Transport of dopamine at the blood-brain barrier of the guinea pig: inhibition by psychotropic drugs and nicotine

Diagnostic procedures for detection of infectious laryngotracheitis virus in tracheas of experimentally infected chickens, including the indirect fluorescent antibody test (IFAT), immunoperoxidase (IP), virus isolation (VI), histopathology, polymerase chain reaction (PCR), and DNA hybridization, were performed and compared. Using VI as a reference, we calculated the sensitivity and specificity of the tests. The sensitivities of IP, IFAT, histopathology, PCR, and hybridization were 100%, 93%, 7%, 27%, and 0%, respectively, and the specificities of IP, IFAT, histopathology, PCR, and hybridization were 93%, 93%, 100%, 100%, and 100%, respectively. Histopathology, PCR, and hybridization were more specific but lacked sensitivity compared to IP and IFAT. IP and IFAT were equally specific, but IP was more sensitive than IFAT. Based on these results, IP performed better than any other test.     

Heat-stabilized albumin microspheres as a sustained drug delivery system for the antimetabolite, 5-fluorouracil

A nested polymerase chain reaction (PCR) test was developed to examine infection with the bovine lentivirus, bovine immunodeficiency-like virus (BIV), in cattle. Primers were designed to amplify 2 separate regions of the pol and env segments of the BIV genome. Two calves were experimentally infected with an isolate derived from the original strain of BIV, R29, or with a recent field isolate, FL491. Serial blood samples were collected and examined by virus isolation, protein immunoblot, and nested PCR. The nested PCR test detected BIV infection by 3 days after inoculation, earlier than the other 2 methods, and continued to identify infected cattle 9 to 15.5 months after inoculation, even when results from virus isolation and serology became negative. Nested PCR also detected multiple-size env products in samples obtained later in the infection from the calf that received FL491, giving evidence that viral quasispecies were selected during in vivo replication of the virus. Results indicated that the nested PCR test is more sensitive than virus isolation or serology for the detection of BIV infection in cattle.     

Development of PCR tests for the detection of bovine herpesvirus-1, bovine respiratory syncytial viruses and pestiviruses

The development of PCR assays for detection of BHV-1, BRSV, BVDV and another pestiviruses is summarized. A polymerase chain reaction assay based on primers selected from the viral gI glycoprotein gene detected 3 fg pure BHV-1 DNA, 0.1-1.0 TCID50 or a single infected cell. No amplification was observed with DNA from BHV-2, BHV-3, BHV-4, OHV-1 or OHV-2. However, a fragment of the correct size (468 bp) was amplified using DNA from herpesviruses isolated from reindeer, red deer and goat. The PCR assay was able to detect virus in nasal swabs 1-14 days after experimental infection of cattle and there was a good correlation when PCR was compared to virus isolation for the detection of BHV-1 in clinical field samples. Detection of BHV-1 in fetal bovine serum and semen samples was also successful. PCR detecting a broad range of BVDV, BDV and HCV was developed. Of six sets of primers selected from different parts of the pestivirus genome the best results were provided by a pair 324/326 from the highly conserved 5'-non-coding region which gave an amplification with all 129 isolates tested. This panel consisted of 79 isolates from cattle, 33 from pigs and 17 from sheep. Differentiation between viruses was achieved by cleavage of the PCR-amplified products (288 bp) with the restriction endonucleases AvaI and BglI. The BVDV products were cleaved by AvaI, HCV by BglI and AvaI. Both enzymes, AvaI and BglI, did not cut the BDV products. A nested polymerase chain reaction assay was developed for the detection of bovine respiratory syncytial virus (BRSV). Primers were selected from the gene encoding the F fusion protein. The sensitivity of PCR assay was 0.1 TCID50. No cross reaction was observed with nine heterologous respiratory viruses. PCR products of bovine and human RSV strains were discriminated using endonuclease ScaI, which specifically cleaved products of BRSV. PCR assay detected BRSV in nasal swabs collected from cattle in the acute stage of respiratory disease. In vitro amplification detected 31 positive samples of 35 while immunofluorescence only 23 samples.     

Pharmacokinetic study in rats after single intravenous and oral administrations of [14C]-ITF-296

Fifteen mycoplasma-free chickens were contact exposed to five chickens that had been experimentally infected with one of three different strains (two field strains and one laboratory strain) of Mycoplasma synoviae (MS). Culture and polymerase chain reaction (PCR) were positive by 3 days postinoculation (PI) in the experimentally infected birds. Lateral transmission was found by 7-14 days postexposure. Positive serum plate agglutination (SPA) results were detected 3-4 wk after positive culture and/or PCR in individual birds. By 42 days PI, all the birds in the groups exposed to field strain K1858 or K3344 had become infected as determined by culture and PCR, whereas only half of the birds in the group exposed to laboratory strain WUV1853 had become infected. Because of the unanticipated lack of seroconversion to hemagglutination inhibition (HI) and enzyme-linked immunosorbent assay (ELISA) in infected chickens, the study was extended. Each group was split into two groups of 10 birds each, one of which was vaccinated with a live B1/LaSota Newcastle disease (ND) vaccine virus to determine if a viral respiratory challenge might incite a stronger antibody response to the mycoplasma infection. All the birds were tested for seroconversion 14 and 21 days later. Of the birds vaccinated for ND, a slightly greater number were MS positive by SPA than the nonvaccinated birds. This effect was not present 21 days after vaccination, and there was no significant difference in the MS HI results from these groups, suggesting that the viral respiratory infection had little direct impact on seroconversion. The virulent field strain (K3344) elicited a stronger MS antibody response than the other strains. All results from the MS ELISA were negative in all groups through 9 wk. Positive results from PCR analysis correlated well with culture results, whereas serologic tests did not detect MS infection for several weeks. Monitoring programs solely dependent on seroconversion may be inadequate for diagnosis and control of mycoplasma infections.     

The carboxyl-terminal 120-residue polypeptide of infectious bronchitis virus nucleocapsid induces cytotoxic T lymphocytes and protects chickens from acute infection

Specific cytotoxic T-lymphocyte (CTL) responses to nucleocapsid of infectious bronchitis virus (IBV) were identified by using target cells infected with a Semliki Forest virus (SFV) vector. Effector cells for CTL assays were collected from chickens infected with the Gray strain of IBV or inoculated with a DNA plasmid encoding nucleocapsid proteins. IBV-specific CTL epitopes were mapped within the carboxyl-terminal 120 amino acids of the nucleocapsid protein. CTL lysis of target cells infected with SFV encoding nucleocapsid was major histocompatibility complex restricted and mediated by CD8+ T cells. In addition, splenic T cells collected from chickens inoculated in the breast muscle with a DNA plasmid encoding this CTL epitope(s) recognized target cells infected with wild-type virus or an SFV vector encoding nucleocapsid proteins. CTL activity of splenic T cells collected from chicks immunized with a DNA plasmid encoding CTL epitopes was cross-reactive, in that lysis of target cells infected with serologically distinct strains of IBV was dose responsive in a manner similar to that for lysis of target cells infected with the homologous strain of IBV. Furthermore, chickens immunized with a DNA plasmid encoding a CTL epitope(s) were protected from acute viral infection.     

In vitro generation of human cytomegalovirus pp65 antigenemia, viremia, and leukoDNAemia

Immunocompromised patients with disseminated human cytomegalovirus (HCMV) infection have circulating PMN carrying HCMV pp65 (antigenemia), infectious virus (viremia), and viral DNA (leukoDNAemia). Because HCMV does not fully replicate in PMN, it is generally hypothesized that virions and viral materials are taken up by phagocytosis from fully permissive HCMV-infected endothelial cells. However, no experimental evidence has ever been provided for these PMN-endothelium interactions. PMN from 11 donors were cocultured with endothelial cells infected with an endothelium-adapted HCMV strain and with human fibroblasts infected with low-passaged clinical and laboratory-adapted HCMV strains. pp65-positive PMN were detected after coculture with both HCMV-infected endothelial and fibroblast cells, provided that wild and not laboratory-adapted strains were used. In addition, cocultured PMN carried infectious virus as demonstrated by virus isolation and presence of complete virus particles by electron microscopy. Moreover, high levels of viral DNA were consistently detected by quantitative PCR in cocultured PMN. Thus, we have generated in vitro the three most important viral parameters detected in patients with disseminated HCMV infection (antigenemia, viremia, and leukoDNAemia). The failure of laboratory-adapted HCMV strain to induce this phenomenon demonstrates that important modifications have occurred in attenuated viral strains affecting basic biological functions.     

History and examination of the orofacial pain patient

A polymerase chain reaction (PCR) assay was developed to detect. Chlamydia psittaci DNA in faeces and tissue samples from avian species. Primers were designed to amplify a 264 bp product derived from part of the 5' non-translated region and part of the coding region of the ompA gene which encodes the major outer membrane protein. Amplified sequences were confirmed by Southern hybridization using an internal probe. The sensitivity of the combined assay was found to be between 60 to 600 fg of chlamydial DNA (approximately 6 to 60 genome copies). The specificity of the assay was confirmed since PCR product was not obtained from samples containing several serotypes of C. trachomatis, strains of C. pneumoniae, the type strain of C. pecorum, nor from samples containing microorganisms commonly found in the avian gut flora. In this study, 404 avian faeces and 141 avian tissue samples received by the Central Veterinary Laboratory over a 6 month period were analysed by PCR, antigen detection ELISA and where possible, cell culture isolation. PCR performed favourably compared with ELISA and cell culture, or with ELISA alone. The PCR assay was especially suited to the detection of C. psittaci DNA in avian faeces samples. The test was also useful when applied to tissue samples from small contact birds associated with a case of human psittacosis where ELISA results were negative and chlamydial isolation was a less favourable method due to the need for rapid diagnosis.     

The surgical treatment of disorders in the formation and fusion of the lumbar vertebrae in children

OBJECTIVE: To investigate the value of polymerase chain reaction (PCR) for follow-up patients infected by Chlamydia trachomatis. METHODS: Follow-up specimens were collected from 30 patients. Chlamydia trachomatis positive were detected by PCR and direct fluorescence assay test (DFA) in the 30 patients before therapy. 15 patients were treated with minocycline (100 mg twice daily) for 10 days, and 15 patients were treated with 1.0 g of azithromycine as a single oral dose. RESULTS: After 1-2 weeks of antimicrobial therapy, all patients had negative DFA for Chlamydia trachomatis, but 9 had positive Chlamydia trachomatis DNA as detected by PCR. CONCLUSIONS: The 9 specimens were not confirmed to livae viable organisms of Chlamydia trachomatis. The debris of nonviable Chlamydia trachomatis DNA was excluded from urinogenital tract at about one month.     

A nested PCR for detection of North American isolates of bluetongue virus based on NS1 genome sequence analysis of BTV-17

A nested polymerase chain reaction (PCR)-based assay, for detection of bluetongue virus (BTV) ribonucleic acid in cell culture and tissue samples, was developed. Two pairs of oligonucleotide primers (BTV1 and BTV4 and BTV2 and BTV3), selected from non-structural protein 1 (NS1) gene of BTV-17, were used for the nested PCR in two amplification steps. First a 826-bp product was amplified using an outer primer pair BTV1 and BTV4. The second amplification, using nested or internal primer pair BTV2 and BTV3, produced a 517-bp PCR product. RNA from North American prototype serotypes 2, 10, 11, 13 and 17, propagated in cell cultures, were detected by this nested PCR-based assay. The nested primers BTV2 and BTV3 increased the sensitivity of the BTV PCR assay, and as little as 0.1 fg of BTV RNA (equivalent to 5 viral particles) could be detected. Amplification products were not detected when the PCR-based assay was applied to RNA from a closely related orbivirus, epizootic hemorrhagic disease virus (EHDV) prototype serotypes 1 and 2; total nucleic acid extracts from uninfected BHK-21 cells; or whole blood from calves and deer that were BTV-seronegative and virus isolation negative. Application of this nested BTV PCR-based assay to clinical samples resulted in detection of BTV RNA from a variety of tissues collected from calves and deer with natural and experimental BTV infections. The described BTV PCR-based assay provides a valuable tool to study the epidemiology of BTV infection in susceptible wild ruminants and domestic livestock.     

Comparison of the genomic short regions of a vaccine strain (SA-2) and a virulent strain (CSW-1) of infectious laryngotracheitis virus (Gallid herpesvirus 1)

Restriction enzyme linkage maps were produced for the genomic short region of the virulent infectious laryngotracheitis virus (CSW-1 strain). After comparison with the equivalent restriction enzyme linkage maps for the infectious laryngotracheitis virus SA-2 strain (a vaccine strain), it was determined that the maps for the short regions of the two strains were identical, apart from a single section in each of the inverted terminal repeats. Each inverted terminal repeat of the SA-2 strain was discovered to contain 467 base pairs more DNA than the CSW-1 strain's inverted terminal repeats. This extra DNA was more precisely mapped entirely within the EcoRI fragments D and d of SA-2, which were found to form part of the SmaI fragments U and P of SA-2 and Q and b of SA-2 and to contain one SmaI restriction enzyme site.     

Diethyl ether. 1963

Chlamydia psittaci is a significant cause of conjunctivitis in cats, but can be difficult to diagnose owing to the small number of organisms in conjunctival swabs. In the United Kingdom laboratory diagnosis is based on three techniques: isolation of the infectious organism, amplification of chlamydial DNA by the polymerase chain reaction (PCR) or the detection of anti-chlamydial antibodies by immunofluorescence assay. To determine the most sensitive method these techniques were compared in the field. The PCR based on previously published protocols was less sensitive than isolation, but by modifying the protocol its sensitivity was increased by a factor of 25 to 1250 and it was then more sensitive than isolation. The modified PCR detected chlamydia in samples containing non-infectious organisms. Serology was of limited use in predicting which cats shed C psittaci although seronegative cats were negative by PCR and isolation. The modified PCR was the most sensitive and robust method for confirming C psittaci infection in cases of conjunctivitis in pet cats.     

A simple, rapid and sensitive detection of salmonella in food by polymerase chain reaction

A polymerase chain reaction (PCR) method was developed for detection of salmonella in food. A set of PCR primers was designed to amplify a 199 bp salmonella-specific DNA fragment derived from a repetitive DNA of Salmonella Weltevreden. The assay detected all 52 most prevalent serovars found in contaminated food in Thailand and no cross-reaction was observed with other non-salmonella organisms. The limit of detection was 1 fg of purified target DNA or five bacteria from pure culture. The detection of artificially contaminated food performed following a 6 h enrichment step was three bacteria per gram and the result was obtained within 4 h.     

Reactive pleuropericarditis following laparoscopic fundoplication

Samples of peripheral blood from 76 different donors were detected for the presence of human cytomegalovirus (HCMV) DNA by polymerase chain reaction (PCR). The results showed that HCMVs were present in 39 out of 76 blood donors which reached an infection positive rate of 51.3%. In addition, the sensitivity and specificity on PCR were tested, it also indicated that the sensitivity can reach the level of 5fg.microliter-1. The method is simple, rapid, highly sensitive and specificity. It can be considered as a diagnostic measure in clinical detection of HCMV.     

Detection of herpes simplex virus type 1 shedding in the oral cavity by polymerase chain reaction and enzyme-linked immunosorbent assay at the prodromal stage of recrudescent herpes labialis

Recrudescent herpes labialis (RHL) is a disease caused by herpes simplex virus (HSV), predominantly type 1 (HSV-1). We have monitored HSV-1 shedding in the oral cavity by polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA) using digoxigenin-labeled primers designed to amplify a 278 bp segment of the HSV-1 UL 42 region. Prodromal RHL was confirmed by thermographic imaging in 22 patients. Infectious virus was not detected using tissue culture for virus isolation (0/22). Using PCR and agarose gel electrophoresis, we could detect HSV-1 DNA in 8/22 patients. Using a biotinylated-probe internal to the predicted sequence of the PCR product, HSV-1 DNA was detected in 10/22 patients by ELISA. We conclude that HSV-1 DNA is shed into the oral cavity of patients presenting with sub-clinical RHL and that the PCR-ELISA technique represents a more sensitive method to monitor HSV-1 shedding than conventional tissue culturing or PCR-electrophoresis alone.     

Feline immunodeficiency virus is shed in semen from experimentally and naturally infected cats

Although a laboratory isolate of feline immunodeficiency virus (FIV), FIV-NCSU1, has been transmitted by artificial insemination in domestic cats, transmission by naturally infected males during mating has not been reported. In order to determine whether virus shedding in semen is unique to the NCSU1 isolate, we analyzed electroejaculates from four specific-pathogen-free males infected with another laboratory strain, FIV-Petaluma, and eight random source males with naturally acquired infections. Seminal cell lysates from the cats infected with the Petaluma isolate were screened by nested polymerase chain reaction amplification for FIV gag DNA. Seminal cells and seminal plasma from these FIV-Petaluma cats were further analyzed for the presence of virus by cocultivation with a feline CD4+ T cell line and Crandell feline kidney cells. Electroejaculates from the naturally infected cats were cocultivated with the T cell line. Our results demonstrated that cell-free FIV was present in seminal plasma from two FIV-Petaluma cats and two naturally infected cats. Cell-associated seminal virus was detected in all of the FIV-Petaluma infected cats and one naturally infected cat. Secretion of viral gag p26 antigen, an indication of active viral replication, was evident in cocultures containing motile sperm purified by a swim-up procedure from a FIV-Petaluma cat. These results confirm that FIV shedding in semen is not restricted to a specific virus isolate. Furthermore, swim-up sperm from FIV-infected cats may be infectious in vitro.     

Comparison of plasmid- and chromosome-based polymerase chain reaction assays for detecting Chlamydia trachomatis nucleic acids

Several laboratories have demonstrated that the polymerase chain reaction (PCR) is more sensitive than culture or enzyme immunoassay (EIA) for detecting Chlamydia trachomatis in genitourinary tract specimens when various DNA targets are used for amplification, including the cryptic plasmid, major outer membrane protein (MOMP), or rRNA genes. We compared the performances of five different PCR assays, including assays with two plasmid, two MOMP, and one rRNA targets, by amplifying serial dilutions of C. trachomatis DNA and testing genitourinary tract specimens. By using published procedures, two different plasmid primers had sensitivities of 0.1 fg for C. trachomatis plasmid DNA and 10 fg for total cellular DNA. The sensitivities of the assays with the two MOMP primers were 0.1 and 10 pg, and the sensitivity for the assay with the rRNA primers was 1 pg for cellular DNA. Both plasmid-based assays detected 38 of 38 confirmed Chlamydiazyme-positive specimens, whereas the assays with the MOMP and rRNA primers detected 36 of 38 and 29 of 38 confirmed Chlamydiazyme-positive specimens, respectively. Six of 18 Chlamydiazyme-negative specimens collected from individuals whose specimens were positive by culture or immunofluorescence were positive by both plasmid-based PCRs; 4 of these were positive by PCR with the MOMP primers and 3 were positive by PCR with the rRNA primers. The results obtained with both purified DNA and genitourinary tract specimens indicated that the plasmid-based PCRs are more sensitive than bacterial chromosome-based PCRs for detecting C. trachomatis.     

A polymerase chain reaction assay for detecting snails infected with bilharzia parasites (Schistosoma mansoni) from very early prepatency

In the present study, we adapted a polymerase chain reaction (PCR) assay, previously shown by us to be very sensitive for detecting cercariae in water, for the sensitive detection of Schistosoma mansoni DNA in infected snails from early prepatency. Polymerase chain reaction primers were designed based on the 121-basepair highly repeated sequence we previously identified in the genome of S. mansoni. The DNA was prepared from the snails by a simple alkaline extraction procedure, and the PCR assay enabled a clear differentiation between infected and normal snails. Infected snails were detected as early as one day after penetration of a single miracidium. The high sensitivity of the test enabled identification of a single infected snail even when its DNA was pooled with material from up to 99 uninfected snails, thus demonstrating the possibility of mass diagnosis in pools of snails. The assay has the potential for large-scale determination of prepatent infection prevalence in snails, thus offering new possibilities for the evaluation of schistosomiasis transmission and for schistosomiasis control, as discussed.     

Monitoring genetically modified rhizobia in field soils using the polymerase chain reaction

Monitoring genetically modified (GM) bacterial inoculants after field release using conventional culture methods can be difficult. An alternative is the detection of marker genes in DNA extracted directly from soil, using specific oligonucleotide primers with the polymerase chain reaction (PCR). The PCR was used to monitor survival of two GM Rhizobium leguminosarum bv. viciae inoculants after release in the field at Rothamsted. One strain, RSM2004, is marked by insertion of transposon Tn5; the second strain, CT0370, released at the same site, is modified by chromosomal integration of a single copy of the gene from E. coli conferring GUS activity. Both GM strain provide a realistic case study for the development of PCR-based detection techniques. Specific primers were developed to amplify regions of the Tn5 and GUS genetic markers using PCR and conditions optimized for each primer set to routinely detect a signal from 10 fg of purified template DNA, the equivalent of one cell per reaction. Procedures to improve the sensitivity of detection are described, to detect fewer than 50 cells g-1 soil in soil-extracted DNA.