Expression and antigenic analysis of the recombinant TRP36 protein from Ehrlichia canis São Paulo strain for serologic tests

Ehrlichia canis is the main etiological agent of canine monocytic ehrlichiosis (CME), a globally canine infectious disease. In Brazil, CME is considered to be endemic, and its prevalence can reach 65% in some states. The diagnosis of ehrlichiosis is important for treatment and epidemiological purposes. The E. canis TRP36 (Tandem Repeat Protein) protein elicits the earliest acute-phase antibody response observed during the course of the disease. This study aimed to generate the recombinant TRP36 protein from E. canis São Paulo strain and to evaluate its potential as a tool for the serologic diagnosis of CME. The E. canis São Paulo isolate was cultivated in DH82 lineage cells, and its genomic DNA was obtained. The bacterial DNA fragment encoding the entire ORF of TRP36 was cloned into the pBAD/Thio-TOPO vector and transformed into Escherichia coli DH10B competent cells with the trp36-bearing plasmid for protein expression. To evaluate the protein antigenicity, 16 canine serum samples were previously tested (by PCR and the commercial SNAP®4Dx® serological test). The results were in accordance with the SNAP®4Dx® test. Experiments using this recombinant protein as an antigen, targeting the development of a serologic test based on ELISA methodology, are the next step to produce a reliable, affordable and useful diagnostic tool for CME in Brazil.

Immunogenic Properties of Recombinant Enzymes from Bothrops ammodytoides towards the Generation of Neutralizing Antibodies against Its Own Venom

Bothropic venoms contain enzymes such as metalloproteases, serine-proteases, and phospholipases, which acting by themselves, or in synergism, are the cause of the envenomation symptoms and death. Here, two mRNA transcripts, one that codes for a metalloprotease and another for a serine-protease, were isolated from a Bothrops ammodytoides venom gland. The metalloprotease and serine-protease transcripts were cloned on a pCR®2.1-TOPO vector and consequently expressed in a recombinant way in E. coli (strains Origami and M15, respectively), using pQE30 vectors. The recombinant proteins were named rBamSP_1 and rBamMP_1, and they were formed by an N-terminal fusion protein of 16 amino acid residues, followed by the sequence of the mature proteins. After bacterial expression, each recombinant enzyme was recovered from inclusion bodies and treated with chaotropic agents. The experimental molecular masses for rBamSP_1 and rBamMP_1 agreed with their expected theoretical ones, and their secondary structure spectra obtained by circular dichroism were comparable to that of similar proteins. Additionally, equivalent mixtures of rBamSP_1, rBamMP_1 together with a previous reported recombinant phospholipase, rBamPLA2_1, were used to immunize rabbits to produce serum antibodies, which in turn recognized serine-proteases, metalloproteases and PLA2s from B. ammodytoides and other regional viper venoms. Finally, rabbit antibodies neutralized the 3LD50 of B. ammodytoides venom.

Diplotype Analysis of NUDT15 Variants By Targeted Sequencing in Pediatric Acute Lymphoblastic Leukemia

Background: The NUDT15 polymorphisms, V18_V19insGV and R139C, located on exon 1 and exon 3 were associated with intolerance of 6-mercaptopurine (6MP), which were frequently used during the maintenance therapy of childhood acute lymphoblastic leukemia (ALL). However, these two NUDT15 diplotypes with significant different biological activity, mono-allelic *1/*2 and complex homozygosity *3/*6, could not be distinguished from each other by direct Sanger sequencing of exon 1 and exon 3. We applied targeted sequencing of NUDT15 cDNA to clarify the diplotypes of complex homozygous of NUDT15. Methods: Thirty-five ALL patients who were diagnosed in National Taiwan University Hospital with both V18_V19insGV and R139C of NUDT15 were included in this study. A single amplicon (562 bp) covers all the coding regions of NUDT15 was amplified from cDNA and then sequenced by paired-end sequencing using MiSeq reagent kit v3 (600 cycles). For validation, the amplicons from complex homozygous NUDT5 were cloned into pCR-Blunt II-TOPO vector. Result: By targeted sequencing, thirty-three patients were identified as mono-allelic *1/*2. Two complex homozygous NUDT15, *3/*6 and *2/R34T, were identified in patient No.27 and No.37. We also cloned amplicons from these two patients into vector and the results were the same as targeted sequencing. These two patients with complex homozygous NUDT15 variants tolerated only 1~2 mg/m2 daily mercaptopurine doses. Conclusions: Targeted sequencing of NUDT15 cDNA is a simple method to determine the NUDT15 diplotype and helpful for the identifications of complex homozygous NUDT15, which should be identified before the administration of 6MP to avoid excessive myelosuppression caused by improper of 6MP dosage. Figure: (A) NUDT15 haplotypes. (B) The primers is used for direct Sanger sequencing and cDNA targeted sequencing. (C) The NUDT15 diplotype of patient No.27 is analyzed using cDNA cloning followed by Sanger sequencing. Figure Disclosures No relevant conflicts of interest to declare.

Cloning and optimizing the expression of the DHDPS gene in the Medicago truncatula

Medicago truncatula seeds were cultured and developed in Thua Thien Hue province, Vietnam, and they were used as materials for cloning a DHDPS gene with the encoding of the isozyme dihydrodipicolinate synthase (DHDPS) as well as optimizing the culture conditions for having the highest DHDPS gene expression in Escherichia coli BL21 StarTM (DE3) cells. The results revealed that the coding region of the DHDPS gene from M. truncatula was 100% similar with M. truncatula 4-hydroxy-tetrahydrodipicolinate synthase 2 (DHDPS2) submitted in NCBI (accession number: XM_013589555.2), coding for a long polypeptide of 307 amino acid with the molecular mass of about 33495 Da (Protein ID: XP_013445009.1). The DHDPS gene was successfully expressed in the Escherichia coli BL21 StarTM (DE3) cells with a pET200 / D-TOPO vector, and this produced the DHDPS2 protein with molecular masses of approximately 33.87 kDa (»33.5 kDa of DHDPS2 and 3.7 kDa of fusion fragment of pET 200/D-TOPO vector). The effects of the six different culture mediums of LB, SOB, SOC, YJ, HSG and TB, the induction times of 2h, 4h, 6h, 8h, 10h and 12h, and the inducer concentrations of 0.2 mM; 0.5 mM; 0.7 mM; 1.0 mM; 1.2 mM and 1.5 mM IPTG (Isopropyl â-D-1-thiogalactopyranoside) were also investigated for the purpose of optimising the expression of DHDPS2 in E. coli cells, and it was found that strong expression of recombinant DHDPS2 protein in E. coli. BL21 (DE3) cells occurred on the TB, HSG and YJ culture mediums after 8 hours with 0.2 mM inducible IPTG (BioRad).

1731. Disseminated Metacestode Infection Due to an Unknown Versteria Species

Background A 68-year-old woman with hypogammaglobulinemia and prior treated lymphoma presented with fever and abdominal pain. Evaluation revealed numerous nodules in the lung, eye, brain, and liver (Figure 1). Initial lung and liver biopsies showed necrotizing granulomas with no organisms and negative serology and cultures. After progression while on broad-spectrum antibiotics for 4 months, an open liver biopsy revealed numerous nodular lesions and a mass made up of multifocal coalescing cystic lesions. The mass consisted of a degenerating 3-layered membrane without scoleces characterized by a wavy protuberant ciliated eosinophilic outer layer, subjacent degenerating cells with pyknotic nuclei, and loose connective tissue suggestive of a bladder wall and calcareous corpuscles in a matrix of granulomatous inflammation with areas of necrosis (Figure 2). This was diagnostic of disseminated metacestodes (larval stage) of a cestode (tapeworm). Treatment with praziquantel and albendazole led to improvement of symptoms and lesions. Disseminated cestode infections other than due to Echinococcus species are rare in humans. Sequencing was pursued due to the unusual findings. Methods DNA was extracted from liver tissue followed by targeted amplification of the cestode COX1 gene. PCR products confirmed to be 134 bp, as expected for a cestode COX1 gene, then inserted into a 2.1 Topo vector and cloned. Five separate isolates were sequenced, and 4 were interpretable. The 129-bp consensus sequence is shown in Figure 3. Basic Local Alignment Search Tool (NCBI BLAST) was used to find highly similar sequences. Results The sequence matched to Versteria sp. (T. mustelae) COX1 gene from a mink in Oregon (accession KT223034) with 98% identity. Conclusion Metacestodes have the propensity to proliferate and rarely disseminate. There is one reported case of Versteria sp. causing a lethal disseminated infection of an orangutan. This is the first report of a Versteria sp. disseminated infection in a human and is singular because the patient survived. The patient likely accidentally ingested ova shed from a tapeworm in a mink or similar mammalian host. Histopathologic assessment is crucial in diagnosing cestode infection. COX1 gene sequencing is useful for cestode identification. Disclosures All authors: No reported disclosures.

First Report of Celery mosaic virus Infecting Celery in Venezuela

Celery mosaic virus (CeMV) is a significant pathogen of celery (Apium graveolens) worldwide (1). In 2005, in a produce market located in Los Salias, Miranda, celery plants with mottling and leaf malformation were noticed. Electron microscopic analysis of leaf-dip preparations from three symptomatic samples revealed flexuous viral particles that were 750 nm long. Infected cells contained pinwheel inclusions typical of those associated with potyvirus infection. Inoculation of healthy celery plants with leaf extracts from four symptomatic plants produced symptoms identical to those first observed. A survey of five produce markets in Miranda was conducted to determine the prevalence of virus infection in celery using serological and molecular analyses. Mottling and malformation of celery leaflets were observed in all the markets visited. Symptoms were noted in all five markets in each of three visits during a 3-month period. A total of 125 postharvested symptomatic plants were collected from five markets on March 29, 2005 and tested for CeMV using double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with antiserum provided by F. Rabenstein, BAX, Aschersleben, Germany. Of the 125 samples collected during the survey, 53% were ELISA positive. Twenty ELISA-positive samples were also tested using reverse transcription-polymerase chain reaction (RT-PCR) with general primers for the family Potyviridae (2). All 20 samples produced an amplicon of the expected size (1.7 kbp) after RT-PCR. Amplicons from three samples were cloned into the pCR-TOPO vector (Invitrogen, Carlsbad, CA). Sequence analysis of one clone revealed more than 98% nt to a CeMV isolate from Australia (GenBank Accession No AF203532). To our knowledge, this is the first report of CeMV in Venezuela. Our results suggest that the disease may be widely spread on celery crops growing in the areas surrounding produce markets in Miranda State. References: (1) A. Brunt et al. Viruses of Plants. CAB International, Wallingford, Oxon, UK. 1996. (2) J. Chen et al. Arch. Virol. 146:757, 2001.