Two main animal pathogenic subspecies of Mycobacterium avium are M. avium avium (Maa) and M. avium paratuberculosis (Map). Their pathogenicity is host-specifi c, Maa causing avian tuberculosis in poultry whereas Map commonly cross-infects to ruminant.Veterinary diagnosis of M. avium infections is microscopic examination of acid-fast bacilli or culture in Löwenstein-Jensen medium,which are time-consuming and low sensitivity. This present study aimed to apply real-time PCR coupled with High-Resolution Melting (HRM) analysis for differential detection of Maa in Thai domestic ducks. Specifi c primer targeting host-expression dependent (hed) region
was designed, PCR product of Maa were amplifi ed from duck’s tissue lesions whereas Map were amplifi ed from cow and deer. HRM real-time PCR was performed and analyzed. Different HRM patterns were showed and melting temperature were analyzed at 83.26 ± 0.12°C for Maa and 84.04 ± 0.09°C for Map. This technique can detect as few as 102 DNA copies and present high specifi city by negative amplifi cation of other pathogenic bacterial species. This technique is sensitive, specifi c, rapid and does not require fl uorescent probes or post-PCR electrophoresis. Our technique is a possible new tool for the detection of Maa and Map infection in tissue specimens.
DIAGNOSTIC ADVANCES IN HAEMOPROTOZOAN INFECTIONS OF LIVESTOCKrinkusarawade
The document discusses diagnostic advances for haemoprotozoan infections in livestock. It covers conventional parasitological diagnosis using blood smears as well as molecular diagnosis techniques like PCR, LAMP, and probes. Immunological diagnosis methods such as ELISA, IFAT, CATT, ICT are also summarized. Molecular tools allow specific and sensitive detection of parasites while serological assays are suitable for chronic infections when parasitemia is low. Advanced diagnostics combined with effective treatment can help control haemoprotozoan infections and drug resistance.
Prevalence Study of Infectious Bovine Keratoconjunctivitisin Dairy cattle und...iosrjce
IOSR Journal of Agriculture and Veterinary Science (IOSR-JAVS) is a double blind peer reviewed International Journal edited by the International Organization of Scientific Research (IOSR). The journal provides a common forum where all aspects of Agricultural and Veterinary Sciences are presented. The journal invites original papers, review articles, technical reports and short communications containing new insight into any aspect Agricultural and Veterinary Sciences that are not published or not being considered for publication elsewhere.
Advances in diagnostic technology allow for more sensitive, specific, rapid and cost-effective diagnosis of diseases. New methods like PCR, real-time PCR, in situ hybridization, biosensors, infrared thermography, and ELISA have improved on classical diagnostic approaches by being able to detect minute amounts of pathogens, identify pathogens rapidly, and differentiate between field strains and vaccine strains. These advanced diagnostic techniques are important for disease control, treatment, and surveillance.
Novel research aimed at finding a cure for AIDS requires animal models responding to human antiretroviral drugs. However, there have been few antiretrovirals cross-active against the simian viruses. In this study, we expanded the arsenal of drugs active against the simian retrovirus SIVmac251 and showed that this virus is inhibited by the protease inhibitor, darunavir, and the CCR5 blocker, maraviroc. Administration of these two drugs in combination with the reverse transcriptase inhibitors, tenofovir and emtricitabine, and the integrase inhibitor, raltegravir, resulted in prolonged plasma viral loads below assay detection limits, and, surprisingly, restricted the viral reservoir, a marker of which is viral DNA. We then decided to employ this multidrug regimen (termed “highly intensified ART”) in order to increase the potency of a previous strategy based on the gold drug auranofin, which recently proved able to restrict the viral reservoir in vivo. A short course of highly intensified ART following the previous treatment resulted, upon therapy suspension, in a remarkably spontaneous control of the infection, that may pave the way to a persistent suppression of viremia in the absence of ART. These results corroborate the robustness of the macaque AIDS model as a vanguard for potentially future treatments for HIV in humans.
This document discusses the development of two diagnostic tools (ELISA and lateral flow device) for the detection of antibodies against ovine and bovine theileriosis. It provides background information on the genus Theileria, which are tick-transmitted protozoan parasites that infect ruminants. It reviews the taxonomy, life cycle, clinical signs, and pathogenic species of Theileria that infect small ruminants. Existing methods for diagnosis of ovine and bovine theileriosis are described. The objectives of this study are to develop a recombinant protein ELISA for detection of T. uilenbergi infection and a lateral flow device for rapid detection of T. annulata infection under field conditions.
1) The study evaluated the performance of the OptiMal malaria rapid diagnostic test under different storage conditions of 25°C, 30°C, and 39°C for 24, 48, and 72 hours.
2) The test detected all 111 positive blood samples except for 2 low parasitemia Plasmodium malariae samples.
3) The study suggests that the OptiMal test can be used for malaria diagnosis in Brazilian regions, though further research is needed to evaluate its performance under different environmental conditions like humidity.
Presentation 6: Vibrio parahaemolyticus: genome plasticity, mobile genetic el...ExternalEvents
http://www.fao.org/documents/card/en/c/28b6bd62-5433-4fad-b5a1-8ac61eb671b1/
International Technical Seminar/Workshops on Acute hepatopancreatic necrosis disease (AHPND)
DIAGNOSTIC ADVANCES IN HAEMOPROTOZOAN INFECTIONS OF LIVESTOCKrinkusarawade
The document discusses diagnostic advances for haemoprotozoan infections in livestock. It covers conventional parasitological diagnosis using blood smears as well as molecular diagnosis techniques like PCR, LAMP, and probes. Immunological diagnosis methods such as ELISA, IFAT, CATT, ICT are also summarized. Molecular tools allow specific and sensitive detection of parasites while serological assays are suitable for chronic infections when parasitemia is low. Advanced diagnostics combined with effective treatment can help control haemoprotozoan infections and drug resistance.
Prevalence Study of Infectious Bovine Keratoconjunctivitisin Dairy cattle und...iosrjce
IOSR Journal of Agriculture and Veterinary Science (IOSR-JAVS) is a double blind peer reviewed International Journal edited by the International Organization of Scientific Research (IOSR). The journal provides a common forum where all aspects of Agricultural and Veterinary Sciences are presented. The journal invites original papers, review articles, technical reports and short communications containing new insight into any aspect Agricultural and Veterinary Sciences that are not published or not being considered for publication elsewhere.
Advances in diagnostic technology allow for more sensitive, specific, rapid and cost-effective diagnosis of diseases. New methods like PCR, real-time PCR, in situ hybridization, biosensors, infrared thermography, and ELISA have improved on classical diagnostic approaches by being able to detect minute amounts of pathogens, identify pathogens rapidly, and differentiate between field strains and vaccine strains. These advanced diagnostic techniques are important for disease control, treatment, and surveillance.
Novel research aimed at finding a cure for AIDS requires animal models responding to human antiretroviral drugs. However, there have been few antiretrovirals cross-active against the simian viruses. In this study, we expanded the arsenal of drugs active against the simian retrovirus SIVmac251 and showed that this virus is inhibited by the protease inhibitor, darunavir, and the CCR5 blocker, maraviroc. Administration of these two drugs in combination with the reverse transcriptase inhibitors, tenofovir and emtricitabine, and the integrase inhibitor, raltegravir, resulted in prolonged plasma viral loads below assay detection limits, and, surprisingly, restricted the viral reservoir, a marker of which is viral DNA. We then decided to employ this multidrug regimen (termed “highly intensified ART”) in order to increase the potency of a previous strategy based on the gold drug auranofin, which recently proved able to restrict the viral reservoir in vivo. A short course of highly intensified ART following the previous treatment resulted, upon therapy suspension, in a remarkably spontaneous control of the infection, that may pave the way to a persistent suppression of viremia in the absence of ART. These results corroborate the robustness of the macaque AIDS model as a vanguard for potentially future treatments for HIV in humans.
This document discusses the development of two diagnostic tools (ELISA and lateral flow device) for the detection of antibodies against ovine and bovine theileriosis. It provides background information on the genus Theileria, which are tick-transmitted protozoan parasites that infect ruminants. It reviews the taxonomy, life cycle, clinical signs, and pathogenic species of Theileria that infect small ruminants. Existing methods for diagnosis of ovine and bovine theileriosis are described. The objectives of this study are to develop a recombinant protein ELISA for detection of T. uilenbergi infection and a lateral flow device for rapid detection of T. annulata infection under field conditions.
1) The study evaluated the performance of the OptiMal malaria rapid diagnostic test under different storage conditions of 25°C, 30°C, and 39°C for 24, 48, and 72 hours.
2) The test detected all 111 positive blood samples except for 2 low parasitemia Plasmodium malariae samples.
3) The study suggests that the OptiMal test can be used for malaria diagnosis in Brazilian regions, though further research is needed to evaluate its performance under different environmental conditions like humidity.
Presentation 6: Vibrio parahaemolyticus: genome plasticity, mobile genetic el...ExternalEvents
http://www.fao.org/documents/card/en/c/28b6bd62-5433-4fad-b5a1-8ac61eb671b1/
International Technical Seminar/Workshops on Acute hepatopancreatic necrosis disease (AHPND)
The document summarizes preliminary research toward developing a method for stably transfecting the malaria parasite Plasmodium vivax. The researchers tested various transfection conditions and found the highest parasite survival rates using the Amaxa Nucleofector program U-033 and pre-loading erythrocytes with DNA. However, no fluorescent parasites were observed, likely due to the low efficiency of Plasmodium transfection. Future work aims to increase parasite numbers for transfection and further optimize methods.
1. The authors developed a quantitative real-time PCR (Q-PCR) method for detecting Rhodococcus equi, an important horse and emerging human pathogen.
2. The method uses two Q-PCR assays, one targeting the chromosomal choE gene to quantify total R. equi, and the other targeting the virulence plasmid gene vapA to detect the "horse-pathogenic" genotype.
3. Testing on 178 R. equi isolates and 77 non-target bacteria showed the assays were 100% sensitive and specific. The method can accurately quantify R. equi down to 1,000 CFU/ml in bronchoalveolar lavage fluid.
— Herpesviruses that infect fishes belong to the Herpesvirales order and Alloherpesvirus family. In these species, the different types of herpesvirus can cause tumors, adenocarcinoma and skin lesions. This study aims detect to presence of herpesvirus in fishes from commercial, recreation or experimental creations of the States of São Paulo and Minas Gerais, Brazil. Organ fragments and lesions of 53 fish species coming of mortality cases were forwarded at Biological Institute for examination by transmission electron microscopy by research of etiological agent. By transmission electron microscopy through negative staining technique, were observed herpes virus-like particles in 46 fishes and through embedding resin technique, in ultrathin sections were visualized herpes virus immature particles, measuring 90-110nm in diameter, located in the nuclei and complete particles measuring 160nm. In the histopathology technique, lesions associated with the virus as corpuscles inclusion, papillomas, and dermal lesions and in the gills were observed in 27 fishes. The evaluated techniques of TEM and the histopathology were effective for the rapid detection of herpesvirus in the examined samples.
Investigation on the Efficacy of Salmonella Bivalent VaccineIOSR Journals
The document describes a study that investigated the efficacy of a Salmonella bivalent vaccine containing Salmonella gallinarum and Salmonella pullorum. Shaver brown chickens were vaccinated and monitored over time. PHA antibody titers were measured in the vaccinated chickens at various time points post-vaccination and were found to increase after primary vaccination, booster dose, and pre-challenge. Chickens that received the bivalent vaccination withstood challenge with virulent S. gallinarum and S. pullorum, demonstrating the vaccine conferred protection. The results indicate the experimental Salmonella bivalent vaccine was immunogenic and provided effective protection against challenge infection in chickens.
Rapid identification of dermatophyte species by 28S rDNA Polymerase Chain Rea...iosrjce
IOSR Journal of Dental and Medical Sciences is one of the speciality Journal in Dental Science and Medical Science published by International Organization of Scientific Research (IOSR). The Journal publishes papers of the highest scientific merit and widest possible scope work in all areas related to medical and dental science. The Journal welcome review articles, leading medical and clinical research articles, technical notes, case reports and others.
This document describes the isolation and characterization of a new giant virus called Cedratvirus. Key points:
- Cedratvirus was isolated from an environmental sample in Algeria using Acanthamoeba castellanii.
- It has an ovoid shape with a cork structure at each end, resembling Pithovirus sibericum but with a unique double cork feature.
- The 589kb genome is most closely related to the pithovirus genomes, sharing over 100 genes, but with only 21% of genes involved in best reciprocal hits, indicating genetic distance from known pithoviruses.
This study established a modified immunohistochemistry method using murine antiserum as the primary antibody to detect mouse hepatitis virus (MHV) and Mycoplasma pulmonis antigens in formalin-fixed tissue sections. Using this method, MHV antigen was detected in the liver, stomach, caecum, colon and spleen of infected mice. Mycoplasma pulmonis antigen was demonstrated on the luminal surface of bronchioles in infected rats. This technique provides a useful method for diagnosing MHV and M. pulmonis infections when commercial antibodies are unavailable or serological diagnosis is not possible due to immunosuppression.
The document discusses Sundiresan's dissertation submitted to Lala Lajpat Rai University of Veterinary and Animal Sciences in partial fulfillment of the requirements for a Master of Veterinary Sciences degree. The dissertation involves VP2 based genotyping of field isolates of canine parvovirus through techniques like PCR amplification, sequencing, and restriction fragment length polymorphism analysis. Certificates from the major advisor and heads of relevant departments confirm that the dissertation is Sundiresan's original work and meets the requirements for the degree.
This study analyzed diagnostic records from the Taiwan National Laboratory Animal Center from 2004 to 2007 to assess the health status of rodent colonies in Taiwan. The key findings were:
1) Demand for pathogen diagnostic services increased steadily each year over the study period, indicating growing awareness of animal health issues.
2) Many mouse colonies tested positive for pathogens such as mouse parvovirus, mouse hepatitis virus, Theiler murine encephalomyelitis virus, and Mycoplasma pulmonis.
3) Nearly 40% of rat colonies tested positive for Mycoplasma pulmonis and rat parvovirus, and some also contained viruses like Kilham rat virus or intestinal parasites.
4
Molecular biomarkers can be used for several purposes in infectious disease research and clinical practice. These include detecting pathogens, measuring antibody responses, identifying markers of virulence, resistance, and disease severity, and understanding human immune responses and genetic susceptibility. Challenges include lack of sensitivity, mobile genetic elements, and changes in RNA sequences. Whole genome sequencing allows investigation of microbial phylogeny, evolution, and virulence factors.
Characteristics of salmonella spp. isolated from wild birds confiscated in il...racheltrans
1) Salmonella was isolated from 3 of 109 wild birds confiscated from illegal wildlife trade in Rio de Janeiro, Brazil, including one strain of Salmonella Typhimurium and two strains of Salmonella Panama.
2) All Salmonella isolates showed resistance to multiple antimicrobial drugs. PFGE analysis found 100% similarity between the Salmonella Typhimurium strain isolated from a bird and strains from a human outbreak in southern Brazil, indicating potential spread between wildlife and humans.
3) The two Salmonella Panama strains isolated from birds in the same catch showed identical genetic fingerprints, suggesting a common source of infection. However, these strains did not match any isolates in reference databases.
This document describes the development of a rapid field-applicable recombinase polymerase amplification (RPA) assay for the detection of Mycoplasma capricolum subsp. capripneumoniae, the bacterium that causes contagious caprine pleuropneumonia (CCPP). The RPA assay was designed to target a specific sequence in the genome of M. capricolum subsp. capripneumoniae and demonstrated high sensitivity and specificity. Testing on clinical samples showed the RPA assay could detect M. capricolum subsp. capripneumoniae directly from goat pleural fluid and lung tissues within 15-20 minutes without prior DNA extraction. The entire CCPP diagnosis using
Presentation 2.5 Ecology, virulence factors and global spread of pathogenic V...ExternalEvents
http://www.fao.org/documents/card/en/c/28b6bd62-5433-4fad-b5a1-8ac61eb671b1/
FAO Second International Technical Seminar/Workshop on Acute hepatopancreatic necrosis disease (AHPND) There is a way forward! FAO Technical Cooperation Programme: TCP/INT/3501 and TCP/INT/3502.
Bovine tuberculosis: Occupational hazard in Abattoir workersiosrjce
IOSR Journal of Dental and Medical Sciences is one of the speciality Journal in Dental Science and Medical Science published by International Organization of Scientific Research (IOSR). The Journal publishes papers of the highest scientific merit and widest possible scope work in all areas related to medical and dental science. The Journal welcome review articles, leading medical and clinical research articles, technical notes, case reports and others.
For over 10 decades, agents of infectious diseases have been identified through their phenotype directly in specimen and after a growth in culture.
Today, we are in a molecular era, there is an opportunity to detect organisms more rapidly and accurately based on their genetic signatures.
Biomedical science research discovery offers a growing numbers of a nucleic acid amplification tests (NAATS) among which is polymerase chain reaction (PCR) for detection and identification of bacterial, parasitic, fungi and viral pathogens.
These assays improve patient care, reduce antibiotic usage, enhance test utilization and increase laboratory and hospital efficiency.
In this seminar, we will explore the clinical usefulness and potential of both conventional and real-time PCR assays in Clinical Microbiology.
This study aimed to investigate the bactericidal potential of Mycobacterium tuberculosis targets under various in vivo simulated in vitro conditions and in vivo in mice. Using antisense RNA to inhibit five target genes, the study evaluated target cidality under six physiological conditions in vitro and in vivo. It identified aroK, which encodes shikimate kinase, as an in vivo bactericidal target based on correlations between in vitro and in vivo cidality data. The study suggests that the low pH in vitro model best predicts in vivo cidality and identifies targets with potential for anti-tuberculosis drug development.
Marios Stylianou_Paper II_ Novel High-Throughput Screening Method for Identif...Marios Stylianou
This document describes the development of a new high-throughput screening method to identify inhibitors of the yeast-to-hypha morphological transition in the fungal pathogen Candida albicans. The method uses automated microscopy and image analysis to quantitatively measure morphological parameters that distinguish between yeast and hyphal forms. Parameters like length/width ratio and mean object shape are calculated from images of fluorescently stained cells. Cell viability is also measured to identify inhibitors that block morphological transition without affecting growth. The method is validated using known inhibitors like farnesol and shown to be suitable for high-throughput screening through calculation of the Z-factor metric. This screening method could help identify new antifungal drug candidates that target virulence traits rather
The document provides an overview of tuberculosis (TB) including epidemiology, diagnosis, and laboratory testing. Some key points:
- TB infects millions worldwide each year and is a leading cause of death. Rates are highest in developing countries.
- Diagnosis involves sputum smear microscopy, culture, and molecular testing like PCR. Smear microscopy has low sensitivity but high specificity. Culture is more sensitive but slower.
- Rapid culture methods like BACTEC and MGIT can detect TB in 2-8 days compared to 6-8 weeks for traditional culture.
- Molecular tests like PCR that detect TB DNA sequences like IS6110 can identify TB in smear-negative cases and distinguish TB from
Gene transfer in the liver using recombinant adeno-associated virusJonathan G. Godwin
This document describes methods for delivering gene transfer vectors to the liver using recombinant adeno-associated virus (rAAV). It discusses that intravenous injection of rAAV serotypes results in efficient transduction of the liver. The document provides protocols for preparing rAAV vector samples and delivering the vectors to mouse liver through lateral tail vein injection, retro-orbital sinus injection, or portal vein injection. It notes that tail vein injection is the most common method but retro-orbital sinus injection can be used as an alternative for young or neonatal mice. Precise vector dosing and injection technique are emphasized for achieving effective liver-directed gene transfer.
Paratuberculosis (PTB) remains one of the most obstacles limit animal breeding sector all over the world. The current study aimed to detect the etiology of PTB in tissues of clinically suspected small ruminants using histopathological and real-time polymerase chain reaction (RT-PCR) methods. Clinical examination showed 10 (26.4%) PTB suspected cases out of the total (38) examined animals. The suspected cases were euthanized, necropsied, gross lesions were recorded and tissue samples were collected for histopathological and molecular procedures. Grossly intestinal and mesenteric lymph nodes thickening, corrugations and edematous swellings were recorded. Semi-thin sections of the intestine and mesenteric lymph nodes stained with toluidine blue demonstrated MAP organism inside epithelium cells and macrophages. RT-PCR detected MAP IS900 gene in all suspected cases (100%), thus we recommend using RT-PCR as a rapid sensitive method in the diagnosis of PTB.
Key-words: Paratuberculosis, Mycobacterium, Semi thin sections, Toluidine blue, IS900 gene
Seroepidemiology for MERS coronavirus using microneutralisation and pseudopar...Ranawaka A.P.M Perera
We describe a novel spike pseudoparticle neutralisation
assay (ppNT) for seroepidemiological studies on
Middle East respiratory syndrome coronavirus (MERSCoV)
and apply this assay together with conventional
microneutralisation (MN) tests to investigate 1,343
human and 625 animal sera. The sera were collected
in Egypt as a region adjacent to areas where MERS has
been described, and in Hong Kong, China as a control
region. Sera from dromedary camels had a high prevalence
of antibody reactive to MERS-CoV by MERS NT
(93.6%) and MERS ppNT (98.2%) assay. The antibody
titres ranged up to 1,280 and higher in MN assays
and 10,240 and higher in ppNT assays. No other
investigated species had any antibody reactivity to
MERS-CoV. While seropositivity does not exclude the
possibility of infection with a closely related virus, our
data highlight the need to attempt detection of MERSCoV
or related coronaviruses in dromedary camels. The
data show excellent correlation between the conventional
MN assay and the novel ppNT assay. The newly
developed ppNT assay does not require Biosafety Level
3 containment and is thus a relatively high-throughput
assay, well suited for large-scale seroepidemiology
studies which are needed to better understand the
ecology and epidemiology of MERS-CoV.
The document summarizes preliminary research toward developing a method for stably transfecting the malaria parasite Plasmodium vivax. The researchers tested various transfection conditions and found the highest parasite survival rates using the Amaxa Nucleofector program U-033 and pre-loading erythrocytes with DNA. However, no fluorescent parasites were observed, likely due to the low efficiency of Plasmodium transfection. Future work aims to increase parasite numbers for transfection and further optimize methods.
1. The authors developed a quantitative real-time PCR (Q-PCR) method for detecting Rhodococcus equi, an important horse and emerging human pathogen.
2. The method uses two Q-PCR assays, one targeting the chromosomal choE gene to quantify total R. equi, and the other targeting the virulence plasmid gene vapA to detect the "horse-pathogenic" genotype.
3. Testing on 178 R. equi isolates and 77 non-target bacteria showed the assays were 100% sensitive and specific. The method can accurately quantify R. equi down to 1,000 CFU/ml in bronchoalveolar lavage fluid.
— Herpesviruses that infect fishes belong to the Herpesvirales order and Alloherpesvirus family. In these species, the different types of herpesvirus can cause tumors, adenocarcinoma and skin lesions. This study aims detect to presence of herpesvirus in fishes from commercial, recreation or experimental creations of the States of São Paulo and Minas Gerais, Brazil. Organ fragments and lesions of 53 fish species coming of mortality cases were forwarded at Biological Institute for examination by transmission electron microscopy by research of etiological agent. By transmission electron microscopy through negative staining technique, were observed herpes virus-like particles in 46 fishes and through embedding resin technique, in ultrathin sections were visualized herpes virus immature particles, measuring 90-110nm in diameter, located in the nuclei and complete particles measuring 160nm. In the histopathology technique, lesions associated with the virus as corpuscles inclusion, papillomas, and dermal lesions and in the gills were observed in 27 fishes. The evaluated techniques of TEM and the histopathology were effective for the rapid detection of herpesvirus in the examined samples.
Investigation on the Efficacy of Salmonella Bivalent VaccineIOSR Journals
The document describes a study that investigated the efficacy of a Salmonella bivalent vaccine containing Salmonella gallinarum and Salmonella pullorum. Shaver brown chickens were vaccinated and monitored over time. PHA antibody titers were measured in the vaccinated chickens at various time points post-vaccination and were found to increase after primary vaccination, booster dose, and pre-challenge. Chickens that received the bivalent vaccination withstood challenge with virulent S. gallinarum and S. pullorum, demonstrating the vaccine conferred protection. The results indicate the experimental Salmonella bivalent vaccine was immunogenic and provided effective protection against challenge infection in chickens.
Rapid identification of dermatophyte species by 28S rDNA Polymerase Chain Rea...iosrjce
IOSR Journal of Dental and Medical Sciences is one of the speciality Journal in Dental Science and Medical Science published by International Organization of Scientific Research (IOSR). The Journal publishes papers of the highest scientific merit and widest possible scope work in all areas related to medical and dental science. The Journal welcome review articles, leading medical and clinical research articles, technical notes, case reports and others.
This document describes the isolation and characterization of a new giant virus called Cedratvirus. Key points:
- Cedratvirus was isolated from an environmental sample in Algeria using Acanthamoeba castellanii.
- It has an ovoid shape with a cork structure at each end, resembling Pithovirus sibericum but with a unique double cork feature.
- The 589kb genome is most closely related to the pithovirus genomes, sharing over 100 genes, but with only 21% of genes involved in best reciprocal hits, indicating genetic distance from known pithoviruses.
This study established a modified immunohistochemistry method using murine antiserum as the primary antibody to detect mouse hepatitis virus (MHV) and Mycoplasma pulmonis antigens in formalin-fixed tissue sections. Using this method, MHV antigen was detected in the liver, stomach, caecum, colon and spleen of infected mice. Mycoplasma pulmonis antigen was demonstrated on the luminal surface of bronchioles in infected rats. This technique provides a useful method for diagnosing MHV and M. pulmonis infections when commercial antibodies are unavailable or serological diagnosis is not possible due to immunosuppression.
The document discusses Sundiresan's dissertation submitted to Lala Lajpat Rai University of Veterinary and Animal Sciences in partial fulfillment of the requirements for a Master of Veterinary Sciences degree. The dissertation involves VP2 based genotyping of field isolates of canine parvovirus through techniques like PCR amplification, sequencing, and restriction fragment length polymorphism analysis. Certificates from the major advisor and heads of relevant departments confirm that the dissertation is Sundiresan's original work and meets the requirements for the degree.
This study analyzed diagnostic records from the Taiwan National Laboratory Animal Center from 2004 to 2007 to assess the health status of rodent colonies in Taiwan. The key findings were:
1) Demand for pathogen diagnostic services increased steadily each year over the study period, indicating growing awareness of animal health issues.
2) Many mouse colonies tested positive for pathogens such as mouse parvovirus, mouse hepatitis virus, Theiler murine encephalomyelitis virus, and Mycoplasma pulmonis.
3) Nearly 40% of rat colonies tested positive for Mycoplasma pulmonis and rat parvovirus, and some also contained viruses like Kilham rat virus or intestinal parasites.
4
Molecular biomarkers can be used for several purposes in infectious disease research and clinical practice. These include detecting pathogens, measuring antibody responses, identifying markers of virulence, resistance, and disease severity, and understanding human immune responses and genetic susceptibility. Challenges include lack of sensitivity, mobile genetic elements, and changes in RNA sequences. Whole genome sequencing allows investigation of microbial phylogeny, evolution, and virulence factors.
Characteristics of salmonella spp. isolated from wild birds confiscated in il...racheltrans
1) Salmonella was isolated from 3 of 109 wild birds confiscated from illegal wildlife trade in Rio de Janeiro, Brazil, including one strain of Salmonella Typhimurium and two strains of Salmonella Panama.
2) All Salmonella isolates showed resistance to multiple antimicrobial drugs. PFGE analysis found 100% similarity between the Salmonella Typhimurium strain isolated from a bird and strains from a human outbreak in southern Brazil, indicating potential spread between wildlife and humans.
3) The two Salmonella Panama strains isolated from birds in the same catch showed identical genetic fingerprints, suggesting a common source of infection. However, these strains did not match any isolates in reference databases.
This document describes the development of a rapid field-applicable recombinase polymerase amplification (RPA) assay for the detection of Mycoplasma capricolum subsp. capripneumoniae, the bacterium that causes contagious caprine pleuropneumonia (CCPP). The RPA assay was designed to target a specific sequence in the genome of M. capricolum subsp. capripneumoniae and demonstrated high sensitivity and specificity. Testing on clinical samples showed the RPA assay could detect M. capricolum subsp. capripneumoniae directly from goat pleural fluid and lung tissues within 15-20 minutes without prior DNA extraction. The entire CCPP diagnosis using
Presentation 2.5 Ecology, virulence factors and global spread of pathogenic V...ExternalEvents
http://www.fao.org/documents/card/en/c/28b6bd62-5433-4fad-b5a1-8ac61eb671b1/
FAO Second International Technical Seminar/Workshop on Acute hepatopancreatic necrosis disease (AHPND) There is a way forward! FAO Technical Cooperation Programme: TCP/INT/3501 and TCP/INT/3502.
Bovine tuberculosis: Occupational hazard in Abattoir workersiosrjce
IOSR Journal of Dental and Medical Sciences is one of the speciality Journal in Dental Science and Medical Science published by International Organization of Scientific Research (IOSR). The Journal publishes papers of the highest scientific merit and widest possible scope work in all areas related to medical and dental science. The Journal welcome review articles, leading medical and clinical research articles, technical notes, case reports and others.
For over 10 decades, agents of infectious diseases have been identified through their phenotype directly in specimen and after a growth in culture.
Today, we are in a molecular era, there is an opportunity to detect organisms more rapidly and accurately based on their genetic signatures.
Biomedical science research discovery offers a growing numbers of a nucleic acid amplification tests (NAATS) among which is polymerase chain reaction (PCR) for detection and identification of bacterial, parasitic, fungi and viral pathogens.
These assays improve patient care, reduce antibiotic usage, enhance test utilization and increase laboratory and hospital efficiency.
In this seminar, we will explore the clinical usefulness and potential of both conventional and real-time PCR assays in Clinical Microbiology.
This study aimed to investigate the bactericidal potential of Mycobacterium tuberculosis targets under various in vivo simulated in vitro conditions and in vivo in mice. Using antisense RNA to inhibit five target genes, the study evaluated target cidality under six physiological conditions in vitro and in vivo. It identified aroK, which encodes shikimate kinase, as an in vivo bactericidal target based on correlations between in vitro and in vivo cidality data. The study suggests that the low pH in vitro model best predicts in vivo cidality and identifies targets with potential for anti-tuberculosis drug development.
Marios Stylianou_Paper II_ Novel High-Throughput Screening Method for Identif...Marios Stylianou
This document describes the development of a new high-throughput screening method to identify inhibitors of the yeast-to-hypha morphological transition in the fungal pathogen Candida albicans. The method uses automated microscopy and image analysis to quantitatively measure morphological parameters that distinguish between yeast and hyphal forms. Parameters like length/width ratio and mean object shape are calculated from images of fluorescently stained cells. Cell viability is also measured to identify inhibitors that block morphological transition without affecting growth. The method is validated using known inhibitors like farnesol and shown to be suitable for high-throughput screening through calculation of the Z-factor metric. This screening method could help identify new antifungal drug candidates that target virulence traits rather
The document provides an overview of tuberculosis (TB) including epidemiology, diagnosis, and laboratory testing. Some key points:
- TB infects millions worldwide each year and is a leading cause of death. Rates are highest in developing countries.
- Diagnosis involves sputum smear microscopy, culture, and molecular testing like PCR. Smear microscopy has low sensitivity but high specificity. Culture is more sensitive but slower.
- Rapid culture methods like BACTEC and MGIT can detect TB in 2-8 days compared to 6-8 weeks for traditional culture.
- Molecular tests like PCR that detect TB DNA sequences like IS6110 can identify TB in smear-negative cases and distinguish TB from
Gene transfer in the liver using recombinant adeno-associated virusJonathan G. Godwin
This document describes methods for delivering gene transfer vectors to the liver using recombinant adeno-associated virus (rAAV). It discusses that intravenous injection of rAAV serotypes results in efficient transduction of the liver. The document provides protocols for preparing rAAV vector samples and delivering the vectors to mouse liver through lateral tail vein injection, retro-orbital sinus injection, or portal vein injection. It notes that tail vein injection is the most common method but retro-orbital sinus injection can be used as an alternative for young or neonatal mice. Precise vector dosing and injection technique are emphasized for achieving effective liver-directed gene transfer.
Paratuberculosis (PTB) remains one of the most obstacles limit animal breeding sector all over the world. The current study aimed to detect the etiology of PTB in tissues of clinically suspected small ruminants using histopathological and real-time polymerase chain reaction (RT-PCR) methods. Clinical examination showed 10 (26.4%) PTB suspected cases out of the total (38) examined animals. The suspected cases were euthanized, necropsied, gross lesions were recorded and tissue samples were collected for histopathological and molecular procedures. Grossly intestinal and mesenteric lymph nodes thickening, corrugations and edematous swellings were recorded. Semi-thin sections of the intestine and mesenteric lymph nodes stained with toluidine blue demonstrated MAP organism inside epithelium cells and macrophages. RT-PCR detected MAP IS900 gene in all suspected cases (100%), thus we recommend using RT-PCR as a rapid sensitive method in the diagnosis of PTB.
Key-words: Paratuberculosis, Mycobacterium, Semi thin sections, Toluidine blue, IS900 gene
Seroepidemiology for MERS coronavirus using microneutralisation and pseudopar...Ranawaka A.P.M Perera
We describe a novel spike pseudoparticle neutralisation
assay (ppNT) for seroepidemiological studies on
Middle East respiratory syndrome coronavirus (MERSCoV)
and apply this assay together with conventional
microneutralisation (MN) tests to investigate 1,343
human and 625 animal sera. The sera were collected
in Egypt as a region adjacent to areas where MERS has
been described, and in Hong Kong, China as a control
region. Sera from dromedary camels had a high prevalence
of antibody reactive to MERS-CoV by MERS NT
(93.6%) and MERS ppNT (98.2%) assay. The antibody
titres ranged up to 1,280 and higher in MN assays
and 10,240 and higher in ppNT assays. No other
investigated species had any antibody reactivity to
MERS-CoV. While seropositivity does not exclude the
possibility of infection with a closely related virus, our
data highlight the need to attempt detection of MERSCoV
or related coronaviruses in dromedary camels. The
data show excellent correlation between the conventional
MN assay and the novel ppNT assay. The newly
developed ppNT assay does not require Biosafety Level
3 containment and is thus a relatively high-throughput
assay, well suited for large-scale seroepidemiology
studies which are needed to better understand the
ecology and epidemiology of MERS-CoV.
This document summarizes information about malaria vaccines. It discusses how malaria is caused by Plasmodium parasites and transmitted by mosquitoes. Four species can infect humans. Current vaccines target different stages of the parasite's life cycle, including pre-erythrocytic, blood, and sexual stages. Challenges to vaccine development include the parasite's ability to evade the immune system through antigenic variation. Several candidate vaccines are discussed that target different stages, but none have achieved high levels of efficacy and durability.
Prevalence of Moraxella ovis Infection in Goats under the Ladang Angkat Progr...iosrjce
IOSR Journal of Agriculture and Veterinary Science (IOSR-JAVS) is a double blind peer reviewed International Journal edited by the International Organization of Scientific Research (IOSR). The journal provides a common forum where all aspects of Agricultural and Veterinary Sciences are presented. The journal invites original papers, review articles, technical reports and short communications containing new insight into any aspect Agricultural and Veterinary Sciences that are not published or not being considered for publication elsewhere.
This document describes the development of a PCR-RFLP assay to identify Plasmodium species and variants of P. vivax infecting Anopheles mosquitoes. Specific primers were designed that target regions of the circumsporozoite gene to distinguish P. falciparum, P. malariae, and P. vivax variants VK210, VK247, and P. vivax-like. The assay was tested on artificially infected mosquitoes and showed good agreement with nested PCR. The PCR-RFLP method provides a sensitive way to detect Plasmodium species and variants, which can help understand malaria transmission dynamics.
Objective: To generate preliminary information about of enteroviruses and Enterovirus 71 (EV71) in patients with aseptic meningitis in Khartoum State, Sudan.
Method: Cerebrospinal fluid specimens were collected from 89 aseptic meningitis patients from different Khartoum Hospitals
(Mohammed Alamin Hamid Hospital, Soba Teaching Hospital, Omdurman Military Hospital, Alban Gadeed Teaching Hospital and Police Hospital) within February to May 2015. Among these 89 patients, 43 (48%) were males and 46 (52%) were females. The patient’s age ranged between 1 day and 30 years old. The collected specimens were assayed to detect enteroviruses and EV71 RNA using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) technique
This study evaluated four recombinant proteins representing the 19 kDa C-terminal region of the Plasmodium vivax merozoite surface protein-1 (MSP119) for detecting P. vivax antibodies in human serum samples. The sensitivity of an ELISA using the recombinant proteins to detect antibodies in 200 samples from individuals with P. vivax infection ranged from 90-93.5%. Specificity using control samples without P. vivax exposure was 98.3-100%. The study demonstrates the potential of an ELISA using recombinant MSP119 proteins for serological detection of P. vivax infection.
The document summarizes a study on the prevalence of canine parvovirus (CPV) in domestic dogs living around Serengeti National Park in Tanzania. Blood samples were collected from 77 asymptomatic domestic dogs and tested for CPV using PCR. The results found that 10.4% of samples were positive for CPV, with 6.5% positive for the CPV-2a strain and 3.9% for CPV-2b. This suggests that domestic dogs can act as reservoirs for CPV transmission to other dogs and wildlife in the area.
1) The document discusses the use of Loop-Mediated Isothermal Amplification (LAMP) to detect and amplify DNA from several agricultural viruses, including avian influenza virus, West Nile virus, fish iridovirus, white spot syndrome virus, Japanese yam mosaic virus, tomato yellow leaf curl virus, and tomato spotted wilt virus.
2) LAMP methods were found to be more rapid and sensitive than PCR for detecting avian influenza virus and West Nile virus. LAMP could also detect fish iridovirus, white spot syndrome virus, Japanese yam mosaic virus, tomato yellow leaf curl virus, and tomato spotted wilt virus from infected samples.
3) In
Molecular Study of Cutaneous Leishmaniasis Human Reservoirs and Infections in...CrimsonpublishersCJMI
Molecular Study of Cutaneous Leishmaniasis Human Reservoirs and Infections in Bastak by Houshang Jamali in Cohesive Journal of Microbiology & Infectious Disease
This study investigated the prevalence of Toxoplasma gondii infection in 197 pets and stray cats in 4 districts of Khyber Pakhtunkhwa, Pakistan using serological (ELISA) and molecular (PCR) techniques. The results showed that T. gondii infection was significantly higher in stray cats (74.6%) compared to pet cats (25.4%). Infection rates also varied significantly between districts and were highest in Kohat (95.5%). Older cats (>4 years) had significantly higher infection rates (91.66%) than younger cats. Chronic and reactivated chronic infections (58.37%) were more common than acute infections. This research suggests T. gondii is widely
Johne's disease (JD) or Paratuberculosis (PTB) has gained a great attention by many industrial countries for its severing economic losses and possibly zoonotic concerns. In the current study conventional clinical and direct microscopic examination compared to real time polymerase chain reaction (RT-PCR) were used to diagnose JD in clinically suspected small ruminants. Clinical examination revealed 130 (8.7%) suspected cases that showed history of emaciation and diarrhea out of the total examined (1500) animals. Direct microscopy of Ziehl-Neelsen (ZN) stained smears (130) revealed 62 (47.7%) acid fast bacteria resembled Mycobacterium avium subsp. paratuberculosis (MAP). RT-PCR insertion sequence gene (IS900) detected MAP in 25 (65.8%) out of 38 fecal samples harbored acid fast bacilli. We concluded and recommended that RT-PCR considers the most rapid confirmatory method for screening and diagnosis of the MAP in comparison to low specific conventional phenotypic methods, which still remained valuable techniques in the diagnosis of JD in developing countries.
Key-words: Johne's disease, Paratuberculosis, Acid fast bacteria, Ziehl-Neelsen stain, IS900 gene
This document discusses various methods for diagnosing malaria, including clinical diagnosis, microscopy, rapid diagnostic tests (RDTs), serological tests, polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), microarrays, and flow cytometry (FCM). Microscopy remains the gold standard but requires expertise and specialized equipment. RDTs provide rapid results using minimal equipment but cannot quantify parasites. Molecular methods like PCR and LAMP can detect low parasite levels but require specialized labs. Different methods have varying detection limits, time requirements, expertise needed, and costs. The optimal method depends on the situation and resources available.
Customizable pcr microplate array for differential identification of multiple...Tiensae Teshome
1. Customizable PCR-microplate arrays were developed that allow for the simultaneous identification of 10 foodborne pathogens and biothreat agents using pathogen-specific primers.
2. The arrays were tested using genomic DNA from 38 pathogen strains, and specifically identified all pathogens present.
3. In tests with food matrices, the arrays showed detection limits as low as 9 cfu/g for Salmonella Typhimurium in beef hot dogs and 78 cfu/ml in milk. Such microplate arrays could serve as tools for rapid identification of these pathogens during outbreak investigations or for confirmation purposes.
Smith TC, Male MJ, Harper AL, Kroeger J, Tinkler G, Moritz-Korolev E, Herwaldt L, Diekema D. High prevalence of MRSA found in Midwestern US Swine and Swine workers. PLoS ONE, 4(1):e4258, 2009.
This study found that rat tissues from farms in the Netherlands tested positive for the pla gene, which is a marker for Yersinia pestis. The pla gene sequences from rats were nearly identical to Y. pestis pla but further analysis identified adjacent sequences similar to bacterial replication genes. Attempts to culture or detect other Y. pestis markers from rat tissues were unsuccessful. The findings suggest there are unknown bacteria in rats that contain a pla homolog, which could produce false positive results in Y. pestis detection assays that only target the pla gene. Methods to confirm the presence of Y. pestis should include additional gene targets.
Comparison of primer sets for amplification of Major Piroplasm Surface Protei...Sudhakar Goud Karpurapu
This document compares the efficiency of two primer sets for detecting Theileria orientalis, the causative agent of oriental theileriosis, in cattle. Blood samples from 32 cattle showing clinical signs of the disease were examined microscopically and using two PCR primer sets that target the major piroplasm surface protein gene of T. orientalis. Microscopy found 22 cattle positive, while primer set 1 identified 23 positive and primer set 2 identified all 32 cattle as positive. The results suggest PCR using primer set 2 is more sensitive for detection of T. orientalis infection compared to microscopy or primer set 1.
RNA Extraction of Peste Des Petits Ruminants Virus (PPRV) from Clinical Sampl...ZULKIFAL HUSSAIN
This document describes a study that evaluated two RNA extraction methods, Tri-reagent and Acid guanidinium thiocyanate–phenol–chloroform (AGPC), for detecting Peste Des Petits Ruminants Virus (PPRV) in clinical samples. RNA was extracted from 10 tissue samples that tested positive for PPRV using Immuno-capture ELISA. Both extraction methods produced RNA of sufficient quality and purity for downstream applications. The study found that both Tri-reagent and AGPC are effective methods for extracting RNA from PPRV samples and can enable accurate diagnosis of the disease. Rapid detection of PPRV through nucleic acid-based methods like these helps control outbreaks by facilitating early
This study aimed to identify pinworm species infecting a colony of Syrian hamsters and determine if transmission could occur to immunodeficient mice. The pinworm was identified as Syphacia mesocriceti based on morphology and 28S rDNA sequencing, though dimensions were slightly larger than reported. Weekly transfers of infected hamster bedding to sentinel hamsters and mice showed no transmission to mice by tape tests or PCR over 5 weeks, suggesting S. mesocriceti is host-specific.
Similar to American Journal of Current & Applied Research in Microbiology (20)
A 5-year old boy, with an established diagnosis of a topic
dermatitis, previously treated by topical corticosteroids and emollient cream with a good improvement, developed widespread papules on his legs, hands and forearm that appeared 5 months ago.
Methods: Retrospectively, the file records of the patients who underwent sleeve gastrectomy were examined. Demographic features, Body Mass Index (BMI), the mouth opening, Mallampati score, thyromental distance, sternomental distance, neck circumference measurements and videolaryngoscopic examination results were recorded Results: In a total of 140 consecutive patients (58 male, 82 female) were included in the study. The mean age of the study participants was 35.40 ± 9.78 and the mean BMI of the patients was 44.33 ± 7.52 kg/m2
. The mean mouth opening of the patients was 4.82 ± 0.54 cm
and the mean neck circumference was 43.52 ± 4.66 cm. The mean thyromental distance was 8.02 ± 1.00 cm and the mean sternomental distance was16.58 ± 1.53 cm. Difficult intubation was determined in 8 (5.7%) patients. In logistic regression analysis, age (p : 0.446), gender (p : 0.371), BMI (p : 0.947), snoring (p : 0.567), sleep apnea (p : 0.218), mouth opening (p : 0.687), thyromental distance (p :0.557), sternomental (p : 0.596) and neck circumference (p : 0.838) were not the independent predictors of difficult intubation. However, Mallampati score (p : 0.001) and preoperative direct laryngoscopy findings (p : 0.037) performed in outpatient clinic were the significant
predictors of difficult intubation. Interestingly, all patients with grade 4 laryngoscopy findings had difficult intubation.
Introduction: Laparoscopic surgery has been performed in Mexico since 1989, but no reports about training tendencies exist. We conducted a national survey in 2015, and here we report the results concerning training characteristics during the surgical residence of the respondents. Materials and Methods: A prospective study was conducted through a survey questioning demographic data, laparoscopic training during pre and post surgical residency and other of areas of laparoscopic practice. The sample was calculated and survey piloted before
application. Special interest in this report was placed on type and quality of training received. Data are reported in percentages.
Heterotopic Ossification (HO) is defined as pathological bone formation at locations where bone normally does not exist. The
presence of HO has been found to be a rare complication after stroke in several studies, whereas there are only sporadic references relating HO to Cerebral Palsy (CP) and few for CP and stroke. No effective treatment for HO has yet been found, whereas the cellular and molecular mechanisms have not been completely understood. Therefore, increased awareness among physicians is required, as a challenge for early diagnosis and treatment. A case of a male patient with CP, who developed HO on the paretichip joint following an ischemic stroke is presented.
Objectives: To assess the practice of food hygiene and safety, and its associated factors among street food vendors in urban areas of Shashemane, West Arsi Zone, Oromia Ethiopia, 2019.
Methods: Cross-sectional study design was applied from December 28, 2019 to January 27, 2020. Data was collected from 120 food handlers, which were selected by purposive sampling techniques. Information was gathered from interview and field observation by conducting food safety survey and using questionnaires via face to face interview. The collected data was entered using Epi Data 3.1 and finally, it was analyzed using SPSS VERSION 20.
A Division I football player experienced acute posterior leg pain while playing. An ultrasound examination revealed an unusual injury - a complete rupture of the plantaris tendon mid-substance. This type of isolated plantaris tendon injury has rarely been reported. Ultrasound was useful for diagnosis and guided rehabilitation by monitoring healing over time. The athlete was able to return to full competition within 3 weeks through a progressive rehabilitation program focused on restoring range of motion and strength. This case suggests isolated plantaris tendon injuries may allow for faster return to play than other potential causes of posterior leg pain.
Type 1 Diabetes (T1D), is a severe disease, representing 5-10% of all reported cases of diabetes worldwide. Fulminant Type 1 Diabetes Mellitus (FT1D) is a subtype of type 1 diabetes mellitus that is largely characterized by the abrupt onset of Diabetic Ketoacidosis (DKA) and severe hyperglycemia without insulin defi ciency. Viral infections have been hypothesized to play a major role in the pathogenesis of Fulminant Type 1 Diabetes Mellitus (FT1D) through the complete and rapid destruction of pancreatic beta cells. Coxsackie viral infection has been detected in islets of 50% of the pancreatic tissue recovered from recent-onset Type 1 Diabetes (T1D) patients. In this report we have highlighted a case where the patient developed a Group B Coxsackie virus infection culminating in the development of Fulminant Type 1 Diabetes Mellitus (FT1D).
Methods: Cercariae are released by infected water snails. To determine the occurrence of cercariae-emitting snails in SchleswigHolstein, 155 public bathing places were visited and searched for fresh water snails. Family and genus of the collected snails were determined and the snails were examined for the shedding of cercariae, using a standard method and a newly developed method.
Femoral hernias, comprise 2% to 4% of all hernias in the inguinal region, and occur most commonly in women. Th ey present typically with a mass below the level of the inguinal ligament. The sac may contain preperitoneal fat, omentum, small bowel, or other structures and have a high rate of incarceration and strangulation due to the small size of the hernia neck orifice, requiring emergency surgery. We present the case of a 54-year-old female patient with intestinal occlusion due to incarcerated femoral hernia, repaired by laparoscopic approach, that gave the patient the opportunity to attend her daughter’s wedding the same day.
Small Supernumerary Marker Chromosome (sSMC) is a rare genetic condition marked by the presence of an extra chromosome to the 46 human chromosomes. This case report describes a 4 year old child with SSMC on the 46th chromosome. The child presented with delayed speech and language development, seizures and mild developmental delay. Speech and Language evaluation was carried out and management options are discussed.
A catheter is a thin tube made from medical grade materials that serve a broad range of functions, but mainly catheters are medical devices that can be inserted in the body to treat disease or perform surgical procedures. Catheters have been inserted into body cavities, ducts, or vessels to allow for drainage, administration of therapeutic fluids or gases, operational access for surgery. Catheters help perform tasks in various systems such as cardiovascular, urological, gastrointestinal, neurovascular, and ophthalmic systems. A dataset of 12 patients with varying “weights” and “heights” was recorded along with the lengths of their catheter tubes. This data set was found from two revered statistical textbooks on linear regression and the Department of Scientific Computing at Florida State University. This data set was not able to be linked to any particular clinical or experimental research studies, but the data set can be used to help catheter manufacturers and medical professionals better decide on what particular catheter lengths to use for patients knowing only their height & weight. These research insights could be helpful to healthcare professionals that have patients with incomplete or no healthcare records
to decide what catheter length to use. The main investigative inquiry that needed to be answered was how does patient weight & height influence catheter length together and separately? We conducted linear regression and other statistical analysis procedures in R program & Microsoft Excel and discovered that this data exhibited a quality called multi collinearity. With multi collinearity, all predictors (2 or more
independent variables) are not significant in an all encompassing linear aggression, but the predictors might be significant in their own individual linear regressions. Individual linear regression analyses were conducted for both patient height & weight to see how much they both contribute to varying catheter length. Patient weight was found to be more impatful than patient height in relationship to catheter length, even though height and weight are a classical example of multi collinearity predictors.
Bovine mastitis has a negative impact through economic losses in the dairy sector across the globe. A cross sectional study was carried out from September 2015 to July 2016 to determine the prevalence of bovine mastitis, associated risk factors and isolation of major causative bacteria in lactating dairy cows in selected districts of central highland of Ethiopia. A total of 304 lactating cows selected randomly from five districts were screened by California Mastitis Test (CMT) for subclinical mastitis. Based on CMT result and clinical examination, over all prevalence of mastitis at cow level was 70.62% (214/304).
Two hundred fourteen milk samples collected from CMT positive cows were cultured for isolation of major causative bacteria. From 214 milk samples,187 were culture positive and the most prevalent isolates were Staphylococcus aureus 42.25% (79/187) followed by Streptococcus agalactiae 14.43%
(27/187). Other bacterial isolates were included Coagulase Negative Staphylococcus species 12.83% (24/187), Streptococcus dysgalactiae 5.88% (11/187), Escherichia coli 13.38% (25/187) and Entrococcus feacalis 11.23% (21/187) were also isolated. Moreover, age, parity number, visible teat abnormalities,husbandry practice, barn fl oor status and milking hygiene were considered as risk factors for the occurrence of bovine mastitis and they were found significantly associated with the occurrence of mastitis (p < 0.05). The findings of this study warrants the need for strategic approach including dairy extension that focus on enhancing dairy farmers’ awareness and practice of hygienic milking, regular screening for subclinical mastitis, dry cow therapy and culling of chronically infected cows.
A 36-year-old female developed right upper quadrant pain and nausea after taking the herbal supplement kratom for two weeks to manage back pain. Laboratory tests showed elevated liver enzymes. A liver biopsy ruled out other causes and determined she had drug-induced liver injury from kratom use. Her symptoms and liver enzymes gradually returned to normal over six weeks after stopping kratom. The case report discusses kratom's potential for hepatotoxicity and advises clinicians to consider its effects on patient health.
The assessment, diagnosis and treatment of critically ill patients is extremely challenging. Patients often deteriorate whilst being
reviewed and their rapidly changing pathophysiology barrages healthcare professionals with new data. Furthermore, comprehensive assessments must be postponed until the patient has been stabilised. So, important data and interventions are often missed in the heat of the moment. In emergency situations, suboptimal management decisions may cause signifi cant morbidity and mortality. Fortunately, standardisation and careful design of documentation (i.e. proformas and checklists) can enhance patient safety. So, I have developed a series of checklist proformas to guide the assessment of critically ill patients. These proformas also promote the systematic recording and presentation of information to facilitate the retrieval of the precise data required for the management for critically ill patients. The proformas have been modifi ed extensively over the last twenty years based on my personal experience and extensive consultation with colleagues in several world-renowned centres of excellence. The proformas were originally developed for use in the intensive therapy unit
or high dependency unit. However, they have been adapted for use by outreach teams reviewing patients admitted outside of critical care areas. The use of these tools can direct eff orts to provide appropriate organ support and provides a framework for diagnostic reasoning.
This review article discusses microvascular and macrovascular disease in systemic hypertension. It summarizes that:
1) Cardiac imaging plays a crucial role in risk stratifying hypertensive patients and identifying management strategies by properly diagnosing microvascular and coronary artery disease.
2) The nitric oxide synthase (eNOS) G298 gene allele may be a marker for microvascular angina in hypertensive patients, as studies have found it to be more prevalent in hypertensive patients with chest pain and reversible myocardial defects but normal coronary arteries.
3) Both structural changes like capillary rarefaction and functional changes like endothelial dysfunction can cause microvascular dysfunction and angina in hypertensive individuals in the absence of
This study characterized dengue infections in Pakistan by analyzing hematological and serological markers in 154 suspected dengue cases and 146 control patients with other febrile illnesses. NS1 antigen was detected in 55% of dengue cases, IgM antibodies in 30%, and both in 15%. Control groups primarily had malaria (71%) and enteric fever (20%). Hematological markers (platelet count, hematocrit, WBC) measured before and after treatment showed significant differences for platelet count and hematocrit but not WBC count between the groups. Analysis of clinical symptoms and serological/hematological markers helps diagnose dengue, assess prognosis, and inform prevention efforts to reduce morbidity, mortality and spread of the disease.
Researchers from Utrecht recently published yet another paper on the use of Magnetic Resonance Imaging (MRI)demonstrating an additional failed attempt to understand the importance of qualitative versus quantitative imaging, and anatomic versus physiologic imaging. Th e implications of this failure here cannot be overstated.
Introduction: Stroke is an even more dramatic major public health problem in young people. Goal of the study: Contribute to the knowledge of strokes in young people. Methodology: This was a retrospective study carried out over a period of 02 years (January 2017 to December 2018) including the files of patients aged 18 to 49 years hospitalized for any suspected case of stroke in the Neurology department of the University Hospital
Center of the Sino-Central African Friendship (CHUSCA) of Bangui.
Background: This report describes a unique case of a patient that developed psychotic symptoms believed to be secondary
to a tentorial meningioma with associated hydrocephalus. These psychotic symptoms subsequently abated with placement of a
ventriculoperitoneal shunt. Case description: 60-year-old female was admitted to an inpatient psychiatric facility on a psychiatric involuntary commitment petition due to progressive paranoia, homicidal ideation and psychosis. The work up showed a calcified six cm tentorial meningioma with associated hydrocephalus. The patient initially rejected treatment but later became amenable to placement of Ventriculoperitoneal Shunt
(VPS).
Introduction: Adjuvant chemotherapy such as S-I is thought to prolong the life expectancy of patients with gastric cancer. The
number of older patients with gastric cancer has recently been increasing. Here we examined the prognosis of older patients with stage II or III gastric cancer.
Methods: The study cohort comprises 658 patients with stage II or III gastric cancer who underwent curative surgery from 1994 to 2014 in our institution. From 1994 to 2003 was considered the early phase, whereas from 2004 to 2014 was considered the late phase. The patients were classifi ed by age into under 65 years (Non-Elderly [NE]); 65-74 years (Early Elderly [EE]); and over 74 years (Late Elderly [LE] groups.
More from SciRes Literature LLC. | Open Access Journals (20)
Rasamanikya is a excellent preparation in the field of Rasashastra, it is used in various Kushtha Roga, Shwasa, Vicharchika, Bhagandara, Vatarakta, and Phiranga Roga. In this article Preparation& Comparative analytical profile for both Formulationon i.e Rasamanikya prepared by Kushmanda swarasa & Churnodhaka Shodita Haratala. The study aims to provide insights into the comparative efficacy and analytical aspects of these formulations for enhanced therapeutic outcomes.
These lecture slides, by Dr Sidra Arshad, offer a quick overview of the physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar lead (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
6. Describe the flow of current around the heart during the cardiac cycle
7. Discuss the placement and polarity of the leads of electrocardiograph
8. Describe the normal electrocardiograms recorded from the limb leads and explain the physiological basis of the different records that are obtained
9. Define mean electrical vector (axis) of the heart and give the normal range
10. Define the mean QRS vector
11. Describe the axes of leads (hexagonal reference system)
12. Comprehend the vectorial analysis of the normal ECG
13. Determine the mean electrical axis of the ventricular QRS and appreciate the mean axis deviation
14. Explain the concepts of current of injury, J point, and their significance
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. Chapter 3, Cardiology Explained, https://www.ncbi.nlm.nih.gov/books/NBK2214/
7. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
Local Advanced Lung Cancer: Artificial Intelligence, Synergetics, Complex Sys...Oleg Kshivets
Overall life span (LS) was 1671.7±1721.6 days and cumulative 5YS reached 62.4%, 10 years – 50.4%, 20 years – 44.6%. 94 LCP lived more than 5 years without cancer (LS=2958.6±1723.6 days), 22 – more than 10 years (LS=5571±1841.8 days). 67 LCP died because of LC (LS=471.9±344 days). AT significantly improved 5YS (68% vs. 53.7%) (P=0.028 by log-rank test). Cox modeling displayed that 5YS of LCP significantly depended on: N0-N12, T3-4, blood cell circuit, cell ratio factors (ratio between cancer cells-CC and blood cells subpopulations), LC cell dynamics, recalcification time, heparin tolerance, prothrombin index, protein, AT, procedure type (P=0.000-0.031). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and N0-12 (rank=1), thrombocytes/CC (rank=2), segmented neutrophils/CC (3), eosinophils/CC (4), erythrocytes/CC (5), healthy cells/CC (6), lymphocytes/CC (7), stick neutrophils/CC (8), leucocytes/CC (9), monocytes/CC (10). Correct prediction of 5YS was 100% by neural networks computing (error=0.000; area under ROC curve=1.0).
Muktapishti is a traditional Ayurvedic preparation made from Shoditha Mukta (Purified Pearl), is believed to help regulate thyroid function and reduce symptoms of hyperthyroidism due to its cooling and balancing properties. Clinical evidence on its efficacy remains limited, necessitating further research to validate its therapeutic benefits.
These lecture slides, by Dr Sidra Arshad, offer a simplified look into the mechanisms involved in the regulation of respiration:
Learning objectives:
1. Describe the organisation of respiratory center
2. Describe the nervous control of inspiration and respiratory rhythm
3. Describe the functions of the dorsal and respiratory groups of neurons
4. Describe the influences of the Pneumotaxic and Apneustic centers
5. Explain the role of Hering-Breur inflation reflex in regulation of inspiration
6. Explain the role of central chemoreceptors in regulation of respiration
7. Explain the role of peripheral chemoreceptors in regulation of respiration
8. Explain the regulation of respiration during exercise
9. Integrate the respiratory regulatory mechanisms
10. Describe the Cheyne-Stokes breathing
Study Resources:
1. Chapter 42, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 36, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 13, Human Physiology by Lauralee Sherwood, 9th edition
TEST BANK For Community Health Nursing A Canadian Perspective, 5th Edition by...Donc Test
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Integrating Ayurveda into Parkinson’s Management: A Holistic ApproachAyurveda ForAll
Explore the benefits of combining Ayurveda with conventional Parkinson's treatments. Learn how a holistic approach can manage symptoms, enhance well-being, and balance body energies. Discover the steps to safely integrate Ayurvedic practices into your Parkinson’s care plan, including expert guidance on diet, herbal remedies, and lifestyle modifications.
8 Surprising Reasons To Meditate 40 Minutes A Day That Can Change Your Life.pptxHolistified Wellness
We’re talking about Vedic Meditation, a form of meditation that has been around for at least 5,000 years. Back then, the people who lived in the Indus Valley, now known as India and Pakistan, practised meditation as a fundamental part of daily life. This knowledge that has given us yoga and Ayurveda, was known as Veda, hence the name Vedic. And though there are some written records, the practice has been passed down verbally from generation to generation.
Cell Therapy Expansion and Challenges in Autoimmune DiseaseHealth Advances
There is increasing confidence that cell therapies will soon play a role in the treatment of autoimmune disorders, but the extent of this impact remains to be seen. Early readouts on autologous CAR-Ts in lupus are encouraging, but manufacturing and cost limitations are likely to restrict access to highly refractory patients. Allogeneic CAR-Ts have the potential to broaden access to earlier lines of treatment due to their inherent cost benefits, however they will need to demonstrate comparable or improved efficacy to established modalities.
In addition to infrastructure and capacity constraints, CAR-Ts face a very different risk-benefit dynamic in autoimmune compared to oncology, highlighting the need for tolerable therapies with low adverse event risk. CAR-NK and Treg-based therapies are also being developed in certain autoimmune disorders and may demonstrate favorable safety profiles. Several novel non-cell therapies such as bispecific antibodies, nanobodies, and RNAi drugs, may also offer future alternative competitive solutions with variable value propositions.
Widespread adoption of cell therapies will not only require strong efficacy and safety data, but also adapted pricing and access strategies. At oncology-based price points, CAR-Ts are unlikely to achieve broad market access in autoimmune disorders, with eligible patient populations that are potentially orders of magnitude greater than the number of currently addressable cancer patients. Developers have made strides towards reducing cell therapy COGS while improving manufacturing efficiency, but payors will inevitably restrict access until more sustainable pricing is achieved.
Despite these headwinds, industry leaders and investors remain confident that cell therapies are poised to address significant unmet need in patients suffering from autoimmune disorders. However, the extent of this impact on the treatment landscape remains to be seen, as the industry rapidly approaches an inflection point.
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INTRODUCTION
Mycobacterium avium was recognised as a cause of tuberculosis
primarily in chickens, ducks and other fowls, but can also infect to an
extensive range of different animal species [1]. At least 20 different
types of M. avium have been identified which cause tuberculosis in
most avian species [2]. Avian tuberculosis generally is transmitted
by direct contact with infected birds and can susceptible to human
and bovine after ingestion of contaminated feed and water. All avian
species are susceptible to infection by M. avium. Humans, most
livestock species, and other mammals can also become infected. The
susceptibility is high in domestic fowl (Gallus domesticus), pheasants,
wild birds, swine, rabbit and human with immunocompromised [3].
In human, there are many authenticated cases of M. avium infection
although humans are considered highly resistant to this pathogenic
species. Infection is more likely to occur in persons with pre-existent
diseases, especially those involving the lungs, and in immune systems
impaired-person [4].
Infection with M. avium causes serious problems in tropical
countries in Asia particularly Thailand where a number of infected-
poultry including chickens and ducks was reported. There are two
main pathogenic subspecies of M. avium were recognized as an
important problem are M. avium subsp. avium (Maa) and M. avium
subsp. paratuberculosis (Map).
Several regions in Thailand were presented the overlapping
of these two subspecies infections. Avian tuberculosis is one of the
most important diseases that affect both domestic and pet birds, the
disease is most often caused by Mycobacterium avium belonging
Maa whereas Map is the etiologic agent of Johne’s disease causing
chronic diarrhea, malnutrition, and muscular wasting in ruminants
[5,6]. Maa and MAP are closely related and similar by morphology or
genetics information, and both can infect to the human in the term
of M. Avium Complex (MAC). Notably, MAC comprising M. avium
subsp. avium, M. avium subsp. paratuberculosis and M. avium subsp.
Silvaticum or even M. intracellulare, may also infect different animal
species like cattle, deer, horses, swine, and exotic species besides
causing infection in immunocompromised human [7].
Microbiological methods based on microscopic examination
of Acid-Fast Bacilli (AFB) presented the low sensitivity regarding
to staining procedures and proficiency of technician. Regularly, the
bacterial culture in Lowenstein–Jensen medium is specific to M.
avium but it’s time consuming (6-8 weeks). They are some definite
weak points from routine detection protocol, the stained AFB as well
as culture colonies of Maa and Map cannot distinguish each other.
Furthermore, they have low sensitivity, especially in early or lightly
infected cases.
High Resolution Melting (HRM) analysis of DNA is a simple
solution for genotyping, scanning of mutation and other aspects
on sequence analysis. The melting profile of DNA, a product from
PCR amplification, depends on GC content, length, and which is
monitored with saturating dyes that fluorescent in the presence of
double-stranded DNA [8,9]. In the field of veterinary and animal
science, HRM has been used for detection or differentiation of
various pathogenic microorganisms for example, in viruses, bacteria,
or parasites [10-12]. For mycobacterial, HRM analysis was applied
in many aspects, for example, to differential identification at the
species level in clinical isolates or in laboratory standard strains
from American Type Culture Collection (ATCC) and the Culture
Collection [13,14].
For the studies in mycobacterium avium such as HRM was applied
for identifying the polymorphisms in Map types I, II, and III [15], for
sub-typing Map by analysis of Short Sequence Repeats (SSR) loci [16],
differentiating Map cattle- and sheep-type [17], and genotyping the
members of M. Avium-Intracellulare (MAI) complex [18]. However,
real-time Polymerase Chain Reaction (real-time PCR) with HRM
for differentiation of Maa and Map in animal lesion specimens has
not been reported yet. In the present study, we developed a real-time
PCR coupled with HRM to differential detection of Maa and Map
in animal lesion specimens including poultry (ducks) and ruminant
(cow and deer) hosts. In addition, we also validate the diagnostic
specificity and sensitivity of this approach.
MATERIALS AND METHODS
Sample collection and research ethic
The animal experiments and related-protocols in this study were
approved, the authors were certified by the Institutional Animal Care
and Use Committee (Thai-IACUC) cooperated with the Institute
of Animal for Scientific Purposes Development (IAD) of Thailand
(permit number. U1-05294-2559) and Ethical Principle for the Use
and Care of Animals in Scientific Research (ACUC 5/2560), based
on the Ethics of Animal Experimentation of the National Research
Council of Thailand.
Forty-one DNA samples of M. avium infected-domestic ducks
were obtained from Veterinary Research and Development Centre
(VRDC)-Lower Northern Region, Phitsanulok, Thailand which
they were portions of the leftover specimens received from routine
laboratory diagnosis. Among 41 DNA samples from ducks, they were
ABSTRACT
Two main animal pathogenic subspecies of Mycobacterium avium are M. avium avium (Maa) and M. avium paratuberculosis (Map).
Their pathogenicity is host-specific, Maa causing avian tuberculosis in poultry whereas Map commonly cross-infects to ruminant.
Veterinary diagnosis of M. avium infections is microscopic examination of acid-fast bacilli or culture in Löwenstein-Jensen medium,
which are time-consuming and low sensitivity. This present study aimed to apply real-time PCR coupled with High-Resolution Melting
(HRM) analysis for differential detection of Maa in Thai domestic ducks. Specific primer targeting host-expression dependent (hed) region
was designed, PCR product of Maa were amplified from duck’s tissue lesions whereas Map were amplified from cow and deer. HRM
real-time PCR was performed and analyzed. Different HRM patterns were showed and melting temperature were analyzed at 83.26 ±
0.12°C for Maa and 84.04 ± 0.09°C for Map. This technique can detect as few as 102
DNA copies and present high specificity by negative
amplification of other pathogenic bacterial species. This technique is sensitive, specific, rapid and does not require fluorescent probes or
post-PCR electrophoresis. Our technique is a possible new tool for the detection of Maa and Map infection in tissue specimens.
Keywords: Duck; High resolution melting analysis; Mycobacterium Avium; Subspecies
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isolated from lesions including wings (n = 19), lungs (n = 9), intestines
(n = 11) and kidneys (n = 2). Two DNA samples of M. avium infected
cow and deer were kindly provided from National Institute of Animal
Health of Thailand which were recorded as isolated from the lesions
at lungs of both infected animals. DNA samples of Pseudomonas
aeruginosa, Enterococcus faecalis, Escherichia coli, Staphylococcus
aureus, Pasteurella multocida type a, Salmonella Group B (derived
from microbiology laboratory of VRDC-Lower Northern Region).
For negative control, three DNA samples from non-infected ducks
and two DNA samples from non-infected cow were used. All DNA
samples were stored at -80°C until usedDNA amplification by
Polymerase Chain Reaction (PCR)
For specific DNA amplification, a pair of specific primers were
designed to targeting the host-expression dependent (hed) region of
Maa (GenBank accession number: AJ011837) and Map (GenBank
accession number: AJ011838). For the PCR amplification, forward
primer was Mav100-F (5’- ATG AAA GCC ATA CCC GAC GTC
CCT -3’) and reverse primer was Mav100-R (5’- TGC GGT ACT
CGA TCA TGC TGT CCT -3’). Platinum®
Taq DNA Polymerase
(Invitrogen, Carlsbad, CA, USA) was used for PCR amplification by
the conventional PCR. The total reaction volume was 25 μl with 10x
PCR buffer, 50 mM MgCl2
, 10 mM dNTP mix, 10 μM of forward and
reverse primers, and Platinum®
Taq DNA Polymerase. Using the PCR
conditions of 1 cycle for pre-incubation at 95°C for 5 min, 30 cycles
of amplification step at 95°C for 30 sec, 57°C for 30 sec, and 72°C for
30 sec.
Cloning and sequencing of control DNA
For DNA cloning, sequencing and construction of control
plasmids, Positive control plasmids of Maa and Map were
constructed by cloning of the relevant PCR products into the
pCR®
2.1-TOPO®
vector (Invitrogen, Carlsbad, CA, USA), according
to the manufacturer’s instructions and were subsequently propagated
in Escherichia coli DH5-alpha strain (Real Biotech Corp., Taipei,
Taiwan). After amplification and agarose gel electrophoresis, the
bands of PCR product were purified by HiYield™ Gel/PCR DNA Mini
Kit, (Real Biotech Corp., Taipei, Taiwan) and the nucleotide sequence
were determined by 3500 Genetic Analyzer (Applied Biosystems, Inc.
[Hitachi Ltd], Tokyo, Japan).
High-resolution melting analysis
For Real-time PCR and HRM analysis, LightCycler ®
96 Real-Time
PCR System for detection and analysis were used for amplification
and quantification. A LightCycler®
480 High Resolution Melting
Master (Roche Applied Science, Mannheim, Germany) was used as
recommended by the manufacturer, including Master Mix (FastStart
Taq DNA Polymerase, reaction buffer, dNTP mix and HRM dye),
MgCl2
and H2
O PCR-grade. The PCR mixture was prepared to
contain 1x HRM Master, 2.2 mM MgCl2
, 0.8 μM of each 2 primers
(Mav100-F and Mav100-R). The total reaction volume was 20 μl. The
PCR cycling for HRM curve acquisition was run under the following
conditions: 1 cycle for pre-incubation at 95°C for 10 min, 45 cycles
of amplification step at 95°C for 10 sec, 57°C for 8 sec, and 72°C for
15 sec. After amplification, the PCR products were melted by raising
the temperature from 60°C to 95°C, with an increment of 0.1°C/sec.
After, the amplification curve was monitored real-time, the melting
temperatures (Tm) and melting curve were determined and analyzed.
Evaluation of sensitivity and specificity of detection
For determination of analytical sensitivity, the ten-folded serial
dilutionfrom109
to1copiesofMaaandMappositivecontrolplasmids
were prepared. For determination of analytical specificity, DNA
samples of P. aeruginosa, E. faecalis, Escherichia coli, Staphylococcus
aureus, Pasteurella multocida type a, Salmonella Group B were
used. They were subsequently analysed by Real-time PCR and HRM
analysis as described above. The diagnostic values were calculated
using standard methods [19].
RESULTS
Specific amplification of Maa and Map DNA
For PCR amplification and sequencing analysis of M. avium
DNA. Using the primer targeting the region that described in material
and methods, we successfully amplified a predicted 76 bp and 100 bp
product specific from DNA of Maa and Map, respectively (Figure 1).
After purification and DNA sequencing, the obtained sequences
were matched to the sequences of genes from which the primers were
designed. There are some deletions occur in the region of Maa DNA
that amplified from duck’s infected-tissue compared with 100 bp of
Map DNA from infected tissue of the cow and the deer (Figure 2).
For HRM real-time PCR for differential detection of Maa and
Map, the method yielded the positive results for all 41 Maa-infected
and 2 Map-infected DNA samples. After amplification, the melting
temperatures (Tm) and melting curve were analysed. Representative
melting temperatures were 83.26 ± 0.12°C for Maa and 84.04 ± 0.09°C
for Map (Figure 3A-C). DNA from Maa and Map could be obviously
distinguished by the specific melting temperatures determined by the
difference plots (Figure 3A), the normalized melting curves (Figure
3B), and the melting peaks (Figure 3C).
To standardization of the HRM real-time PCR, there is no
fluorescent signal were detected when evaluated with the no DNA
control of each experiments, especially in 3 DNA samples from
non-infected ducks and 2 DNA samples from non-infected cow. For
analytical specificity of HRM real-time PCR, there are no positive
Figure 1: PCR amplification of Maa and Map DNA. The 1.5% agarose gel
electrophoresis presents 76 bp and 100 bp PCR products indicating Maa
and Map DNA, respectively. For each land, M: 100 bp DNA ladder; N:
Negative control (no DNA template); P1 and P2: DNA samples from Maa
infected ducks; R1 and R2: DNA samples from Map infected cow and deer,
respectively.
Figure 2: Primer design and alignment with Maa and Map sequences
obtained from DNA sequencing. Alignment of Mav100 forward and reverse
primers with the sequence obtained from sequencing of amplification
products from DNA samples of Maa infected ducks (P1 and P2), Map infected
cow (R1) and Map infected deer (R2). The arrows indicate the direction of
each primer for PCR amplification.
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result from amplicon at specific melting temperatures was perceived
when various DNAs from other pathogenic bacterial species were
analysed which demonstrates the specificity of this method (Figure
3D). In addition, the analytical sensitivity was determined using
ten-fold serial dilutions (109
- 1 copies) of the Maa and Map positive
control plasmids. The sensitivity was illustrated by DNA of both
Maa and Map could be detected at the lowest concentration used
(102
copies) when considering 45 cycles as the cut-off detection limit
(Figure 3E-F).
DISCUSSION
This is the report of HRM real-time PCR to distinguish between
Maa and Map in DNA samples extracted from tissue lesion specimens
of infected animals in a single assay. We present the application for
homogenous genotyping of M. avium subspecies without fluorescent
labelled-probe and post-PCR electrophoresis. Therefore, the method
consequently reducing the expense on a cost per sample and the
time consuming. Simplification of interpretation is obtained by
different sequences from different subspecies represent a change in
the temperature in analysed-melting peak. The HRM real-time PCR
offers a new alternative for rapid, sensitive and subspecies specific
for differentiation and detection of two main pathogenic M. avium
subspecies in poultry and ruminant hosts. The high sensitivity and
specificity suggest the present test will be of diagnostic value. The
analytical sensitivity in this study was only 100 copies of Maa and
Map DNA. Moreover, real-time PCR with HRM produced no cross
reaction between Maa and Map or various DNAs from other bacterial
species that usually infected duck and cow indicating high specificity.
There are several studies mention the application of HRM
real-time PCR for mycobacterial. Recently in 2017, the researchers
defined comparison of melting curves of different non-tuberculosis
mycobacteria and presented the melting curves corresponding to
the 6 unique-specific HRM groups (M. tuberculosis complex, M.
kansasii, M. simiae, M. fortuitum, M. abscessus–M. chelonae group,
and M. avium complex) for the mycobacterial rpoBC locus in several
collected clinical specimens [13].
Previously, the standardization of HRM real-time PCR were
verified by laboratory standard of Mycobacterial strains [14]. In that
study the DNA were amplified by primer targeting 16S rRNA gene,
the Tm values for M. tuberculosis and M. bovis of the were 86.00 ±
0.00°C. M. smegmatis, M. ulcerans and M. xenopi, the Tm values were
86.40 ± 0.00°C, 86.20 ± 0.00°C and 86.60 ± 0.00°C, respectively. The
Tm values for M. avium, M. avium subspecies paratuberculosis and
M. intracellulare of the M. Avium Complex (MAC) were 86.20 ±
0.00°C, 86.60 ± 0.00°C and 86.80 ± 0.00°C, respectively. In our present
study, we use the primer targeting at hed region and the Tm values at
83.26 ± 0.12°C for Maa and 84.04 ± 0.09°C for Map were obtained.
We presented practically almost 1°C for distinguish of Maa and Map
whereas previous study is only 0.4°C. In addition, DNA samples of
the related pathogenic bacterial species in this group of animals, the
test did not give rise to an identifiable Tm or melting peak of Maa and
Map, indicating the specificity of this method.
Regarding to the samples those sent from local farmers were
infected animals with the lesions were appeared, the technique
optimized for detection of the infection by lesion samples should
be appropriated. However, even this study attempted to apply the
techniques for detection of Mycobacterium avium infection in
lesion specimens, but further verifying by blood or another animal
specimen was very challenged. In addition, because the primer was
designed to target the sequence of Maa and Map directly. Therefore,
the technique can be applied to detect these Mycobacterium avium
subspecies infection in other animals or even in human.
In conclusion, this study highlights a HRM real-time PCR for
the differentiation of the 2 main pathogenic subspecies of M. avium,
Maa and Map. We successfully developed the rapid method for this
approach,especiallyappropriatesfortheDNAsamplesextractedfrom
M. avium infected lesion tissues from animal. This HRM real-time
PCR can potentially be used for subspecies-specific epidemiological
surveys of M. avium in domestic livestock, especially in Thailand
where M. avium infections are usual informed.
ACKNOWLEDGEMENTS
This study was financially supported by the grants from Naresuan
University (grant number: R2558C034) and the National Research
Council of Thailand (grant number: R2559B066).
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