demonstrates linearity (R2 = 0.971 to 0.999; range) throughout a large range of five log10 plasmid concentrations (102 to 106 titrated in host DNA), low variability (CV = 11.2–23.2%; n = 11 separate reactions), and the lower limit of detection is 10 plasmid copies. Quantification of parasite rDNA among ten cod families with differential infections resulted in a positive correlation with gold standard xenoma enumeration/confirmation (Pearson Correlation, r = 0.71), thereby validating effectiveness of qPCR in the absence of parasitological expertise. Furthermore, three fish lacking morphologically discernible spleen xenomas were identified as false negatives based on qPCR detection of 141–242 rDNA copies per 200 ng of spleen DNA, thereby confirming infection. This finding extends qPCR assay utility to detection of even subclinical infections. This qPCR assay improves diagnostic and analytical sensitivity in detecting L. morhua infections, facilitates empirical assessment of preventative/therapeutic treatment of microsporidian infections, and supports studies to identify biomarkers that promote selection of broodstock fishes that resist microsporidian disease.

Molecular detection of Hepatozoon spp. in non-hematophagous bats in Brazil

Even though Hepatozoon spp. has been molecularly detected in several wild animals in Brazil, there is no report on the occurrence of Hepatozoon spp. DNA in bats in Brazil. This study aimed at detecting Hepatozoon, in addition to ectoparasites, in non-hematophagous bats sampled in central-western Brazil using blood smears, hematoxylin-eosin (HE)-staining liver/spleen preparations and molecular and phylogenetic techniques. A total of 135 spleen, 127 liver, and 133 blood samples were collected from 135 non-hematophagous bats from 12 different species in two different sites in Campo Grande, Mato Grosso do Sul state, in the Brazilian Cerrado region. Spleen and blood DNA samples were submitted to two conventional PCR protocols for Hepatozoon spp. based on 18S rRNA. No Hepatozoon spp. gamonts or meronts were observed in blood smears and HE-stained-liver preparations, respectively. While none of the spleen samples was positive for Hepatozoon spp. in the PCR assays, 5 (3 %) blood samples contained 18S rRNA Hepatozoon DNA, including 2/37 (5 %) Artibeus lituratus, 2/32 (6 %) A. planirostris, and 1/23 (4 %) Platyrrhinus lineatus. Out of 5 bats positive for Hepatozoon spp., 3 were parasitized by either Macronyssidae/Spinturnicidae mites or Streblidae flies. BLAST analysis showed that the sequences detected in bats had >99 % identity with Hepatozoon sequences detected in amphibians and reptiles from Brazil, including Hepatozoon caimani detected in Caiman crocodilus. The phylogenetic inferences estimated by the Maximum Likelihood and Bayesian methods clustered the Hepatozoon sequences detected in Brazilian bats with those detected in reptiles and amphibians.

Wild mammals as potential silent reservoirs of Leishmania infantum in a Mediterranean area

A molecular survey of wild mammals was performed to assess their potential as reservoirs of L. infantum. A total of 156 specimens of wild mammalian fauna were obtained for analysis from areas in Catalonia with a reported incidence of canine leishmaniasis. They consisted of 124 small mammals: 35 Mus spretus (Muridae); 64 Erinaceus europaeus (Erinaceidae), 25 Sciurus vulgaris (Sciuridae) and 32 carnivores: 11 Vulpes vulpes (Canidae), 1 Felis catus (Felidae), 15 Meles meles, 4 Martes foina and 1 Mustela vison (Mustelidae). The analysis was performed on samples of liver, spleen, skin (ear) and blood extracted from the heart. Leishmania DNA was determined by a qPCR and specific anti-Leishmania antibodies were detected by an in-house (ELISA).Among the 156 specimens studied, 29.48% were positive in at least one of the samples studied and considered infected. In M. spretus, Leishmania DNA was detected in the liver, spleen or skin of 37.1% of 35 specimens, and 2 of the 13 specimens tested serologically were positive (15.38%). In E. europaeus, 34.4% of the 64 specimens were infected. Leishmania DNA was detected in 19/51 spleens and 5/50 skins; 2 of the 37 specimens analysed in both spleen and skin gave positive results in both samples. Serology was positive in 12.8% (6/47) by ELISA; 3 specimens were positive by both ELISA and qPCR. In S. vulgaris, Leishmania DNA was detected in 5 of 25 specimens (20%). Of the 32 carnivore specimens analysed, Leishmania DNA was detected in both samples studied (spleen and liver) of 4 (12.5%) (2 M. foina, 1 M. vison and 1 F. catus), which were not studied serologically.The data obtained indicate that small mammals, above all wild rodents and carnivores, could act as naturally infected hosts of L. infantum in this endemic area. Among the rodents, M. spretus stands out with the highest prevalence of infection. In E. europaeus, the presence of L. infantum DNA in spleen and skin, and antibodies in heart blood, reported here for the first time, indicates this small mammal could be a possible reservoir. Additionally, S. vulgaris, not previously studied as an L. infantum reservoir, showed non-negligible prevalence values, indicating a potential role in leishmaniasis transmission.

Highly prevalent bartonellae and other vector-borne pathogens in small mammal species from the Czech Republic and Germany

BackgroundRodents are important reservoirs for zoonotic vector-borne agents. Thus, the distribution of rodents and their vicinity to humans and companion animals may have an important impact on human and animal health. However, the reservoir potential of some rodent genera, e.g. Microtus, has not yet been precisely examined concerning tick-borne pathogens in Central Europe. Therefore, we examined small mammals from Germany and the Czech Republic for the following vector-borne pathogens: Babesia spp., Bartonella spp., Anaplasma phagocytophilum, “Candidatus Neoehrlichia mikurensis” (CNM) and Coxiella burnetii. Spleen DNA from 321 small mammals belonging to four genera, Myodes (n = 78), Apodemus (n = 56), Microtus (n = 149), Sorex (n = 38), collected during 2014 in Germany and the Czech Republic were available for this study. DNA samples were examined for the presence of Babesia and Bartonella DNA by conventional PCR targeting the 18S rRNA gene and the 16S–23S rRNA intergenic spacer region, respectively. For the detection of CNM, A. phagocytophilum and C. burnetii real-time PCR assays were performed.ResultsBartonella spp. DNA was detected in 216 specimens (67.3%) with 102/174 (58.6%) positive in Germany and 114/147 (77.6%) in the Czech Republic. The prevalence in each genus was 44.9% for Myodes, 63.2% for Sorex, 77.2% for Microtus and 75% for Apodemus. Four Bartonella species, i.e. Bartonella sp. N40, B. grahamii, B. taylorii and B. doshiae, as well as uncultured bartonellae, were detected. The Bartonella species diversity was higher in rodents than in shrews. In total, 27/321 (8.4%) small mammals were positive for CNM and 3/321 (0.9%) for A. phagocytophilum (S. coronatus and M. glareolus). All samples were negative for Babesia spp. and Coxiella spp.ConclusionsWhile the detected high prevalence for Bartonella in Apodemus and Myodes spp. is confirmatory with previous findings, the prevalence in Microtus spp. was unexpectedly high. This indicates that individuals belonging to this genus may be regarded as potential reservoirs. Interestingly, only Sorex spp. and M. glareolus were positive for A. phagocytophilum in the present study, suggesting a possible importance of the latter for the maintenance of certain A. phagocytophilum strains in nature.

Development of a Multiplexed Assay for Detection of Leishmania donovani and Leishmania infantum Protein Biomarkers in Urine Samples of Patients with Visceral Leishmaniasis.

Visceral leishmaniasis (VL) is a serious and fatal disease caused by the parasites Leishmania infantum and Leishmania donovani The gold standard diagnostic test for VL is the demonstration of parasites or their DNA in spleen, lymph node, or bone marrow aspirates. Serological tests exist but cannot distinguish active VL from either prior exposure to the parasites or previously treated VL disease. Using mass spectroscopy, we have previously identified three L. infantum protein biomarkers (Li-isd1, Li-txn1, and Li-ntf2) in the urine of VL patients and developed a sensitive and specific urine-based antigen detection assay for the diagnosis of VL that occurs in Brazil (where VL is caused by L. infantum). However, unpublished observations from our laboratory at DetectoGen showed that these biomarkers were detected in only 55% to 60% of VL patients from India and Kenya, where the disease is caused by L. donovani Here, we report the discovery and characterization of two new biomarkers of L. donovani (Ld-mao1 and Ld-ppi1) present in the urine of VL patients from these two countries. Capture enzyme-linked immunosorbent assays using specific rabbit IgG and chicken IgY were developed, and the assays had sensitivities of 44.4% and 28.8% for the detection of Ld-mao1 and Ld-ppi1, respectively. In contrast, a multiplexed assay designed to simultaneously detect all five leishmanial biomarkers markedly increased the assay sensitivity to 82.2%. These results validate the utility of leishmanial protein biomarkers found in the urine of VL patients as powerful tools for the development of an accurate diagnostic test for this disease.

Identification of a Novel Gammaherpesvirus in Canada lynx (Lynx canadensis).

Gammaherpesviruses (GHVs) infect many animal species and are associated with lymphoproliferative disorders in some. Previously, we identified several novel GHVs in North American felids; however, a GHV had never been identified in Canada lynx (Lynx canadensis). We, therefore, hypothesized the existence of an unidentified GHV in lynx. Using degenerate nested and subsequently virus-specific PCR, we amplified and sequenced 3.4 kb of DNA from a novel GHV in lynx, which we named Lynx canadensis gammaherpesvirus 1 (LcaGHV1). Phylogenetic analysis determined that LcaGHV1 is a distinct GHV species belonging to the genus Percavirus. We then estimated the prevalence of LcaGHV1 in lynx by developing a PCR-based assay and detected LcaGHV1 DNA in 36% (95% CI: 22–53%) of lynx spleen DNA samples from Maine, USA and 17% (95% CI: 8–31%) from Newfoundland, Canada. The LcaGHV1 DNA sequences from Maine and Newfoundland lynx were nearly identical to each other (two nucleotide substitutions in 3.4 kb), suggesting that the unique lynx subspecies present on the island of Newfoundland (Lynx canadensis subsolanus) is infected with virus that very closely resembles virus found in mainland lynx. The potential ecologic and pathologic consequences of this novel virus for Canada lynx populations warrant further study.