Specific Antisera Research Articles

Prevention of nosocomial Acinetobacter baumannii infections with a conserved immunogenic fimbrial protein.

Acinetobacter baumannii, one of the most life-threatening nosocomial drug-resistant pathogens, imposes high morbidity and mortality rates, thus highlighting immunization-based treatments or prevention measures. The selection of appropriate antigens can elicit protective immunity. The gene encoding a fimbrial protein introduced via reverse vaccinology was cloned, expressed and evaluated for immunogenicity in a murine model. Mice immunized with the recombinant protein were challenged with A.baumannii ATCC 19606. Adherence to A549 cell line of specific anti-sera treated A.baumannii was also assessed. Passive immunity was evaluated in a murine pneumonia model. Indirect ELISA showed a high specific antibody titre. Adherence of A.baumannii to A549 cell line decreased by 40% after incubation with 1:250 dilution of specific anti-sera. All the actively immunized mice survived. Bacterial load in the spleen and liver of the immunized mice was 3-fold lower than those of the control. The number of bacteria in the lungs of passively immunized mice was about 6-fold lower than the control mice. The fimbrial protein could be considered as a promising protective immunogen against A.baumannii.

First Report of Impatiens Necrotic Spot Virus Infecting Lettuce in Greece

HomePlant DiseaseVol. 104, No. 10First Report of Impatiens Necrotic Spot Virus Infecting Lettuce in Greece PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Impatiens Necrotic Spot Virus Infecting Lettuce in GreeceD. Beris, I. Malandraki, O. Kektsidou, N. Vassilakos, and C. VarveriD. Beris†Corresponding author: D. Beris; E-mail Address: d.mperi@bpi.grhttp://orcid.org/0000-0003-3308-8742Laboratory of Virology, Department of Phytopathology, Benaki Phytopathological Institute, Kifissia, Athens 14561, GreeceSearch for more papers by this author, I. Malandrakihttp://orcid.org/0000-0001-5623-5261Laboratory of Virology, Department of Phytopathology, Benaki Phytopathological Institute, Kifissia, Athens 14561, GreeceSearch for more papers by this author, O. KektsidouLaboratory of Virology, Department of Phytopathology, Benaki Phytopathological Institute, Kifissia, Athens 14561, GreeceSearch for more papers by this author, N. VassilakosLaboratory of Virology, Department of Phytopathology, Benaki Phytopathological Institute, Kifissia, Athens 14561, GreeceSearch for more papers by this author, and C. VarveriLaboratory of Virology, Department of Phytopathology, Benaki Phytopathological Institute, Kifissia, Athens 14561, GreeceSearch for more papers by this authorAffiliationsAuthors and Affiliations D. Beris † I. Malandraki O. Kektsidou N. Vassilakos C. Varveri Laboratory of Virology, Department of Phytopathology, Benaki Phytopathological Institute, Kifissia, Athens 14561, Greece Published Online:18 Aug 2020https://doi.org/10.1094/PDIS-11-19-2316-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat During the autumn of 2018 and the winter of 2019, virus-like disease symptoms were observed in four fields of lettuce (Lactuca sativa) in Kalamos, Marathonas, and Leonidio regions of central and south-eastern Greece. In all cases, the disease affected 30 to 40% of the plants. Symptoms consisted of brown ringspots, necrotic lesions, vein necrosis, and leaf distortion resembling those associated with tospovirus infections. Six symptomatic plants (four in 2018 and two in 2019) were collected from four different fields and were tested for the presence of tomato spotted wilt virus (TSWV), tomato chlorotic spot virus (TCSV), impatiens necrotic spot virus (INSV), groundnut ringspot virus (GRSV), and chrysanthemum stem necrosis virus (CSNV) with double antibody sandwich ELISA specific and generic polyclonal antisera, respectively (Loewe Biochemica, Germany). All samples were found negative for TSWV but positive for other Tospovirus infection with the generic ELISA test. Total RNA was purified from all six symptomatic samples and reverse transcription polymerase chain reaction (RT-PCR) was performed with the gM410/gM870c primer pair (Chen et al. 2012) targeting the conserved NSm genomic region of tospoviruses. Direct Sanger sequencing of the 500-bp PCR products obtained from all six samples revealed the presence of INSV Tospovirus, and showed 97.5 to 98.7% identity at nucleotide level with the respective region of the Chinese isolate HDL (GU112503). To further characterize our INSV isolate, total RNA isolated with NucleoZOL reagent (Macherey-Nagel, Germany) from one symptomatic lettuce was subjected to high-throughput sequencing (HTS) on a Next Seq platform (Illumina, U.S.A.) by using the TruSeq Stranded mRNA Library Prep kit (Illumina) for the construction of the poly-A selected HTS library. The analysis generated approximately 14,000,000 single-end reads of 75-bp length that were analyzed with Geneious bioinformatics software (Biomatters, New Zealand). After the de novo assembly of the unique reads (7,448,124 reads), contigs (in total 108,234 contigs ranging in size from 150 to 12,000 bp) were annotated with BLAST, and the presence of INSV was confirmed. Assembled nucleotide sequences of S (MN553562) and M (MN553561) segments exhibited 88 to 98% coverage and 99% identity at nucleotide level with an Italian isolate (DQ425096 and DQ425095, respectively). Nucleotide sequence of the L segment also showed 64% coverage with 88% identity with the Italian isolate (DQ425094). Furthermore, HTS analysis revealed the presence of lettuce big-vein associated virus (LBVaV, Rhabdoviridae, Varicosavirus genus). To isolate INSV, Nicotiana tabacum, an insusceptible host of LBVaV, was used. Infected N. tabacum tissues were used to reproduce symptoms in virus-tested-negative lettuce plants after mechanical inoculation. In the latter plants, INSV presence was confirmed with RT-PCR assays targeting specific regions of the three INSV segments by respective primer pairs designed based on the HTS analysis. Sequences of the three PCR products were 100% identical with the HTS-assembled INSV segments of the respective regions and confirmed the presence of all virus segments. INSV was first described in Impatiens spp. plants (Law and Moyer 1990) and is an important pathogen for several crops including ornamental plants, tomato, and lettuce (Kuo et al. 2014). The relatively high incidence of the virus in different regions of Greece suggests that INSV is an emerging pest for lettuce cultivation in Greece. To the best of our knowledge, this is the first report of INSV in Greece.The author(s) declare no conflict of interest.

Heterologous expression of pepper mild mottle virus coat protein encoding region and its application in immuno-diagnostics

Pepper mild mottle virus (PMMoV), a tobamovirus of family Virgaviridae affects the quality and quantity of Capsicum. PMMoV is highly contagious, capable of transmitting through infected seeds and soil. Symptoms are more severe when crop is infected at young stage but remain unnoticed when infection takes place at maturity. Therefore, cost effective diagnostic techniques are required for timely and accurate detection of virus. In present study, coat protein encoding region of PMMoV-HP1 isolate was cloned into expression vector system, pET28a and expressed in BL21, a protease deficient strain of Escherichia coli. The PMMoV-HP1 pathotype was identified as PMMoV-P12 on the basis of coat protein amino acid sequence analysis in our previous study. The overexpression of recombinant coat protein of 26kDa, corresponding to the expected 6X Histidine tag fused recombinant protein was purified using Ni-NTA columns from insoluble fraction. For antisera production, the purified recombinant protein was dialyzed ~ 24h to remove urea and then used for raising polyclonal antisera. The specificity and sensitivity of antiserum obtained was demonstrated using different dilutions of antiserum for western blot assay and direct antigen coating enzyme linked immunosorbent assay (DAC-ELISA). In Western blot assay, the test antiserum reacted strongly both with PMMoV-CP in purified protein and native CP in crude sap from PMMoV infected pepper plants, whereas no reaction was observed with healthy plant sap. In DAC-ELISA antiserum dilution up to 1:1000 was capable of detecting the virus in infected sample. The absence of any cross reactivity of test antiserum was confirmed against tobacco mosaic virus, cucumber mosaic virus, tomato spotted wilt virus, pepper veinal mottle virus, potato virus Y and tomato yellow leaf curl virus antigen, known to infect capsicum.

Invasive Streptococcus pyogenes infections (2011–2018): EMM-type and clinical presentation

IntroductionStreptococcus pyogenes (S. pyogenes) is an important human pathogen that is responsible for a broad range of infections, from uncomplicated to more severe and invasive diseases with high morbidity/mortality. The M protein (emm type) is a critical virulence factor. Several studies have shown an increased incidence of invasive S. pyogenes disease. This was associated with an increase in the prevalence of M1 and M3 types, well-recognised virulent M types. The aim of the present study was to confirm the resurgence of invasive S. pyogenes disease during 2011–2018 and to identify the relationship between specific M types with disease presentation. Material and methodsIsolates were confirmed using standard techniques: colony morphology, β-haemolysis, biochemical tests, and agglutination with specific antisera (DiaMondiaL Strep Kit, DiaMondiaL, Langenhagen, Germany). The antibiotic sensitivity was performed using microdilution (Vitek®2 Compact, bioMeriéux, Inc., Durham, NC). Molecular analysis included the determination of the emm gene and superantigen profile. ResultsA total of 29 invasive isolates were collected (2011–2018) from blood (16), pleural fluid (9), synovial fluid (3), and cerebrospinal fluid (1). One strain per year was isolated between 2011 and 2013, with 2, 5, 4, 6, and 9 strains being isolated between 2014 and 2018, respectively. The most frequent clinical presentations were bacteraemia and pneumonia (10 and 9 cases). The predominant types were M1 (11 isolates) and M3 (3 isolates). A correlation was found between M1 and M3 types, and pneumonia (6/7 cases) and deep soft tissue infections (3/3 cases). ConclusionsAn increased incidence of invasive S. pyogenes disease was observed during the study period, with M1 and M3 types being those most commonly isolated and associated with pneumonia and deep soft tissue infections.

Expression of 1B capsid protein of Foot-and-mouth disease virus (FMDV) using baculovirus expression system and its validation in detecting SAT 2- specific antisera.

Foot-and-mouth disease virus (FMDV) is one of the most devastating animal viruses that affect livestock worldwide. The 1B capsid of FMDV has been widely used to detect and confirm the infection. In the present study, the sequence coding for 1B subunit of FMDV capsid was expressed in insect cells using the baculovirus expression system under the polyhedrin (polh) promoter. The expression of 1B capsid protein was validated in the culture filtrate of insect cells using SDS-PAGE and western blotting. The culture filtrate containing recombinant 1B capsid (r1B) was used as a coated antigen in an indirect enzyme-linked immunosorbent assay (ELISA). The antigenicity and specificity of r1B against SAT 2 serotype-specific antibodies were assessed. Our results revealed that a protein concentration as low as 25 ng could detect SAT 2-specific antibodies in ELISA. The results highlight the application of insect cells developed r1B protein in the detection of FMDV. Further studies are required to determine the ability of r1B to detect other FMDV serotypes.

Low prevalence red blood cell antigens: transfusions, babies, and changing demographics

See article on page 870–874, in this issue

Novel molecular aspects of the CRISPR backbone protein ‘Cas7’ from cyanobacteria

The cyanobacterium Anabaena PCC 7120 shows the presence of Type I-D CRISPR system that can potentially confer adaptive immunity. The Cas7 protein (Alr1562), which forms the backbone of the type I-D surveillance complex, was characterized from Anabaena. Alr1562, showed the presence of the non-canonical RNA recognition motif and two intrinsically disordered regions (IDRs). When overexpressed in E. coli, the Alr1562 protein was soluble and could be purified by affinity chromatography, however, deletion of IDRs rendered Alr1562 completely insoluble. The purified Alr1562 was present in the dimeric or a RNA-associated higher oligomeric form, which appeared as spiral structures under electron microscope. With RNaseA and NaCl treatment, the higher oligomeric form converted to the lower oligomeric form, indicating that oligomerization occurred due to the association of Alr1562 with RNA. The secondary structure of both these forms was largely similar, resembling that of a partially folded protein. The dimeric Alr1562 was more prone to temperature-dependent aggregation than the higher oligomeric form. In vitro, the Alr1562 bound more specifically to a minimal CRISPR unit than to the non-specific RNA. Residues required for binding of Alr1562 to RNA, identified by protein modeling-based approaches, were mutated for functional validation. Interestingly, these mutant proteins, showing reduced ability to bind RNA were predominantly present in dimeric form. Alr1562 was detected with specific antiserum in Anabaena, suggesting that the type I-D system is expressed and may be functional in vivo. This is the first report that describes the characterization of a Cas protein from any photosynthetic organism.

SUN-442 A CLINICAL ANALYSIS OF LIGHT-CHAIN AMYLOIDOSIS WITH RENAL

AL amyloidosis is a monoclonal plasma cells disorderwhich has a variety of symptoms. The common sites of involvementinclude kidney, liver, cardiac, peripheral neuropathy, musculoskeletal and skin. The kidney is involved in two-thirds of patients with AL amyloidosis. The disease manifests with albuminuria, evolving in nephrotic syndrome and progressing to renal failure eventually leading to end-stage renal disease if unrecognized or ineffectively treated. This was a retrospective study of patients with biopsy-proven renal amyloidosis over a period of ten years (2007–2016) at our hospital. A total of 31 cases of renal amyloidosis were diagnosed in the study period. Theimmunohistochemistry staining and immunofluorescencemicroscopy using specific antisera for AAand AL amyloid deposits were used to identify AL or AA type. AL amyloidosis was found in 29 % (9 cases) of all patients who underwent renal biopsy in a 10-year period. Biopsyindications for the patients during the initial diagnosis were proteinuria or renal dysfunction. The median age of patients was 59.67±9.01 years and 55.5% of patients were male. One patient(11%) with AL was younger than 40 years. All patients had different degrees of proteinuria, with amean level of 4.4 ± 3.9 g/24 h.Nephroticsyndrome was presented in 55% (5/9) ofpatients. The median of serum creatinine was 327μmol/L(range: 78–751 μmol/L). Eight (8/9, 88.8%) patientspresented with renal insufficiency at diagnosis, and only 3(33%) patients presented with serum creatinine level lowerthan 150 μmol/L. Five ( 55.5%) patients were found with overt anemia (hemoglobin lower than 100g/L) at diagnosis. Eight (88.8%) of the patients with echocardiographicexamination results had cardiac involvement. Hepatomegaly was detected in two (22.2%) patients. The involved light chain was lambda in over 70% patients. In AL amyloidosis, theprimary type was the most frequent (45%), followed bythe myeloma-associated type (33 %) and monoclonal gammapathy (22 %). During the follow-up period, 2 patients died, and one patient developed ESRD requiring dialysis therapy. Prevalence of AL amyloidosisnoted in our study was higher than previous Tunisian studies.

New natural host of Tobacco mosaic virus on three cucurbits in Java, Indonesia

Tobacco mosaic virus (TMV) is a newly emerging virus on cucumber in Java. Regardless the importance of TMV on Cucurbitaceae, little is known about the existence of TMV in Cucurbitaceae plants. The study aimed to detect and identify TMV on bitter gourd, ridged gourd, and watermelon. Symptomatic leaves were collected from several cucurbit cultivations at 5 regencies in West Java, Central Java and East Java provinces. The frequency of virus on samples were determined serologically by DIBA method using specific antisera to 6 viruses (CMV, CABYV, PRSV, SqMV, TMV, and ZYMV). Nucleic acid detection was conducted by RT-PCR using universal primer for Tobamovirus followed by DNA sequencing to confirm virus identity. The results revealed that samples were infected by 6 viruses with frequency varied and multiple infections occurred. The frequency of TMV infection ranged from 5.9 to 89.6%. RT-PCR successfully amplified DNA fragment with size ± 800 bp from bitter gourd, ridged gourd, and watermelon. The similarity of coat protein gene among isolates reached up to 100%. These TMV isolates showed highest nucleotide and amino acid similarity with cucumber isolate from Brebes regency and other isolates infecting several different crops from other countries. This is a new report on the existence of TMV on different cucurbit species in the fields.

Protective Potential of Conjugated P. aeruginosa LPS -PLGA Nanoparticles in Mice as a Nanovaccine.

Pseudomonas aeruginosa has an important role in nosocomial infections. To evaluate biological activity of the detoxified LPS (D-LPS) entrapped into Poly lactic-co-glycolic acid (PLGA) nanoparticles. LPS was extracted and detoxified from the P. aeruginosa strain PAO1. The D-LPS, conjugated to the PLGA nanoparticles with 1-ethyl-3-dimethyl aminopropyl carbodiimide (EDAC) and N-hydroxy-succinimide (NHS). The connection was evaluated by FTIR (Fourier transform infrared), Zetasizer, and Atomic Force Microscope (AFM). The BALB/c mice injected intramuscularly with the D-LPS-PLGA with two-week intervals and then challenged two weeks after the last immunization. The bioactivity of the induced specific antisera and cytokines responses against D-LPS-PLGA antigen was assessed by ELISA. D-LPS-PLGA conjugation was confirmed by FTIR, Zetasizer, and AFM. The ELISA results showed that D-LPS was successful in the stimulation of the humoral immune response. The immune responses raised against the D-LPS-PLGA, significantly decreased bacterial titer in the spleen of the immunized mice after challenge with PAO1 strain in comparison with the control groups. The conjugation of the bacterial LPS to the PLGA nanoparticle increased their functional activity by decrease in bacterial dissemination and increase the killing of opsonized bacteria.

Expression of DOCK10.1 protein revealed with a specific antiserum: insights into regulation of first exon isoforms of DOCK10.

DOCK10, a guanine-nucleotide exchange factor (GEF) for Rho GTPases, represents the example of a gene that gives rise to alternative first exon mRNA isoforms, named DOCK10.1 and DOCK10.2. Expression of human DOCK10.2 protein in cell lines, and its induction by interleukin-4 (IL-4) in normal B lymphocytes and chronic lymphocytic leukemia (CLL) cells, were previously demonstrated using an antiserum raised against a peptide encoded by sequences from exon 1.2. Here, expression of human DOCK10.1 protein was demonstrated using an antiserum raised against a peptide encoded by sequences from exon 1.1. Specificity of the DOCK10.1 and DOCK10.2 antisera for their respective isoforms was demonstrated using transfected human 293T cells. Their specificity for endogenous DOCK10 was strongly suggested by the high significance of the correlations between the levels of their expected signals at the molecular size of 250kDa and the levels of DOCK10.1 and DOCK10.2 mRNAs, respectively, in human hematopoietic cell lines. Specificity of the DOCK10.1 antiserum for DOCK10 was also demostrated in mouse using the DOCK10 knockout model. The DOCK10.1 protein was induced by IL-4 in CLL cells, which demonstrates that the mechanism by which IL-4 regulates DOCK10 is not isoform-specific. Last, to get insights into differential regulation of the DOCK10 isoforms, their protein levels in cell lines were compared with their gene expression profiles retrieved from the Cancer Cell Line Encyclopedia (CCLE), leading to the identification of BCL3 and KLF12 as potential transcriptional regulators of DOCK10.1 and DOCK10.2, respectively.

Identification of Bacteria Contaminating Musca domestica (Diptera: Muscidae) Collected from Animal Husbandries

Background: House flies (Musca domestica L.) may mechanically transmit many microorganisms to humans. Objectives: To identify bacteria contaminating Musca domestica (Diptera: Muscidae) collected from animal husbandries in province of Qom, Iran in the year of 2019. Methods: In the cross-sectional study, house flies were captured by plastic water bottle fly traps and insect nets from four districts of the Qom Province and were immediately transferred to the Department of Medical Entomology, School of Public Health in sterile glass containers, individually. The bacterial isolation of the surface and gut contents of the flies were separately performed by biochemical methods. To confirm Salmonella spp. and Shigella spp., antigen-antibody agglutination reactions were carried out by specific antisera. Results: A total of 23 bacterial species were identified from 160 house fly external surfaces and/or digestive tract were isolated. The most frequently isolated bacteria from the external body and the digestive tract of 160 flies were E. coli (73.8%) and P. aeruginosa (100%), respectively. The most frequently isolated bacteria were P. aeruginosa, Salmonella serogroup D, S. dysenteriae, E. coli, C. freundii, S. aureus, and S. epidermidis. Conclusions: House flies are important in the mechanical transmission of a different range of the pathogens. In addition, because of the geographical features of Qom and isolated pathogens, it seems that the possibility of mechanical disease transmission by the flies can be increased.

Serological relationship between a novel ovine pestivirus and classical swine fever virus.

The genus Pestivirus comprises globally distributed members of the family Flaviviridae, which cause severe losses in livestock. The most common species of the genus are bovine viral diarrhoea virus type 1 (BVDV-1) and type 2 (BVDV-2), classical swine fever virus (CSFV) and border disease virus (BDV). Recently, a novel ovine pestivirus was repeatedly detected in aborted lamb foetuses on a farm located in the Brescia Province (Italy). Complete genome characterization of this isolate showed that it was highly divergent from known pestivirus species and that it was genetically closely related to CSFV. The aim of this study was to determine the serological relatedness between the identified novel pestivirus and BVDV, BDV and CSFV selected strains for which homologous serum was available, by antigenic characterization performed using cross-neutralization assays. The serological relatedness was expressed as the coefficient of antigenic similarity (R). Both field and specific antisera raised against the ovine pestivirus neutralized the CSFV reference strain Diepholz with titres significantly higher than those specific for the BDV and BVDV strains. Furthermore, the calculated R values clearly indicated that the novel ovine pestivirus is antigenically more related to CSFV than to ruminant pestiviruses, in agreement with the results of the genomic analysis. This would have severe consequences on CSFV serology in the event of a switch to porcine hosts with implications for CSFV surveillance and porcine health management.

Assessment of pregnancy-associated glycoprotein profile in milk for early pregnancy diagnosis in goats.

ObjectiveThis study was conducted to assess the level of pregnancy-associated glycoprotein (PAG) in whole and skim milk samples, and its suitability for early pregnancy diagnosis in goats.MethodsA two-step sandwich enzyme-linked immunosorbent assay (ELISA) system for estimation of milk PAG was developed and validated, which employed caprine-PAG specific polyclonal antisera. Whole and skim milk samples (n = 210 each) from fifteen multiparous goats were collected on alternate days from d 10 to d 30, and thereafter weekly till d 51 post-mating. PAG levels in milk samples were estimated by ELISA and the pregnancies were confirmed at d40 post-mating by transrectal ultrasonography (TRUS).ResultsThe level of PAG in whole and skim milk samples of both pregnant and non-pregnant goats remained below the threshold values until d 24 after mating. Thereafter, PAG concentration in whole and skim milk increased steadily in pregnant goats, whereas it continued below the threshold in non-pregnant does. The PAG profiles in whole and skim milk of pregnant goats were almost similar and exhibited strong positive relationship (r = 0.891; p<0.001). Day 26 post-mating was identified as the first time-point for significantly (p<0.05) higher milk PAG concentration in pregnant goats than to non-pregnant goats. When compared to TRUS examination for pregnancy diagnosis, the accuracy and specificity of PAG ELISA using whole and skim milk samples were 94.5% and 95.4%; and 95.3% and 100%, respectively. The high values of area-under-curve (0.904 [whole milk] and 0.922 [skim milk]), demonstrate outstanding discrimination ability of the milk assays. Among the sampling dates chosen, d 37 post-mating was identified as the best suitable time point for collection of milk samples to detect pregnancy in goats.ConclusionThe PAG concentration in whole and skim milk of goats collected between days 26 and 51 post-breeding can be used for the accurate prediction of pregnancy and may be useful for assisting management decisions in goat flocks.

Expression of Transferrin and Albumin in the Sperm-Storage Tubules of Japanese Quail and their Possible Involvement in Long-Term Sperm Storage.

Because of the presence of sperm storage tubules (SSTs) in the utero-vaginal junction (UVJ) in the oviduct, once ejaculated sperm enter the female reproductive tract, they can survive for a prolonged period in domestic birds; however, the specific mechanisms involved in sperm maintenance within the SST remain to be elucidated. In this study, we showed that transferrin (TF) and albumin (ALB) are expressed in SSTs. When UVJ extracts were subjected to size-exclusion column chromatography, we obtained fractions that extend sperm longevity in vitro. LC-MS/MS analysis of the two major proteins in the fractions identified these proteins as TF and ALB. Immunohistochemical analysis using specific antisera against TF and ALB indicated that both proteins were localized not only in the SSTs, but also in the surface epithelium of the UVJ. When the ejaculated sperm were incubated with either purified TF or ALB, sperm viability increased after 24 h. These results indicated that oviductal TF and ALB are involved in the process of sperm storage in SSTs and may open a new approach for technological improvement to prolong sperm longevity in vitro.

Localization and distribution of CCK-8, NPY, Leu-ENK-, and Ghrelin- in the digestive tract of Prochilodus lineatus (Valenciennes, 1836).

This study describes the histological characteristics and distribution of gastrointestinal tract endocrine cells (ECs) of Prochilodus lineatus (detritivorous fish) using immunohistochemical procedures. The digestive tract of P. lineatus was divided into seven portions: stomach (cardial and pyloric), pyloric caeca, and intestine (anterior, glandular, middle and posterior). A pool of specific antisera against cholecystokinin (CCK-8), -neuropeptide Y (NPY), -ghrelin (Ghre) and -leu-enkephalin (Leu-ENK) to identify ECs were used. According to the morphological characteristics of ECs, two different types were identified and classified as open or closed-type. The number of ECs varied throughout the gastrointestinal tract, though a high abundance was found in the anterior intestine and pyloric caeca. A large number of ECs immunoreactive to CCK-8 and NPY were recorded in the anterior, glandular and middle intestine. ECs immunopositive to Leu-ENK were distributed in the stomach and pyloric caeca. For Ghre, immunopositive ECs were restricted to the glandular intestine. The results of the present study indicate that P. lineatus presents an ECs distribution pattern with species-specific particularities. However, CCK showed a distribution similar to that of omnivores, which is possibly related to local signaling functions in order to achieve the correct digestion of the various organisms found in the detritus.

Purification, characterization and identification of rat brain cytosolic tyrosine transaminase as glutamine Transaminase-K

The current study was undertaken to investigate the spectrum of tyrosine transaminases enzymes in a cytosolic fraction of rat brain and to specifically purify and characterize a previously identified cytosolic brain enzyme possessing tyrosine/glyoxylate transaminase activity. Based upon extensive biochemical and immunochemical characterization of purified brain tyrosine/glyoxylate transaminase, we concluded the purified enzyme is glutamine transaminase-K (EC 2.6.1.64). This conclusion was based on: 1.) a concurrent enrichment in the tyrosine/glyoxylate and glutamine/phenylpyruvate transaminase activities during purification, 2.) demonstration of a co-substrate specificity for amino acids and α-keto acids that was highly consistent with published information for glutamine transaminase-K, 3.) results from detailed kinetic analysis, 4.) glutamine was a potent inhibitor of in vitro tyrosine/glyoxylate transamination, 5.) biochemical characterization, including pH optimum of 8.5 and spectrophotometric analysis and 6.) immunoanalytical analysis using a specific antiserum to rat renal glutamine transaminase-k. In addition, immunochemical characterization of a crude soluble extract of whole brain suggests that the in vitro tyrosine transaminase activity for several different α-keto acid co-substrates likely reflect the activity of glutamine transaminase-K. In conclusion, this investigation confirmed the presence of multiple tyrosine transaminase enzymes in a cytosolic extract of rat brain. Moreover, we concluded glutamine transaminase-K represents a predominant cytosolic enzyme in rat brain that's capable of catalyzing in vitro transamination of p-tyrosine and other aromatic amino acids, including the neurotransmitter precursors L-dopa and 5-hydroxytryptophan. The purified transaminase possesses a broad co-substrate specificity with preferential reactivity with α-keto acids derived from neutral aliphatic and aromatic amino acids. Lastly, we identified a heterogeneous regional distribution of tyrosine/glyoxylate transaminase (glutamine transaminase-K) in rat brain with a significantly higher level of in vitro activity in cerebellum.

Molecular Characterization of Begomovirus on Cucumber in Java

A survey on several cucumber cultivation areas in West Java, Central Java, Yogyakarta, and East Java found many plants showing typical Begomovirus symptoms such as yellow mosaic, cupping, and vein banding. This study was aimed to determine disease frequency, detection and molecular characterization of the causal virus of those symptoms on cucumber in Java. Sampling was conducted by purposive sampling by collecting 50 symptomatic plants from each location in West Java (Indramayu, Subang, and Bogor), Central Java (Brebes and Klaten), Yogyakarta (Kulon Progo), and East Java (Nganjuk, Kediri, and Tulungagung). The detection and disease frequency was determined based on DIBA test using a specific antiserum of Tomato leaf curl New Delhi virus (ToLCNDV) and Squash leaf curl virus (SLCV). The identification of nucleic acid was conducted by PCR using specific primer of ToLCNDV and SLCV, DNA cloning, and sequencing. The results of serological detection showed the disease frequency of ToLCNDV and SLCV ranged from 92.77-100% and 78.33-93.3%, respectively. PCR using specific primer of ToLCNDV successfully amplified the coat protein gene at a size of 600 bp from all samples. Homology nucleotide and amino acid sequences among ToLCNDV Java isolate ranging from 95.6-99.2% and 99.7-100%. ToLCNDV isolates Java had highest nucleotide and amino acid sequences similarity with cucumber isolate from Klaten, Indonesia (AB613825) ranging from 96.1-98.1% and 99.7-100%, and was considered as “Indonesia” strain. SLCV not amplified on all samples by PCR using specific primer, indicating it might not present yet on cucumber in Java.

Serotyping strains of Haemophilus influenzae isolated from clinical specimens and investigating their susceptibilities towards certain antimicrobials

Aim: Haemophilus influenzae is an agent of severe pediatric infections, and a significant cause of mortality and morbidity in children. Its capsulated strains are grouped from ‘a’ to ‘f’ in six groups, the most invasive among which is Type B. The other serotypes are also capable of causing severe infections and invasive diseases. Antibiotic sensitivity testing proves to be useful in directing course of treatment in view of the beta-lactamase-negative and ampicillin-resistant strains as well as the ever-increasing production of beta-lactamase. In this study it has been aimed to serotype Haemophilus influenzae strains isolated from various clinical specimens, and to investigate their status of beta-lactamase production and susceptibilities towards antimicrobials. Materials and Methods: 100 samples of H. influenzae proliferating in various clinical specimens were included in the study. The bacteria were identified according to their requirements of X and V factors, or through the usage of API NH. Detection of beta-lactamase production was made with nitrocefin disks. Serotyping was performed via slide agglutination method with specific antiserums. Susceptibilities towards various antimicrobials were specified with gradient test in Haemophilus Test Medium. Results: 59 of the specimens were sputum, 23 were nasotracheal aspirates, 3 were bronchoalveolar lavages, 7 were middle ear fluids, 3 were cerebrospinal fluids, 2 were eye fluids, 2 were pleural fluids, and 1 was a blood sample. The ranking was made without keeping age groups in regard and turned out as a>b>d>THi>c>e. Serotype f was not found among the strains. Type b was isolated from all of sterile body cavity fluids. Beta-lactamase positivity was found as 2% and the BLNAR ratio was found as 2%. All were found as susceptible towards cefotaxime and siprofloxacin while 66.6% were susceptible towards azithromycin, and 93% were susceptible towards TMP-SMX. Conclusion: It will be appropriate to perform standardized susceptibility tests in routine in order to monitor antimicrobial resistance. Serotyping is important as inclusion of H. influenzae type b in vaccination programs may cause an increase in infections with non-Hib isolates.

Salmonella Serotyping; Comparison of the Traditional Method to a Microarray-Based Method and an in silico Platform Using Whole Genome Sequencing Data.

Salmonella is one of the most common causes of food-borne diseases worldwide. While Salmonella molecular subtyping by Whole Genome Sequencing (WGS) is increasingly used for outbreak and source tracking investigations, serotyping remains as a first-line characterization of Salmonella isolates. The traditional phenotypic method for serotyping is logistically challenging, as it requires the use of more than 150 specific antisera and well trained personnel to interpret the results. Consequently, it is not a routine method for the majority of laboratories. Several rapid molecular methods targeting O and H loci or surrogate genomic markers have been developed as alternative solutions. With the expansion of WGS, in silico Salmonella serotype prediction using WGS data is available. Here, we compared a microarray method using molecular markers, the Check and Trace Salmonella assay (CTS) and a WGS-based serotype prediction tool that targets molecular determinants of serotype (SeqSero) to the traditional phenotypic method using 100 strains representing 45 common and uncommon serotypes. Compared to the traditional method, the CTS assay correctly serotyped 97% of the strains, four strains gave a double serotype prediction. Among the inconclusive data, one strain was not predicted and two strains were incorrectly identified. SeqSero was evaluated with two versions (SeqSero 1 and the alpha test version of SeqSero 2). The correct antigenic formula was predicted by SeqSero 1 for 96 and 95% of strains using raw reads and assembly, respectively. However, 34 and 33% of these predictions included multiple serotypes by raw reads and assembly. With raw reads, one strain was not identified and three strains were discordant with phenotypic serotyping result. With assembly, three strains were not predicted and two strains were incorrectly predicted. While still under development, SeqSero 2 maintained the accuracy of antigenic formula prediction at 98% and reduced multiple serotype prediction rate to 13%. One strain had no prediction and one strain was incorrectly predicted. Our study indicates that the CTS assay is a good alternative for routine laboratories as it is an easy to use method with a short turn-around-time. SeqSero is a reliable replacement for phenotypic serotyping if WGS is routinely implemented.