Positive Amplification Products Research Articles

Prevalence of Anaplasma infections in sheep and goats in Anhui Province in 2020

To investigate the prevalence and molecular characteristics of Anaplasma infections in sheep and goats in Anhui Province in 2020, so as to provide insights into ovine anaplasmosis prevention and control. A total of 355 fresh blood samples were collected from 7 sheep and goat farms in Linquan County of Fuyang City, Lixin County of Bozhou City, Yu'an District of Lu'an City, Wangjiang County of Anqing City, Nanling County of Wuhu City, and Tianchang City and Fengyang County of Chuzhou City in Anhui Province from June to December 2020. A. bovis and A. phagocytophilum 16S ribosomal RNA (16S rRNA) gene, A. ovis major surface protein 4 (MSP4) gene and A. capra citric acid synthase (gltA) gene were amplified using PCR assay in all blood samples, and the prevalence of A. bovis, A. phagocytophilum, A. ovis and A. capra infections was calculated in sheep and goats. In addition, the positive amplification products were sequenced and subjected to genetic evolutionary analysis. The overall prevalence of Anaplasma infections was 17.5% (62/355) in sheep and goats in Anhui Province, and the prevalence of A. bovis, A. phagocytophilum, A. ovis and A. capra infections was 2.8% (10/355), 2.5% (9/355), 2.5% (9/355), and 7.0% (25/355), while the prevalence of A. bovis and A. phagocytophilum, A. phagocytophilum and A. ovis, A. phagocytophilum and A. capra and A. bovis, A. phagocytophilum and A. ovis co-infections was 0.8% (3/355), 1.1% (4/355), 0.3% (1/355) and 0.3% (1/355), respectively. No Anaplasma was detected in the sheep and goat farms in Fengyang County, while at least three Anaplasma species were detected in other sheep and goat farms, with co-infections of multiple Anaplasma species identified. The prevalence of Anaplasma infections was 14.7% (24/163) in goats and 19.8% (38/192) in sheep, and the prevalence of Anaplasma infections was 31.0% (31/100) in goats and sheep under 6 months of age, and 12.2% (31/255) in goats and sheep at ages of 6 months and older, respectively. A. bovis, A. phagocytophilum, A. ovis and A. capra were identified in sheep and goats of different breeds and ages. Multiple Anaplasma species infections were commonly prevalent in goats and sheep in Anhui Province in 2020, notably A. phagocytophilum, A. ovis and A. capra, which have zoonotic risks. Improved surveillance and prevention and control of Anaplasma infections are required in sheep and goats in Anhui Province.

Molecular epidemiological investigation of piroplasms carried by pet cats and dogs in an animal hospital in Guiyang, China.

Piroplasmosis is a zoonotic disease mainly caused by the Babesia and Theileria parasites. Piroplasmosis is often a subclinical infection in dogs and cats that is difficult to detect and is often suspected when clinical signs such as anemia are present. It has been reported to be prevalent in China. However, molecular evidence of the disease has not been reported in pet dogs and cats in Guiyang. In this study, we collected 307 anticoagulated blood samples from an animal hospital in the Wudang District of Guiyang during the period March 2021 to November 2021 and extracted DNA from the samples. The 18S rDNA gene was amplified using PCR, and the positive amplification product was sequenced. The sequences were then analyzed for homology and phylogeny. Of the 307 samples collected, 164 were feline and 143 were canine, with a total of 23 amplifying a target band of approximately 400 bp. The percentage of positives of piroplasms infection in pet cats was 4.27% (7/164), with the pathogens being T. uilenbergi (3) and T. luwenshuni (4). One Colpodella sp. and two undetermined species were also detected in the cat samples. The percentage of positives of piroplasms infection in pet dogs was 7.69% (11/143), with the pathogen being T. uilenbergi (11). One Colpodella sp. was also detected in the dog samples. The results confirmed that T. uilenbergi and T. luwenshuni are prevalent in pet cats and dogs in this area. In addition, the study found a rare zoonotic pathogen, Colpodella sp., in cats and dogs. Therefore, this study is expected to serve as a valuable reference for decision-making regarding animal health management and public health work.

Prevalence and genotyping of Cryptosporidium spp. and Giardia lamblia in dogs and cats from a pet hospital in Shanghai Municipality

To investigate the prevalence and genotypes of Cryptosporidium spp. and Giardia lamblia in dogs and cats from a pet hospital in Shanghai Municipality. A total of 145 fresh fecal samples were collected from pet dogs and cats in a pet hospital in Shanghai Municipality during the period from November 2021 to June 2022, including 99 dog fecal samples and 46 cat fecal samples. The small subunit ribosomal ribonucleic acid (SSU rRNA) gene of Cryptosporidium and the triose phosphate isomerase (TPI) gene of G. lamblia were amplified using nested PCR assay, and the positive amplification products were sequenced from both directions. The sequence assembly was performed using the software Clustal X 2.1, and sequence alignment was conducted using BLAST. A phylogenetic tree was created with the Neighbor-Joining method using MEGA 11.0 to identify parasite species or genotype. The overall prevalence of Cryptosporidium and G. lamblia was 20.00% (29/145) in 145 pet dog and cat fecal samples, with the prevalence of 0.69% (1/145) and 19.31% (28/145) in Cryptosporidium and G. lamblia, respectively. G. lamblia was only detected in dog fecal samples, with prevalence of 18.18% (18/99), while the detection rates of Cryptosporidium and G. lamblia were 2.17% (1/46) and 21.74% (10/46) in cat fecal samples. Nucleotide sequence analysis showed that one Cryptosporidium positive sample was characterized as C. felis, and 28 G. lamblia positive samples were all characterized as Giardia assemblage A, which showed 100% sequence homology with human isolates of Giardia. Phylogenetic analysis revealed that the sequences obtained in this study belonged to the same branch with the reported Giardia assemblage A. Cryptosporidium and G. lamblia infection was prevalent in pet dogs and cats from the study pet hospital in Shanghai Municipality, and there is a zoonotic risk for the species and genotype. Intensified surveillance of Cryptosporidium and Giardia infection is recommended in pets and their owners, and improved management of pet keeping is required.

First Report of Laurel Wilt Caused by Harringtonia lauricola (previously Raffaelea lauricola) on Northern Spicebush in Kentucky and Tennessee.

Laurel wilt (LW) is a vascular disease caused by the fungus Harringtonia lauricola (previously Raffaelea lauricola) and transmitted by its primary vector, the redbay ambrosia beetle (Xyleborus glabratus, RAB), both of which were first detected in the United States (US) in 2002, likely introduced from their native range in Asia (Fraedrich et al. 2008; Harrington et al. 2008). LW has since spread across the southeastern US causing the death of millions of native redbay, sassafras, silk bay, swamp bay and other native Lauraceae species (Hughes et al. 2017). Detection of LW on the deciduous understory shrub northern spicebush (Lindera benzoin) was previously reported in South Carolina (Fraedrich et al. 2016) and Louisiana (Olatinwo et al. 2021) and is hereby confirmed in Kentucky and Tennessee. Spicebush plants displaying typical LW symptoms were observed in September 2020 on a property spanning the KY/TN border (Christian Co., KY and Montgomery Co., TN). Several dense stands of spicebush exhibited leaf wilt, early fall leaf coloration, dead leaves on branches, and black streaks of discolored xylem. LW was already confirmed on sassafras in the area (Loyd et al. 2020), and there were abundant dead sassafras nearby. Ambrosia beetle boring dust was observed and callow female RABs emerged from containerized bolts of spicebush collected from the site, indicating that the vector used spicebush as a brood host. Samples of spicebush sapwood tissue collected from two symptomatic plants were plated onto CSMA (cycloheximide-streptomycin malt extract agar) medium. The cultures were grown at room temperature in ambient light, and a fungus was recovered and further transferred onto MEA (malt extract agar) and PDA (potato dextrose agar) media. The morphology of the two fungal isolates, referred to as LW415 and LW416, matched the typical white mucoid growth and ovoid conidia of H. lauricola (Harrington et al. 2008). DNA was extracted from conidia harvested from two-week-old MEA cultures using a modified method of Dreaden et al. (2014). The identity of the fungus was confirmed by performing PCR with the H. lauricola-specific microsatellite primer sets IFW and CHK (Dreaden et al. 2014, Parra et al. 2020). A positive amplification product was obtained for LW415 and LW416 for both primer sets, validating their identification as H. lauricola. To confirm pathogenicity, four spicebush seedlings (mean height 22.5 cm; mean ground line diameter 3.3 mm) were inoculated: two with H. lauricola isolate LW415 grown on PDA for two weeks at room temperature in the dark, and two were mock-inoculated with sterile PDA as a control. A scalpel was used to nick the spicebush stem at a bud about 5 cm above groundline, and a 3 mm2 agar plug was placed in the wound and wrapped with parafilm. The spicebush seedlings were maintained in a growth chamber with an average temperature of 24°C and a 15 h photoperiod. Wilt symptoms were evident on inoculated seedlings after two weeks, while the control plants remained healthy. Four weeks post-inoculation, black staining of the vascular tissue was present in the symptomatic seedlings, and a fungus matching the morphology of H. lauricola was consistently recovered, while no fungus was isolated from the control plants. These results provide additional evidence that northern spicebush populations may be threatened by LW and could serve as a reservoir for the pathogen and vector (Gramling 2010). The spread of LW and RAB on spicebush may gain importance as preferred hosts (e.g., sassafras) are killed.

Rapid identification of Ralstonia pickettii using PCR-nucleic acid test strips

To develop a method for rapid detection of Ralstonia pickettii in water for pharmaceutical purpose using PCR-nucleic acid test strips. The genomic DNA of Ralstonia pickettii was extracted by boiling method. A pair of specific primers targeting the 16S rDNA with FITC and biotin labeling of the 5' ends was designed and cloned into competent E. coli DH5α cells. The nucleic acid test strips were assembled, and the workload of streptavidin labeled with colloidal gold and antibody concentration in the reaction system was optimized. After verification of the reaction mechanism and assessment of the test sensitivity, specificity and stability, the test strip was used for detecting 7 known strains of Ralstonia pickettii detected in pharmaceutical water, and an evolutionary tree was constructed to analyze the source of contamination. The genomic DNA extracted by boiling method had a purity between 1.8 and 2.0, and the PCR products showed a 100% similarity of with Ralstonia pickettii 16S rDNA registered in GenBank. Using the colloidal gold amplification principle, in every 100 μL colloidal gold solution, 3.5 μL streptavidin was added; the detection line on nitrocellulose membrane was 2.0 mg·mL-1 anti FITC antibody, and the quality control line was 1.2 mg · mL-1 biotinylated BSA, and they generate a red band after binding with positive amplification product. Specificity test of the assembled test strip yielded consistent result with agarose gel electrophoresis without cross reaction with Acinetobacter, Aeromonas, Pseudomonas, or Leclercia adecarboxylata. Sensitivity test of the strip showed a lower detection limit for DNA concentration of 10-5 ng/μL, with a sensitivity 1000 times that of agarose gel electrophoresis. The test strip still had good performance after storage for 3, 6, 9 and 12 months. We successfully developed a PCR-nucleic acid test strip for convenient and cost-effective detection of Ralstonia pickettii with good specificity and sensitivity and low cost to facilitate daily monitoring of pharmaceutical water contaminations.

Molecular and epidemiological characterization of human adenoviruses infection among children with acute diarrhea in Shandong Province, China

BackgroundHuman adenovirus (HAdV) had been recognized as one of the most common enteric viruses associated with acute diarrhea in children. The present study was carried out to demonstrate the molecular and epidemiological characterization of HAdV Infections among children in Shandong province in China between July 2017 and June 2018.MethodsFecal specimens were collected from children under 5 years old with acute diarrhea. DNA was extracted from the stool specimens and adenovirus DNA was detected by PCR amplification with specific primers. The amplification products were subjected to electrophoresis and visualized on a UV transilluminator. All positive RT-PCR amplification products were sequenced and the obtained sequences analyzed by MEGA (version 7.0). Demographic information and clinical manifestation data were also analyzed.ResultsIn total, 656 fecal specimens were collected and the overall positive rate of HAdV was 7.47%. HAdV infections were detected in all age groups, in which children aged 13–24 months presented the highest positive rate. Seasonal pattern could be observed with a peak in December, January and February. Diarrhea, vomiting, dehydration and fever were the main clinical manifestations, in which vomiting was the most common accompanied symptom. By phylogenetic analysis, four species (A, B, C, and F) were detected and seven different serotypes were identified. HAdV-41 (48.98%, 24/49) was the most common serotype followed by HAdV-3 (18.37%, 9/49), HAdV-31 (14.29%, 7/49), HAdV-7 (8.16%, 4/49), HAdV-40 (4.08%, 2/49), HAdV-1 (4.08%, 2/49) and HAdV-2 (2.04%, 1/49).ConclusionThis study indicated that HAdV infection was an important cause of acute diarrhea among children under 5 years old in Shandong province. The results will contribute to (a) increase understanding of the role of HAdV in diarrheal children and enhance identification of the predominant diarrhea pathogen for diagnosis; (b) avoid abuse of antibiotics; (c) monitor the change of prevalent HAdV serotypes and promote vaccine development and vaccination.

Serological Specificity in the Detection of Syphilis among Pregnant Women Attending Antenatal in Some Hospitals in Mubi Metropolis, Adamawa State, Nigeria

Syphilis is a sexually transmitted infection (STI) caused by the Treponema pallidum (spirochetes). Syphilis remains a major cause of reproductive morbidity and poor pregnant outcomes in developing countries. This Research work seeks to determine the specificity of serological VDRL test for syphilis against the use of High Vaginal Swab in molecular detection of syphilis among pregnant women attending antenatal in some selected hospitals and clinics in Mubi North and South L.G.A. Adamawa State, Nigeria to subvert the challenges, hence finding lasting solution to the Reproductive and mental health challenges posed by secondary Syphillis. A total of 120 blood samples were collected from 120 consented pregnant women in batches from General hospital, Mubi, Sabon layi clinic, Lokuwa PHC, Lamurde maternity, Kwaja PHC and Alheri Nursing hospital all in Mubi North and South Local Government, during their Antenatal days. In all, 30(25.0 %) of the samples were seropositive, with the highest prevalence recorded as 11(9.17 %) of the 20 samples collected from Kwajah PHC in Mubi South L.G.A. The HVS of pregnant with positive serological test results were subjected to molecular nPCR test to amplify the tpp47 gene of Treponema pallidum. The only amplified fragment which arouse from the positive as the only positive amplification products was analyzed for specificity in an agarose gel developed 0.01 % ethidum bromide for ease of visualization of the band of fragment weight 260 bp tpp47 gene of Treponema pallidum under U.V since all HVS samples showed no bands of amplification, no further sequencing was technically necessary.

Loop-mediated isothermal amplification: a rapid detection method for rice actin and nopaline synthase promoters in genetically modified crops

The commonly employed promoters in transgenic constructs such as Cauliflower Mosaic Virus 35S and Figwort Mosaic Virus promoters are not necessarily present in all the approved/upcoming genetically modified (GM) events. GM events of cotton and maize with rice actin (P-ract) and nopaline synthase (P-nos) promoters have been approved globally. The scope of screening assays is expanded to a diversified range of promoters being employed in GM crops. Loop-mediated isothermal amplification (LAMP) is a method of choice for screening of genetically modified organisms (GMOs) due to ease-of-use, rapidity and applicability to crude DNA samples. An efficient LAMP-based screening method was developed targeting P-ract and P-nos for better coverage of GM events approved globally. LAMP reactions were isothermally incubated at 63 °C for 30 min for P-ract and 65 °C for 60 min for P-nos. Positive amplification products were visualized by change in colour from orange to green on addition of SYBR® Green 1 dye. Specificity was confirmed using defined sets including both the targets and non-targets. The developed assays showed acceptable specificity and sensitivity with limit of detection of 0.05% for P-ract and 0.01% for P-nos. Practical applicability of assays was confirmed using proficiency test samples of maize for P-ract and spiked samples of cotton for P-nos. These assays could be utilized for efficient GMO testing with less time and low cost inputs.

Prevalence and genetic characteristics of 4CMenB and rLP2086 vaccine candidates among Neisseria meningitidis serogroup B strains, China

ObjectiveTo systematically investigate the prevalence and genetic characteristics of 4CMenB and rLP2086 vaccine candidates among Neisseria meningitidis serogroup B (NmB) in China. MethodsA total of 485 NmB strains isolated in 29 provinces of China between 1968 and 2016 were selected from the culture collection of the national reference laboratory according to the isolation year, location, and source. Multi-locus sequence typing (MLST) and porA gene sequencing were performed on all 485 study strains; PCR was used to detect the fHbp, nadA, and nhba gene of 432 strains; positive amplification products from the fHbp and nadA genes from all strains, as well as those of the nhba gene from 172 representative strains, were sequenced. ResultsMLST results showed that the predominant (putative) clonal complexes (CCs) of NmB isolates have changed over time in China. While strains that could not be assigned to existing (p)CCs were the biggest proportion, CC4821 was the most prevalent lineage (36.0%) since 2005. PCR and sequence analysis revealed that the 4CMenB and rLP2086 vaccine candidates were highly diverse. Respectively, 152 PorA genotypes and 83 VR2 sequences were identified with significant diversity within a single CC; the complete nadA gene was found in ten of 432 study strains; fHbp was present in most strains (422/432) with variant 2 predominating (82.9%) in both patient- and carrier- derived isolates; almost all strains harbored the nhba gene while sequences were diverse. ConclusionsWith regards to clonal lineages and vaccine candidate proteins, NmB isolates from China were generally diverse. Further studies should be performed to evaluate the cross-protection of present vaccines against Chinese NmB strains.

Sensitive Visual Detection of AHPND Bacteria Using Loop-Mediated Isothermal Amplification Combined with DNA-Functionalized Gold Nanoparticles as Probes.

Acute hepatopancreatic necrosis disease (AHPND) is a component cause of early mortality syndrome (EMS) of shrimp. In 2013, the causative agent was found to be unique isolates of Vibrio parahaemolyticus (VPAHPND) that contained a 69 kbp plasmid (pAP1) carrying binary Pir-like toxin genes PirvpA and PirvpB. In Thailand, AHPND was first recognized in 2012, prior to knowledge of the causative agent, and it subsequently led to a precipitous drop in shrimp production. After VPAHPND was characterized, a major focus of the AHPND control strategy was to monitor broodstock shrimp and post larvae for freedom from VPAHPND by nucleic acid amplification methods, most of which required use of expensive and sophisticated equipment not readily available in a shrimp farm setting. Here, we describe a simpler but equally sensitive approach for detection of VPAHPND based on loop-mediated isothermal amplification (LAMP) combined with unaided visual reading of positive amplification products using a DNA-functionalized, ssDNA-labled nanogold probe (AuNP). The target for the special set of six LAMP primers used was the VPAHPND PirvpA gene. The LAMP reaction was carried out at 65°C for 45 min followed by addition of the red AuNP solution and further incubation at 65°C for 5 min, allowing any PirvpA gene amplicons present to hybridize with the probe. Hybridization protected the AuNP against aggregation, so that the solution color remained red upon subsequent salt addition (positive test result) while unprotected AuNP aggregated and underwent a color change from red to blue and eventually precipitated (negative result). The total assay time was approximately 50 min. The detection limit (100 CFU) was comparable to that of other commonly-used methods for nested PCR detection of VPAHPND and 100-times more sensitive than 1-step PCR detection methods (104 CFU) that used amplicon detection by electrophoresis or spectrophotometry. There was no cross reaction with DNA templates derived from non-AHPND bacteria commonly found in shrimp ponds (including other Vibrio species). The new method significantly reduced the time, difficulty and cost for molecular detection of VPAHPND in shrimp hatchery and farm settings.

Selection and characterization of naturally occurring high acidification rate Streptococcus thermophilus strains

Among Streptococcus thermophilus cultures, the principle component of yoghurt and cheese starters, a minority of strains forms the group of ‘H’-strains which show an unusually high acidification rate, grow faster and coagulate milk 3–5 hours earlier than the typical S. thermophilus cultures. A large-scale screening study was performed to select ‘H’-strains of S. thermophilus from more than 100 samples of home-made yoghurt, industrial yoghurt starters and single cultures, maintained in the LBB culture collection. Only four strains – LBB.TN1, LBB.M23, LBB.M34 and LBB.M60 – were isolated/selected due to their ability to form large yellowish colonies on milk agar, supplemented with beta-glycerophosphate and bromocresol purple. While in general S. thermophilus is described as a species with limited proteolytic capacity and in contrast to all other tested S. thermophilus cultures, the four selected strains invariably gave positive amplification product with the polymerase chain reaction when primers, specific for the membrane proteinase-coding gene prtS were used. The macrorestriction profiles of the genomic DNA of the four strains confirmed that they are non-isogenic and not related to each other. When grown in milk and compared to the control industrial strain LBB.A, the four strains showed a dramatically faster acidification, coagulating milk within four hours. The application of strain TN1 or M23 as adjunct culture to industrial yoghurt starter LBB.BY5-12 resulted in shortening the fermentation time with more than 30 min.

DNA Binding to Hydroxyapatite: A Potential Mechanism for Preservation of Microbial DNA

IntroductionMolecular methods are increasingly being deployed for analysis of the microbial flora in the root canal. Such methods are based on the assumption that recovered DNA is associated with the active endodontic infection, yet paleomicrobiology research is based on the recovery of ancient DNA from centuries-old tooth and bone samples, which points to considerable longevity of the DNA molecule in these tissues. The main component of dentin and bone is the mineral hydroxyapatite. This study assessed DNA binding to hydroxyapatite and whether this binding affinity stabilizes the DNA molecule in various media. MethodsDNA was extracted from Fusobacterium nucleatum and added to ceramic hydroxyapatite for 90 minutes. The DNA-bound hydroxyapatite was incubated in different media (ie, water, sera, and DNase I) for up to 3 months. At predetermined intervals, the recovery of detectable DNA was assessed by releasing the DNA from the hydroxyapatite using EDTA and evaluating the presence of DNA by gel electrophoresis and polymerase chain reaction (PCR) amplification. ResultsWhen incubated with hydroxyapatite, nonamplified DNA was detectable after 3 months in water, sera, and DNase I. In contrast, DNA incubated in the same media (without hydroxyapatite) decomposed to levels below the detection level of PCR within 3 weeks, with the exception of DNA in sera in which PCR revealed a weak positive amplification product. ConclusionsThese results confirm a specific binding affinity of hydroxyapatite for DNA. Hydroxyapatite-bound DNA is more resistant to decay and less susceptible to degradation by serum and nucleases, which may account for the long-term persistence of DNA in bone and tooth.

Detection and clinical study on coronavirus HKU1 with acute lower respiratory tract infections of hospitalized children in Changsha

In order to understand the epidemiological and virologic characteristics of coronavirus HKU1 infection in hospitalized children with acute respiratory tract infection (ARTI) in Changsha. 1165 nasopharyngeal aspirates (NPA) specimens were collected from hospitalized children with ARTI between September 2007 and August 2008 in Changsha. Specimens were screened for pol gene of coronavirus HKU1 by polymerase chain reaction. All positive amplification products were confirmed by sequencing and compared with those in GenBank. Coronavirus HKU1 were detected in 12 patients (1.03%) out of the 1165 children. The patients were from 8 days to 3 years. The most common clinical diagnosis was bronchopneumonia(83.33%). Similarity of coronavirus HKU1 with those published in the GenBank at nucleotide levels was 98.18% - 100%. Coronavirus HKU1 may be important pathogens in children with acute lower respiratory tract infection. Coronavirus HKU1 infections are common in children under 3 years old. There is no significant difference in the infectious rate between the boys and the girls. The peak of its prevalence is in spring and winter. A single genetic lineage of Coronavirus HKU1 was revealed in human subjects in Changsha.

Expression Profile of Aquaporins in Human Nasolacrimal Duct Epithelium

Purpose: To determine whether the epithelium of the human nasolacrimal ducts contains aquaporins (AQPs), a family of membrane proteins that function as selective pores and are able to transport water, glycerol, and other small solutes across the cell plasma membrane.Methods: Expression of AQPs 0 and 1–10 in human nasolacrimal duct tissue was determined by reverse transcription polymerase chain reaction (RT-PCR). Positive PCR amplification products were verified by direct cDNA sequencing. Western blot analysis was used to detect AQPs 3–5. Antisera specific for AQPs were used in immunohistochemical analysis to determine the presence and distribution of ten AQPs (AQP 0 and 1–9) in epithelia and subepithelial glands of the nasolacrimal ducts. All samples investigated originated from human postmortem tissue.Results: In human nasolacrimal duct samples, AQPs 1, 3, 4, 5, 7, 8, 9, and 10 were identified by RT-PCR. No RT-PCR products were detected for AQPs 0, 2, and 6. All AQPs, which were detected by RT-PCR, were also confirmed by direct sequencing of the cDNA. Immunohistochemical analyses revealed AQPs 1, 3, 5, 7, 8, and 9 in human nasolacrimal duct epithelium and were detected in different cells. Expression of AQP 4 could not be detected immunohistochemically but by Western blot analysis. Protein detection of AQP 10 could not be performed due to the unavailability of an appropriate antibody.Conclusions: The results suggest specific roles for AQPs in water transport through the epithelium of the nasolacrimal ducts and underline the presumption that tear fluid components are selectively absorbed in the nasolacrimal passage.

Prevalence and Clinical and Molecular Characterization of Human Metapneumovirus in Children With Acute Respiratory Infection in China

Human metapneumovirus (HMPV), a newly discovered paramyxovirus, has been associated with acute respiratory tract infections (ARTIs). However, the prevalence and molecular characteristics of HMPV in China are still unclear. A total of 661 nasopharyngeal aspirates (NPA) specimens were collected from 661 children with ARTIs between December 2006 and November 2008. Specimens were screened for HMPV by reverse transcription-polymerase reaction. All positive amplification products were confirmed by sequencing. HMPV was detected in 45 patients (6.80%) of the 661 children. The HMPV-infected patients were from 29 days to 9 years of age. A high incidence of HMPV infection (84.4%) was observed during the winter-spring season. Of the 45 HMPV-positive patients, 25 (55.6%) were co-infected with other respiratory viruses, and respiratory syncytial virus (RSV) was the most common additional respiratory virus. The most common clinical diagnosis was bronchopneumonia (57.8%) and cough (88.9%) was the most common clinical symptom. Phylogenetic analysis of the F gene revealed that 80% of the HMPV detected were A2, 2.2% were A1, and 17.8% were B1. Statistical analyses showed that sex, ages, seasons, and severity of the disease did not correlate with HMPV genotype (P = 0.986, 0.347, 0.660, 0.252), but viral coinfection with HMPV increased hospitalization rates (P = 0.005). HMPV was frequently detected in the pediatric patients with ARTI in China. RSV was the most common coinfection virus and coinfection increased hospitalization rates. All HMPV subgroups except B2 cocirculated and there was no association found between HMPV genotypes and severity of disease.

Isolation, molecular and biochemical characterization of oil degrading bacteria from contaminated soil at an oil refinery

Biodegradation using microorganisms is considered to be cost-effective and environmentally friendly treatment of oil-contaminated sites. Oil-biodegrading bacterial strains were isolated, identified and characterized from oil contaminated soil samples at oil refinery in Zarqa (Jordan). Thirty four bacterial isolates were grown on mineral salt media supplemented with crude oil, but 16 showed positive biodegradation of diesel. All the 34 bacterial isolates were characterized at the molecular and bio-chemical levels, and showed positive polymerase chain reaction (PCR) amplification product size of 1500 bp when 16s rDNA bacterial universal primers were used. Eighteen bacterial isolates showed positive PCR amplification product size of 150 bp specific for the genus Pseudomonas and 3 bacterial isolates showed positive amplification product size of 1500 bp specific for the genus Acinetobacter. Biochemical and physiological characterization performed on the 34 bacterial isolates, revealed the presence of oil biodegrading bacterial genera and species of Pseudomonas Acidovorans, P. aerugi-nosa, P. alcaligenes, P. fluorescens, P. cepacia, P. mallei, P. maltophilia, P. oleovorans, P. putida, P. stutzeri P. vesicularis, Acinetobacter calcoaceticus, Ac. lowffii, Micro-ococcus luteus , M. varians, M. lylae, M. roseus, Alcaligenes denitrificians, Bacillus megaterium, Comamonas sp., Moraxella sp., Bordetella sp., P. putida, P. stutzeri and P. mallei.

IDENTIFICATION OF NEW TARGET SEQUENCES FOR PCR DETECTION OF VIBRIO PARAHAEMOLYTICUS BY GENOME COMPARISON

ABSTRACT A genome comparison method was used to identify specific target sequences for the polymerase chain reaction (PCR) detection of Vibrio parahaemolyticus, and the CDS value of this bacterium was compared with that of 139 other bacterial genomes. It was found that 20 CDS of V. parahaemolyticus were relatively specific according to their E value in BLAST (a new tool for comparing protein and nucleotide sequences), and four of them were selected for the design of PCR primers. There were positive amplification products of these four pairs of primers from nine V. parahaemolyticus strains, whereas there were no amplification products from nine other Vibrionaceae strains and four non‐Vibrionaceae strains. An evaluation of detection sensitivities revealed that these four pairs of primers can be used in a PCR assay for the detection of V. parahaemolyticus.PRACTICAL APPLICATIONSAn automatic BLAST method was developed in this study, by which species‐specific sequences can be screened out rapidly. In this way, new and specific genes of Vibrio parahaemolyticus were identified to be used as target sequences for PCR detection. In terms of acceptable specificity and sensitivity, the four pairs of primers were selected by screening, which can be applied in PCR assays and other molecular methods. These kinds of methods might become commercial detection products in the new future. In addition, this method for searching specific DNA sequences can also be used for the mining specific sequences in other genus and species, such as Salmonella, Staphylococcus, etc.

Plasmodium falciparumin Ancient Egypt

To the Editor: Malaria is a disease caused by parasites of the genus Plasmodium. The infection is transmitted to humans through the bites of female mosquitoes of the genus Anopheles. Four species of Plasmodium are pathogenic to humans, and each leads to different clinical features: P. falciparum causes severe malaria with undulating high fever (malaria tropica); P. malariae, P. vivax, and P. ovale cause less severe clinical courses of disease with the manifestations of malaria quartana (P. malariae) and malaria tertiana (P. vivax and P. ovale). Literary evidence for malaria infection dates back to the early Greek period when Hippocrates described the typical undulating fever (1), highly suggestive of plasmodial infection. Although it is believed that malaria widely affected early pre-Hippocrates populations, until now only 1 study, which used molecular analysis, clearly identified P. falciparum in a Roman infant dating back to the 5th century AD (2). Two other studies used molecular analysis to identify more recent plasmodial DNA in ancient human remains, i.e., from 100–400 years ago (3,4). A substantial number of nonspecific amplifications in these previous studies raised concerns as to the specificity of current molecular markers for ancient malaria (3,4). In this report, we describe the unambiguous identification of ancient DNA (aDNA) for P. falciparum in ancient Egyptian mummy tissues from ≈4,000 years ago. We analyzed 91 bone tissue samples from ancient Egyptian mummies and skeletons. The Egyptian material derived from the Predynastic to Early Dynastic site of Abydos (n = 7; 3500–2800 BC), a Middle Kingdom tomb in Thebes West (n = 42; 2050–1650 BC), and various tomb complexes in Thebes West, which were built and used between the Middle and New Kingdom until the Late Period (n = 42; c. 2050–500 BC). All samples were first tested for Plasmodium spp. DNA by using the heminested PCR for the 18S rDNA primer targets usually used for malaria identification (5). Direct sequencing was performed on those with positive amplification products. Thereby, a high number of amplification products of various sizes (including the expected size) were detected. However, on sequencing, all amplicons provided nonspecific products. Consequently, in a second set, all material was tested for the P. falciparum chloroquine-resistance transporter gene (pfcrt gene) (6,7), which was also further characterized by direct sequencing. In this second set of experiments, 2 of the 91 ancient Egyptian samples tested positive for the 134-bp fragment of the pfcrt region of P. falciparum (Figure). The specificity of the amplification was verified by sequencing, which showed 99% sequence concordance. The result was verified by parallel analysis in 2 independent laboratories; observations were fully concordant. The 2 positive samples originated from 2 different tomb complexes dating from the New Kingdom until Late Period (1500–500 BC). Each sample was obtained from adults who had osteopathologic evidence of chronic anemia. No positive results were found for the earlier samples from the Predynastic to Early Dynastic or Middle Kingdom periods. Figure PCR amplification of a 134-bp fragment of ancient DNA of Plasmodium falciparum in Egyptian mummies. Lane 1, molecular marker; lanes 10 and 11, 2 negative controls. One (lane 6) of 8 samples shows a positive amplification product (arrow). Specificity of ... Previously, immunologic tests have been used to investigate the presence and incidence of malaria in ancient Egyptian mummies (8,9). Because >40% of all samples and 92% of samples from persons with bone lesions suggestive of chronic anemia tested positive for the P. falciparum histidine-rich protein-2 antigen, doubts as to the specificity of those tests have been raised. Our study unambiguously identified P. falciparum aDNA in Egyptian mummy samples, thereby proving a specific infection by falciparum malaria in ancient Egypt. With respect to the infection incidence, our molecular analysis suggests a more realistic frequency than had been previously suggested by paleoimmunologic methods. Consequently, the aDNA analysis is superior with respect to the reaction specificity, so that the latter should not further be used for that purpose. This report adds another infectious disease to the spectrum of paleomicrobiology in ancient Egypt, thereby further explaining the previously postulated influence of infectious diseases on the low life expectancy for ancient Egyptian populations (10). Molecular detection of pathogen aDNA can be used not only to identify a certain disease, but it may also provide information on disease frequency, evolutionary origin, and pathways.

Clonal Heavy and Light Chain Immunoglobulin DNA in Plasma/Serum of AIDS Lymphoma Patients.

Background DNA tumor markers in plasma and serum such as point mutations, hypermethylation of tumor suppressor genes, and viral sequences have attracted much interest. We investigated clonal Ig DNA rearrangements in the plasma of AIDS-related lymphoma (ARL) patients (pts).Methods We evaluated specimens for Ig rearrangements from pts with ARL using fluorescently-labeled multiplex primers targeting IgH and IgK. PCR products were analyzed by capillary electrophoresis. Re-amplified PCR products from plasma and tumor were cloned and sequenced.Results Clonal IgH DNA spikes were seen in plasma at baseline in 5 of 10 pts. Clonal DNA was detected in plasma in pts with either early or advanced stage disease, with or without marrow involvement. Presence of a plasma clone was associated with high LDH. Serial specimens were available from 4 pts. In 2 pts who died with primary refractory disease, clonal peaks remained present after the administration of chemotherapy. In a pt with disease that initially responded and then relapsed, clonal DNA disappeared with chemotherapy, then reappeared presaging relapse and death. In 4 pts, matching serum specimens were available. Results were similar in both types of specimens with identical sized rearrangements amplified from matched specimens. Furthermore in 2 pts, the IgH amplification products from plasma and tumor were sequenced and confirmed to be identical. Two pts with positive IgH amplification products were also screened for light chains in the plasma and in both, clonal IgK rearrangements were detected. For purposes of comparison, plasma from healthy donors (n=20) and AIDS pts with Kaposi's sarcoma (KS) (n=10) were also surveyed. Clonal Ig DNA rearrangements were not detected in any of these.Conclusions These results are consistent with observations suggesting that free tumor DNA can often be detected in plasma or serum of cancer pts. The absence of clonal Ig DNA in plasma from healthy donors and pts with AIDS KS suggests that detection of such DNA may be highly specific. Identification of the same Ig rearrangement and sequence in plasma and tumor is consistent with this interpretation. Serial monitoring of circulating clonal DNA appears to track—and perhaps anticipate—clinical evidence of disease. These approaches may be especially valuable in ARL where FDG uptake reflective of inflammatory disease or benign lymphoproliferation detected by PET scan often renders interpretation of imaging ambiguous. Similarly, elevated LDH in this population is relatively non-specific.

Identification of Sex-specific Expression Markers in the Giant Tiger Shrimp (Penaeus monodon)

Bulked segregant analysis (BSA) and AFLP were used for isolation of genomic sex determination markers in Penaeus monodon. A total of 256 primer combinations were tested against 6-10 bulked genomic DNA of P. monodon. Five and one candidate female- and male-specific AFLP fragments were identified. Female-specific fragments were cloned and further characterized. SCAR markers derived from FE10M9520, FE10M10725.1, FE10M10725.2 and FE14M16340 provided the positive amplification product in both male and female P. monodon. Further analysis of these markers using SSCP and genome walk analysis indicated that they were not sex-linked. In addition, sex-specific (or differential) expression markers in ovaries and testes of P. monodon were analyzed by RAP-PCR (150 primer combinations). Twenty-one and fourteen RAP-PCR fragments specifically/differentially expressed in ovaries and testes of P. monodon were successfully cloned and sequenced. Expression patterns of 25 transcripts were tested against the first stranded cDNA of ovaries and testes of 3-month-old and broodstock-sized P. monodon (N=5 and N=7-10 for females and N=4 and N=5-7 for males, respectively). Five (FI-4, FI-44, FIII-4, FIII-39 and FIII-58) and two (M457-A01 and MII-51) derived RAP-PCR markers revealed female- and male-specific expression patterns in P. monodon. Surprisingly, MII-5 originally found in testes showed a higher expression level in ovaries than did testes of juvenile shrimps but a temporal female-specific pattern in P. monodon adults.