scholarly journals Detection of equine infectious anemia nucleic acid in asymptomatic carrier horses by nested PCR

2010 ◽  
Vol 4 (6) ◽  
pp. 971-975
Author(s):  
Sunutcha Suntrarachun ◽  
Surasak Akesowan ◽  
Thaweesak Tirawatnapong
Keyword(s):  
Nucleic Acid ◽  
Nested Pcr ◽  
Equine Infectious Anemia Virus ◽  
Direct Detection ◽  
Asymptomatic Carrier ◽  
False Negative ◽  
Clinical Signs ◽  
Acute Stage ◽  
Dna And Rna ◽  
Equine Infectious Anemia

Abstract Background: Equine infectious anemia virus (EIAV) is a lentivirus with an almost worldwide distribution, infecting equids. It causes a persistent infection that is characterized by recurring episodes of fever, anemia, and thrombocytopenia. Most of the horses may control EIAV replication within a year, remaining persistently infected without clinical signs of disease. Objective: Detect EIA nucleic acid from peripheral blood of asymptomatic horses using nested PCR. Materials and method: We used nested PCR, amplifying P26 gag gene of EIAV, for direct detection of viral RNA in plasma and proviral DNA from PBMC in asymptomatic carrier horses in comparison with the Coggins test. EIA nucleic acid was prepared from 20 seropositive and five EIAV seronegative horses. Amplification of 246 bp expected size fragments was obtained using two different sets of primers targeting the P26 gag gene. Results: Among 20 seropositive horses, nine samples were positive for RNA and DNA. The five samples were positive for DNA but not for RNA, which indicates that the virus integrated into the host cell genome with a low level of viral replication. However, six samples were negative for both DNA and RNA. False negative could result due to primer failure caused by gag sequences variation among strains circulating in Thailand when compared with various strains from other parts of the world. EIAV antigens may also be prepared from cell cultures contaminated with other retroviruses thus causing false positives with the Coggins test. Conclusion: Nested PCR can be a useful tool for detecting the presence of EIAV in asymptomatic carrier horses. This may be especially true during the acute stage of the disease where the viremia levels are usually at the highest levels before detectable antibodies appear.

Molecular detection of equine infectious anemia virus in clinically normal, seronegative horses in an endemic area of Mexico

2021 ◽  
pp. 104063872110061
Author(s):  
César I. Romo-Sáenz ◽  
Patricia Tamez-Guerra ◽  
Aymee Olivas-Holguin ◽  
Yareellys Ramos-Zayas ◽  
Nelson Obregón-Macías ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Equine Infectious Anemia Virus ◽  
Antibody Detection ◽  
Economic Losses ◽  
Clinically Normal ◽  
Pcr Product ◽  
Surveillance Programs ◽  
Equine Infectious Anemia ◽  
Anemia Virus ◽  
Equine Industry

Equine infectious anemia (EIA) is a highly infectious disease in members of the Equidae family, caused by equine infectious anemia virus (EIAV). The disease severity ranges from subclinical to acute or chronic, and causes significant economic losses in the equine industry worldwide. Serologic tests for detection of EIAV infection have some concerns given the prolonged seroconversion time. Therefore, molecular methods are needed to improve surveillance programs for this disease. We attempted detection of EIAV in 6 clinical and 42 non-clinical horses in Nuevo Leon State, Mexico, using the agar gel immunodiffusion (AGID) test for antibody detection, and nested and hemi-nested PCR for detection of proviral DNA. We found that 6 of 6, 5 of 6, and 6 of 6 clinical horses were positive by AGID, nested PCR, and hemi-nested PCR, respectively, whereas 0 of 42, 1 of 42, and 9 of 42 non-clinical horses were positive by these tests, respectively. BLAST analysis of the 203-bp 5′-LTR/ tat segment of PCR product revealed 83–93% identity with EIAV isolates in GenBank and reference strains from other countries. By phylogenetic analysis, our Mexican samples were grouped in a different clade than other sequences reported worldwide, indicating that the LRT/ tat region represents an important target for the detection of non-clinical horses.

scholarly journals Development and Characterization of an In Vivo Pathogenic Molecular Clone of Equine Infectious Anemia Virus

1998 ◽  
Vol 72 (2) ◽  
pp. 1383-1393 ◽  
Author(s):  
R. Frank Cook ◽  
Caroline Leroux ◽  
Sheila J. Cook ◽  
Sandra L. Berger ◽  
Drew L. Lichtenstein ◽  
...  
Keyword(s):  
Cell Culture ◽  
Equine Infectious Anemia Virus ◽  
Consensus Sequence ◽  
Clinical Signs ◽  
Single Amino Acid ◽  
Progeny Virus ◽  
Molecular Clone ◽  
Single Amino Acid Residue ◽  
Equine Infectious Anemia ◽  
Anemia Virus

ABSTRACT An infectious nonpathogenic molecular clone (19-2-6A) of equine infectious anemia virus (EIAV) was modified by substitution of a 3.3-kbp fragment amplified by PCR techniques from a pathogenic variant (EIAVPV) of the cell culture-adapted strain of EIAV (EIAVPR). This substitution consisted of coding sequences for 77 amino acids at the carboxyl terminus of the integrase, the S1 (encoding the second exon of tat), S2, and S3 (encoding the second exon of rev) open reading frames, the complete env gene (including the first exon ofrev), and the 3′ long terminal repeat (LTR). Modified 19-2-6A molecular clones were designated EIAVPV3.3, and infection of a single pony (678) with viruses derived from a mixture of five of these molecular clones induced clinical signs of acute equine infectious anemia (EIA) at 23 days postinfection (dpi). As a consequence of this initial study, a single molecular clone, EIAVPV3.3#3 (redesignated EIAVUK), was selected for further study and inoculated into two ponies (613 and 614) and two horses (700 and 764). Pony 614 and the two horses developed febrile responses by 12 dpi, which was accompanied by a 48 to 64% reduction in platelet number, whereas pony 613 did not develop fever (40.6°C) until 76 dpi. EIAV could be isolated from the plasma of these animals by 5 to 7 dpi, and all became seropositive for antibodies to this virus by 21 dpi. Analysis of the complete nucleotide sequence demonstrated that the 3.3-kbp 3′ fragment of EIAVUKdiffered from the consensus sequence of EIAVPV by just a single amino acid residue in the second exon of the rev gene. Complete homology with the EIAVPV consensus sequence was observed in the hypervariable region of the LTR. However, EIAVUK was found to contain an unusual 68-bp nucleotide insertion/duplication in a normally conserved region of the LTR sequence. These results demonstrate that substitution of a 3.3-kbp fragment from the EIAVPV strain into the infectious nonpathogenic molecular clone 19-2-6A leads to the production of progeny virus particles with the ability to induce clinical signs of EIA. Therefore, EIAVUK, which is the first pathogenic, cell culture-adapted molecular clone of EIAV to be described, should be of value in identifying viral determinants of pathogenicity.

scholarly journals Canine ehrlichiosis: clinical, hematological, serological and molecular aspects

2008 ◽  
Vol 38 (3) ◽  
pp. 766-770 ◽  
Author(s):  
Andréa Cristina Higa Nakaghi ◽  
Rosangela Zacarias Machado ◽  
Mirela Tinucci Costa ◽  
Marcos Rogério André ◽  
Cristiane Divan Baldani
Keyword(s):  
Direct Detection ◽  
Clinical Signs ◽  
Chronic Phase ◽  
Antibody Test ◽  
Acute Stage ◽  
Detection Methods ◽  
Serological Tests ◽  
Humoral Immune ◽  
Canine Ehrlichiosis ◽  
Dot Elisa

The aim of the present study was to compare the direct detection methods of Ehrlichia canis (blood smears and nested PCR), serological tests (Dot-ELISA and Immunofluorescent Antibody Test - IFAT), and demonstrate the most suitable test for the diagnosis of different stages of infection. Blood samples and clinical data were collected from 30 dogs examined at the Veterinary Teaching Hospital, UNESP, Jaboticabal, SP, Brazil. The clinical signs most frequently observed were apathy, anorexia, pale mucous membrane, fever, lymphadenopathy, splenomegaly, hemorrhages and uveitis. Evaluating the humoral immune response, 63.3% of the sera were IFAT positive, while 70% were Dot-ELISA positive. By nestedPCR 53.3% of the samples were positive. Comparing these techniques it was concluded that serology and nPCR are the most suitable tests to confirm the diagnosis of canine ehrlichiosis, however it should be always treated as a complementary data to clinical and hematological evaluation. Serology has an important role in the subclinical and in the chronic phase, nPCR is recommended in the acute stage, and, especially, to identify the ehrlichia specie.

scholarly journals Envelope-specific T-helper and cytotoxic T-lymphocyte responses associated with protective immunity to equine infectious anemia virus

2007 ◽  
Vol 88 (4) ◽  
pp. 1324-1336 ◽  
Author(s):  
Tara L. Tagmyer ◽  
Jodi K. Craigo ◽  
Sheila J. Cook ◽  
Charles J. Issel ◽  
Ronald C. Montelaro
Keyword(s):  
Protective Immunity ◽  
Equine Infectious Anemia Virus ◽  
Cytotoxic T Lymphocyte ◽  
Asymptomatic Carrier ◽  
Cytotoxic T Cell ◽  
T Helper ◽  
Equine Infectious Anemia ◽  
Env Protein ◽  
Anemia Virus ◽  
Vaccine Immunity

Equine infectious anemia virus (EIAV) infection of horses provides a valuable model for examining the natural immunological control of lentivirus infection and disease and the mechanisms of protective and enhancing vaccine immunity. We have previously hypothesized that the EIAV envelope (Env) proteins gp90 and gp45 are major determinants of vaccine efficacy, and that the development of protective immunity by attenuated viral vaccines may be associated with the progressive redirection of immune responses from immunodominant, variable Env segments to immunorecessive, conserved Env sequences. Whilst the antibody-neutralization determinants of Env have been defined, there are to date no comprehensive analyses of the lymphoproliferative (T-helper, Th) and cytotoxic T-cell (CTL) epitopes of the EIAV Env proteins. Thus, in the current study, synthetic-peptide methodologies were used to define regions of EIAV Env associated with protective vaccine immunity in a panel of 12 horses inoculated with the attenuated EIAVD9 vaccine and two asymptomatic carrier horses infected experimentally with the virulent EIAVPV strain expressing the same Env protein as the vaccine strain. The results of these studies identified 17 broadly reactive Th peptides and six broadly reactive CTL peptides in the Env proteins of EIAV that were associated with protective immunity. Thus, these data provide for the first time a comprehensive mapping of EIAV Env-specific cellular regions that can be used to examine the development of protective immunity and to evaluate potential cellular immune determinants of protective immunity.

scholarly journals Tissue Sites of Persistent Infection and Active Replication of Equine Infectious Anemia Virus during Acute Disease and Asymptomatic Infection in Experimentally Infected Equids

2000 ◽  
Vol 74 (7) ◽  
pp. 3112-3121 ◽  
Author(s):  
Sharon M. Harrold ◽  
Sheila J. Cook ◽  
R. Frank Cook ◽  
Keith E. Rushlow ◽  
Charles J. Issel ◽  
...  
Keyword(s):  
Viral Infection ◽  
Virus Replication ◽  
Equine Infectious Anemia Virus ◽  
Clinical Symptoms ◽  
Acute Disease ◽  
Viral Dna ◽  
Dna And Rna ◽  
Equine Infectious Anemia ◽  
Anemia Virus

ABSTRACT Equine infectious anemia virus (EIAV) infection of horses is characterized by recurring cycles of disease and viremia that typically progress to an inapparent infection in which clinical symptoms are absent as host immune responses maintain control of virus replication indefinitely. The dynamics of EIAV viremia and its association with disease cycles have been well characterized, but there has been to date no comprehensive quantitative analyses of the specific tissue sites of EIAV infection and replication in experimentally infected equids during acute disease episodes and during asymptomatic infections in long-term inapparent carriers. To characterize the in vivo site(s) of viral infection and replication, we developed a quantitative competitive PCR assay capable of detecting 10 copies of viral DNA and a quantitative competitive reverse transcription-PCR assay with a sensitivity of about 30 copies of viral singly spliced mRNA. Animals were experimentally infected with one of two reference viruses: the animal-passaged field isolate designated EIAVWyo and the virulent cell-adapted strain designated EIAVPV. Tissues and blood cells were isolated during the initial acute disease or from asymptomatic animals and analyzed for viral DNA and RNA levels by the respective quantitative assays. The results of these experiments demonstrated that the appearance of clinical symptoms in experimentally infected equids coincided with rapid widespread seeding of viral infection and replication in a variety of tissues. During acute disease, the predominant cellular site of viral infection and replication was the spleen, which typically accounted for over 90% of the cellular viral burden. In asymptomatic animals, viral DNA and RNA persisted in virtually all tissues tested, but at extremely low levels, a finding indicative of tight but incomplete immune control of EIAV replication. During all disease states, peripheral blood mononuclear cells (PBMC) were found to harbor less than 1% of the cellular viral burden. These quantitative studies demonstrate that tissues, rather than PBMC, constitute the predominant sites of virus replication during acute disease in infected equids and serve as resilient reservoirs of virus infection, even in the presence of highly effective immune responses that maintain a stringent control of virus replication in long-term inapparent carriers. Thus, these observations with EIAV, a predominantly macrophage-tropic lentivirus, highlight the role of tissues in sequestering lentiviral infections from host immune surveillance.

Highly chlorinatedEscherichia colicannot be stained by propidium iodide

2007 ◽  
Vol 53 (5) ◽  
pp. 664-670 ◽  
Author(s):  
M.-H. Phe ◽  
M. Dossot ◽  
H. Guilloteau ◽  
J.-C. Block
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Propidium Iodide ◽  
False Negative ◽  
Sodium Thiosulphate ◽  
Permeable Membrane ◽  
E Coli ◽  
Dna And Rna ◽  
Chlorine Bleach ◽  
Effect Of Chlorine

Several studies have shown that the staining by fluorochromes (DAPI, SYBR Green II, and TOTO-1) of bacteria is altered by chlorination. To evaluate the effect of chlorine (bleach solution) on propidium iodide (PI) staining, we studied Escherichia coli in suspension and biomolecules in solution (DNA, RNA, BSA, palmitic acid, and dextran) first subjected to chlorine and then neutralized by sodium thiosulphate. The suspensions and solutions were subsequently stained with PI. The fluorescence intensity of the PI-stained DNA and RNA in solution dramatically decreased with an increase in the chlorine concentration applied. These results explain the fact that for chlorine concentrations higher than 3 μmol/L Cl2, the E. coli cells were too damaged to be properly stained by PI. In the case of highly chlorinated bacteria, it was impossible to distinguish healthy cells (with a PI-impermeable membrane and undamaged nucleic acids), which were nonfluorescent after PI staining, from cells severely injured by chlorine (with a PI-permeable membrane and damaged nucleic acids) that were also nonfluorescent, as PI penetrated but did not stain chlorinated nucleic acids. Our results suggest that it would be prudent to be cautious in interpreting the results of PI staining, as PI false-negative cells (cells with compromised membranes but not stained by PI because of nucleic acid damage caused by chlorine) are obtained as a result of nucleic acid damage, leading to an underestimation of truly dead bacteria.

scholarly journals Identification and genetic characterization of equine infectious anemia virus in Western Balkans

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Diana Lupulovic ◽  
Sara Savić ◽  
Delphine Gaudaire ◽  
Nicolas Berthet ◽  
Živoslav Grgić ◽  
...  
Keyword(s):  
Equine Infectious Anemia Virus ◽  
Clinical Signs ◽  
Full Genome Sequence ◽  
Enzyme Linked Immunosorbent Assay ◽  
Full Genome ◽  
Western Balkans ◽  
Full Genome Sequencing ◽  
Horse Population ◽  
Equine Infectious Anemia ◽  
Anemia Virus

Abstract Background Equine infectious anemia (EIA) is a viral disease, caused by the Equine Infectious Anemia virus (EIAV) belonging to the Retroviridae family, genus Lentivirus. Horses (or equids) infected with EIAV are lifelong carriers and they remain contagious for other horses even in the absence of clinical signs. So far, EIAV infection has been reported among horses in North and South America, France, Germany, Italy, Hungary and Romania, with no publication regarding the presence of EIAV in horses in Serbia. To determine the circulation of EIAV among, approximately, the 5000 horses of the Vojvodina region, northern part of Serbia, 316 serum undergone serological testing for EIA. Then, identification and full genome sequencing using next generation sequencing was performed from one EIA positive horse. Results the 316 sera were tested with 3 different commercial agar gel immunodiffusion (AGID) tests and two different commercial enzyme-linked immunosorbent assay (ELISA). With the three AGID kits, 311 (98.4%) among the 316 tested sera were negative and only five (1.6%) sera were positive for EIA. Some discrepancies were seen for the two ELISA kits tested since one exhibited the same results as AGID test and the second gave 295 sera with negative results, five with a positive result and 16 with doubtful outcome. Phylogenetic analysis performed using the full genome sequence showed that EIAV characterized from a horse in Serbia is different from those identify so fare around the world and form a distinct and separate group together with another EIAV strain. Conclusions This study demonstrate for the first time that EIAV is circulating at a low level in the horse population from the Northern part of Serbia. Interestingly, phylogenetic data indicates that this EIAV from the western Balkan region of Europe belongs to a new cluster.

scholarly journals Rapid Detection of Pathogens in Wound Exudate via Nucleic Acid Lateral Flow Immunoassay

Biosensors ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 74
Author(s):  
Anna Brunauer ◽  
René D. Verboket ◽  
Daniel M. Kainz ◽  
Felix von Stetten ◽  
Susanna M. Früh
Keyword(s):  
Nucleic Acid ◽  
Rapid Detection ◽  
Direct Detection ◽  
Point Of Care ◽  
False Negative ◽  
Opportunistic Pathogen ◽  
Lateral Flow ◽  
Lateral Flow Immunoassay ◽  
Wound Fluid ◽  
Wound Exudate

The rapid detection of pathogens in infected wounds can significantly improve the clinical outcome. Wound exudate, which can be collected in a non-invasive way, offers an attractive sample material for the detection of pathogens at the point-of-care (POC). Here, we report the development of a nucleic acid lateral flow immunoassay for direct detection of isothermally amplified DNA combined with fast sample preparation. The streamlined protocol was evaluated using human wound exudate spiked with the opportunistic pathogen Pseudomonas aeruginosa that cause severe health issues upon wound colonization. A detection limit of 2.1 × 105 CFU per mL of wound fluid was achieved, and no cross-reaction with other pathogens was observed. Furthermore, we integrated an internal amplification control that excludes false negative results and, in combination with the flow control, ensures the validity of the test result. The paper-based approach with only three simple hands-on steps has a turn-around time of less than 30 min and covers the complete analytical process chain from sample to answer. This newly developed workflow for wound fluid diagnostics has tremendous potential for reliable pathogen POC testing and subsequent target-oriented therapy.

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