F Gene and N Gene based Reverse Transcription PCR for Molecular Characterization of Peste des Petits Ruminants Virus

2020 ◽  
Author(s):  
A K Pandey ◽  
D M Muglikar ◽  
P P Mhase ◽  
M M Pawade ◽  
S N Daphal ◽  
...  
Keyword(s):  
Molecular Detection ◽  
Sandwich Elisa ◽  
Small Ruminants ◽  
N Gene ◽  
Tissue Samples ◽  
F Gene ◽  
Specific Pcr ◽  
Percent Positivity ◽  
Nasal Swabs ◽  
Ppr Virus

Present investigation was undertaken to detect and characterize the PPR virus from different clinical tissue samples of 14 sheep and 17 goats with respiratory disease from Maharashtra, India. All animals were tested by Sandwich ELISA, of which 70.96% were found positive carrying high PPR virus inwhich 12 were sheep and 10 were goats respectively. For confirmation of PPR, molecular detection was performed with RT-PCR using F gene and N gene specific primers. Intestine samples accounted for highest percent positivity (75%) followed by blood (66.66%) and lymph node (62.5%) for presence of virus. Unusually higher positivity was observed in heart, liver and Kidney (60%, each) than normal predilection sites such as lungs (57.84%) and spleen (50%). While nasal swabs and blood were individually processed with F gene and N gene specific PCR, the triturated organs were pooled for processing into ‘Sample A’ comprised of heart, kidney, liver and intestine combined together and ‘Sample B’ comprising of lung, spleen and lymph nodes combined for the molecular detection of PPR yielding the products each of 372bp and 463bp sizes, respectively. Out of total 40 samples tested, 09/12 each from both sample A and B, while 02/10 nasal swabs resulted positive, respectively and all 06 blood samples remained negative. (F as well as N gene PCR methods were found best suitable for detection of PPR virus from tissue samples of small ruminants).

scholarly journals Comparison of nucleotide sequences of recent and previous lineages of peste-des-petits-ruminants viruses of sheep and goats in Nigeria

2016 ◽  
Vol 83 (1) ◽  
Author(s):  
Samuel Mantip ◽  
Melvyn Quan ◽  
David Shamaki ◽  
Moritz Van Vuuren
Keyword(s):  
Phylogenetic Analysis ◽  
Small Ruminants ◽  
Viral Disease ◽  
N Gene ◽  
Peste Des Petits Ruminants ◽  
East African ◽  
Tissue Samples ◽  
Sheep And Goats ◽  
Ecological Zones ◽  
Agro Ecological Zones

Peste-des-petits-ruminants virus (PPRV) is a highly contagious, fatal and economically important viral disease of small ruminants that is still endemic and militates against the production of sheep and goats in endemic areas of the world. The aim of this study was to describe the viral strains within the country. This was carried out by collecting tissue and swab samples from sheep and goats in various agro-ecological zones of Nigeria. The phylogeny of archived PPRV strains or isolates and those circulating and causing recent outbreaks was determined by sequencing of the nucleoprotein (N)-gene. Twenty tissue and swab samples from apparently healthy and sick sheep and goats were collected randomly from 18 states, namely 3 states in each of the 6 agro-ecological zones visited. A total of 360 samples were collected. A total of 35 samples of 360 (9.7%) tested positive by reverse transcriptase–polymerase chain reaction, of which 25 were from oculo-nasal swabs and 10 were from tissue samples. Neighbour-joining phylogenetic analysis using Phylogenetic Analysis Using Parsimony (PAUP) identified four different lineages, that is, lineages I, II, III and IV. Interestingly, the Nigerian strains described in this study grouped in two separate major lineages, that is, lineages II and IV. Strains from Sokoto, Oyo, Plateau and Ondo states grouped according to the historical distribution of PPRV together with the Nigerian 75/1 strain of lineage II, while other strains from Sokoto, Oyo, Plateau, Akwa-Ibom, Adamawa, Kaduna, Lagos, Bauchi, Niger and Kano states grouped together with the East African and Asian strains of lineage IV. This finding confirms that both lineage II and IV strains of PPRV are circulating in Nigeria. Previously, only strains of lineage II were found to be present in the country.

scholarly journals Evaluation of Risk Factors for Peste des Petits Ruminants Virus in Sheep and Goats at the Wildlife-Livestock Interface in Punjab Province, Pakistan

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Aziz-ul-Rahman ◽  
Muhammad Abubakar ◽  
Muhammad Hidayat Rasool ◽  
Shumaila Manzoor ◽  
Muhammad Saqalein ◽  
...  
Keyword(s):  
Risk Factors ◽  
Significant Risk ◽  
Small Ruminants ◽  
Significant Risk Factor ◽  
N Gene ◽  
High Morbidity ◽  
Peste Des Petits Ruminants ◽  
Sheep And Goats ◽  
Wild Ruminants ◽  
Nasal Swabs

Peste des petits ruminants virus (PPRV) is causing infectious disease with high morbidity and mortality rate in domestic and wild small ruminants of Pakistan with valuable economical losses. The present study was carried out to investigate risk factors of PPRV in domestic small ruminants which were present in the vicinity of wildlife parks. A total of 265 sera samples (27 wild ruminants and 238 domesticated small ruminants) from apparently healthy animals from two different wildlife parks were collected and analysed for PPRV antibodies. Also, 20 nasal swabs from domestic small ruminants showing respiratory signs were collected to check for presence of PPRV antigen. Competitive ELISA revealed highest proportions of anti-PPRV antibodies in domestic small ruminants around the Wildlife Park at Lahore (35%) as compared to Faisalabad (13%), with no existence of PPRV antibodies in tested serum of wild ruminants at these parks. Higher seropositivity was observed in females (25.6%) than in males (5.1%) and in goats (34.5%) compared to sheep (11.2%). The results of N-gene based RT-PCR highlight the absence of PPRV due to lack of current PPR outbreak in the region during study period. Even though grazing was not a significant risk factor, there is still a possibility of wildlife-livestock interactions for feed and water reservoirs, resulting in spillover of PPR to wildlife. Keeping in view the high seropositivity and risk of PPR, vaccination should be adopted to avoid circulation of PPRV among wild and domestic small ruminants (sheep and goats).

scholarly journals Isolation of Mycobacterium avium Subsp. Paratuberculosis in the Feces and Tissue of Small Ruminants Using a Non-Automated Liquid Culture Method

Animals ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 20
Author(s):  
Luigi De Grossi ◽  
Davide Santori ◽  
Antonino Barone ◽  
Silvia Abbruzzese ◽  
Matteo Ricchi ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Liquid Culture ◽  
Culture Media ◽  
Small Ruminants ◽  
Culture Method ◽  
Mycobacterium Avium Subsp ◽  
Type I ◽  
Culture Methods ◽  
Specific Pcr ◽  
Mycobacterium Avium Subsp Paratuberculosis

Paratuberculosis is a chronic disease of ruminants caused by Mycobacterium avium subsp. Paratuberculosis (MAP). Since isolation of MAP type I (S) is rarely reported in Italy, our research was aimed at isolating, by an inexpensive liquid culture manual method, this type of MAP isolates. At first, we used an ELISA to point out to serologically positive samples from five flocks. Secondly, we used a fecal direct IS900-qPCR on the ELISA positive samples, in order to detect shedder animals. Feces from IS900-qPCR positive samples were inoculated in solid and liquid culture media. IS900-qPCR was further used to test the growth of MAP isolates in liquid medium, which were further confirmed by f57-qPCR and submitted to typing by specific PCR in order to identify the MAP type. Twenty-eight samples (24 fecal and four tissutal samples) were processed by culture methods, resulting in the isolation of six type I MAP field isolates. Notably, no isolates were recovered by solid media, underlining the utility of this liquid method. Few data about this type of MAP are currently available in Italy, and further analyses should be carried out in order to study the origin and epidemiology of type I strains circulating in Italy.

scholarly journals Performance of Unobserved Self-Collected Nasal Swabs for Detection of SARS-CoV-2 by RT-PCR Utilizing a Remote Specimen Collection Strategy

2021 ◽  
Author(s):  
Ron M Kagan ◽  
Amy A Rogers ◽  
Gwynngelle A Borillo ◽  
Nigel J Clarke ◽  
Elizabeth M Marlowe
Keyword(s):  
Return To Work ◽  
Screening Program ◽  
False Negative ◽  
N Gene ◽  
Rt Pcr ◽  
Pooled Testing ◽  
Specimen Collection ◽  
Asymptomatic Patients ◽  
Nasal Swabs ◽  
The Impact

Abstract Background The use of a remote specimen collection strategy employing a kit designed for unobserved self-collection for SARS-CoV-2 RT-PCR can decrease the use of PPE and exposure risk. To assess the impact of unobserved specimen self-collection on test performance, we examined results from a SARS-CoV-2 qualitative RT-PCR test for self-collected specimens from participants in a return-to-work screening program and assessed the impact of a pooled testing strategy in this cohort. Methods Self-collected anterior nasal swabs from employee return to work programs were tested using the Quest Diagnostics SARS-CoV-2 RT-PCR EUA. The Ct values for the N1 and N3 N-gene targets and a human RNase P (RP) gene control target were tabulated. For comparison, we utilized Ct values from a cohort of HCP-collected specimens from patients with and without COVID-19 symptoms. Results Among 47,923 participants, 1.8% were positive. RP failed to amplify for 13/115,435 (0.011%) specimens. The median (IQR) Cts were 32.7 (25.0-35.7) for N1 and 31.3 (23.8-34.2) for N3. Median Ct values in the self-collected cohort were significantly higher than those of symptomatic, but not asymptomatic patients. Based on Ct values, pooled testing with 4 specimens would have yielded inconclusive results in 67/1,268 (5.2%) specimens but only a single false-negative result. Conclusions Unobserved self-collection of nasal swabs provides adequate sampling for SARS-CoV-2 RT-PCR testing. These findings alleviate concerns of increased false negatives in this context. Specimen pooling could be used for this population as the likelihood of false negative results is very low due when using a sensitive, dual-target methodology.

scholarly journals Isolation ofMycobacterium aviumsubspeciesparatuberculosisfrom non-ruminant wildlife living in the sheds and on the pastures of Greek sheep and goats

2007 ◽  
Vol 136 (5) ◽  
pp. 644-652 ◽  
Author(s):  
M. FLOROU ◽  
L. LEONTIDES ◽  
P. KOSTOULAS ◽  
C. BILLINIS ◽  
M. SOFIA ◽  
...  
Keyword(s):  
Type Strain ◽  
Insertion Sequence ◽  
Small Ruminants ◽  
Dairy Sheep ◽  
Chain Reaction ◽  
Tissue Samples ◽  
Sheep And Goats ◽  
Faecal Samples ◽  
Polymerase Chain ◽  
Type Strains

SUMMARYThis study aimed to: (1) investigate whether non-ruminant wildlife interfacing with dairy sheep and goats of four Greek flocks endemically infected withMycobacterium aviumsubspeciesparatuberculosis(MAP) harboured MAP and (2) genetically compare the strains isolated from the wildlife to those isolated from the small ruminants of these flocks. We cultured and screened, by polymerase chain reaction (PCR), pooled-tissue samples from 327 wild animals of 11 species for the MAP-specific IS900insertion sequence. We also cultured faecal samples from 100 sheep or goats from each of the four flocks. MAP was detected in samples from 11 sheep, 12 goats, two mice, two rats, a hare and a fox. Only one rat had histopathological findings. Genetic typing categorized 21 isolates as cattle-type strains and two, from a house mouse and a goat respectively, as sheep-type strains; this is the first report of a rodent harbouring a sheep-type strain. The MAP types that were most frequently isolated amongst the sheep and goats of each flock were also the ones isolated from sympatric rodents; those isolated from the fox and hare also belonged to the predominant ruminant strains.

scholarly journals Genotypic detection of some virulence factors among Aeromonas hydrophila Isolated from Diarrhea Cases (Iraq)

2018 ◽  
Vol 9 (SPL1) ◽  
Author(s):  
Ilham A Bunyan ◽  
Israa K Obais
Keyword(s):  
Virulence Factors ◽  
Aeromonas Hydrophila ◽  
Molecular Detection ◽  
Virulence Gene ◽  
Molecular Study ◽  
Protease Gene ◽  
Allelic Ladder ◽  
Specific Pcr ◽  
Molecular Length ◽  
Positive Results

The present study included the detection ofsome virulence factorsof Aeromonas hydrophila under molecular level to clinical isolates were taken from patients suffering from diarrhea during the period from July (2017) to October (2017). Molecular detection of Hemolysin gene (ahh) was done for all isolates. The results showed that all isolates (100%) gave positive results for this virulence gene. the positive results were detected by the presence of (130) bp bands when compared with allelic ladder. The genomic DNA of the samples was extracted and bands were observed by performing agarose gel electrophoresis. When PCR was performed,results clearly indicate that all isolated organisms contained serine protease gene and all the amplified products produced a band at the level of (900 bp) when compared with the allelic ladder. Molecular detection of this gene was carried out by using a specific PCR primer were done by comparison with allelic ladder which gave a (309bp) It was found that (Aerolysin) gene present in (12) (75%) of the positive samples. Lip gene was also detected in A. hydrophila samples and found that all 16 samples (100%) gave positive results to this gene which gave molecular length (382) bp. Molecular study was carried out to show the sequence identity of cytotonic enterotoxins gene in Aeromonas spp. to that in A. hydrophila. Analysis of the A. hydrophila genome revealed a number of a putative virulence factors,including a gene that heat-labile cytotonic enterotoxin (alt). Our study showed that all (16) isolates (100%) gave positive results to this gene,which gave molecular length (442)bp. Molecular detection of cytotonic enterotoxins gene (ast) was done for (16) A. hydrophila isolates and the results showed that all isolates have this gene (100%). The positive results for (ast) virulence were detected by the presence of (331) bp band compared with allelic ladder.

scholarly journals First report of Coleus blumei viroid 1 in commercial Coleus blumei in the United States

Plant Disease ◽  
2021 ◽  
Author(s):  
Gardenia Orellana ◽  
Alexander V Karasev
Keyword(s):  
United States ◽  
Leaf Tissue ◽  
The United States ◽  
Universal Primers ◽  
Rt Pcr ◽  
Universal Primer ◽  
Tissue Samples ◽  
First Report ◽  
Specific Pcr ◽  
Coleus Blumei

Coleus scutellarioides (syn. Coleus blumei) is a widely grown evergreen ornamental plant valued for its highly decorative variegated leaves. Six viroids, named Coleus blumei viroid 1 to 6 (CbVd-1 to -6) have been identified in coleus plants in many countries of the world (Nie and Singh 2017), including Canada (Smith et al. 2018). However there have been no reports of Coleus blumei viroids occurring in the U.S.A. (Nie and Singh 2017). In April 2021, leaf tissue samples from 27 cultivars of C. blumei, one plant of each, were submitted to the University of Idaho laboratory from a commercial nursery located in Oregon to screen for the presence of viroids. The sampled plants were selected randomly and no symptoms were apparent in any of the samples. Total nucleic acids were extracted from each sample (Dellaporta et al. 1983) and used in reverse-transcription (RT)-PCR tests (Jiang et al. 2011) for the CbVd-1 and CbVd-5 with the universal primer pair CbVds-P1/P2, which amplifies the complete genome of all members in the genus Coleviroid (Jiang et al. 2011), and two additional primer pairs, CbVd1-F1/R1 and CbVd5-F1/R1, specific for CbVd-1 and CbVd-5, respectively (Smith et al. 2018). Five C. blumei plants (cvs Fire Mountain, Lovebird, Smokey Rose, Marrakesh, and Nutmeg) were positive for a coleviroid based on the observation of the single 250-nt band in the RT-PCR test with CbVds-P1/P2 primers. Two of these CbVd-1 positive plants (cvs Lovebird and Nutmeg) were also positive for CbVd-1 based on the presence of a single 150-nt band in the RT-PCR assay with CbVd1-F1/R1 primers. One plant (cv Jigsaw) was positive for CbVd-1, i.e. showing the 150-nt band in RT-PCR with CbVd1-F1/R1 primers, but did not show the ca. 250-bp band in RT-PCR with primers CbVds-P1/P2. None of the tested plants were positive for CbVd-5, either with the specific, or universal primers. All coleviroid- and CbVd-1-specific PCR products were sequenced directly using the Sanger methodology, and revealed whole genomes for five isolates of CbVd-1 from Oregon, U.S.A. The genomes of the five CbVd-1 isolates displayed 96.9-100% identity among each other and 96.0-100% identity to the CbVd-1 sequences available in GenBank. Because the sequences from cvs Lovebird, Marrakesh, and Nutmeg, were found 100% identical, one sequence was deposited in GenBank (MZ326145). Two other sequences, from cvs Fire Mountain and Smokey Rose, were deposited in the GenBank under accession numbers MZ326144 and MZ326146, respectively. To the best of our knowledge, this is the first report of CbVd-1 in the United States.

Isolation and Molecular Characterization of Newcastle Disease Virus in Layers

2021 ◽  
Author(s):  
Smita Bordoloi ◽  
Anju Nayak ◽  
A.P. Singh ◽  
R.V. Singh ◽  
Kajal Jadav ◽  
...  
Keyword(s):  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Virulent Strain ◽  
Indian Subcontinent ◽  
Economic Losses ◽  
Tissue Samples ◽  
F Gene ◽  
Virulent Newcastle Disease Virus

Background: Newcastle disease (ND) in spite of the availability of vaccines remains a constant threat to poultry producers worldwide. It is prevalent in Indian subcontinent and leads to economic losses. The present study was aimed with isolate and identify virulent Newcastle disease virus (NDV) in layer poultry from field outbreaks.Methods: Total 47 samples consisting of nasal (05), oropharyngeal (13) and cloacal swabs (11) and tissue samples consisting of trachea (07), lungs (06), larynx (05) were collected from layer birds. For isolation of NDV swab and tissue samples were inoculated in 9-11 days old embryonated eggs via allantoic cavity route. After preparing the viral inoculum, 47 suspected samples (29 swab and 18 tissue samples) were inoculated in 141 embryonated eggs to isolate the virus.Result: Out of 47 samples 10 (21.27%) samples were positive for HA activity. All the 10 isolates showing HA activity subjected to Reverse-Transcriptase PCR of F gene and 6 were found positive in RT-PCR for F1 gene. The PCR amplified product showed amplicon at 356 bp and 254 bp positive for F1 and F2 gene, respectively. On basis of F gene, 06 (50%) isolates were considered as virulent Newcastle Disease Virus. One isolate sequence was submitted at NCBI with accession MT890653 On phylogenetic analysis MT890653 designated as Class II/ genotype II/ virulent strain and had the motif 112R-R-R-K-R-F117 at the cleavage site of the fusion protein.

Sandwich ELISA detection of Mycoplasma bovis in pneumonic calf lungs and nasal swabs

1994 ◽  
Vol 135 (22) ◽  
pp. 531-532 ◽  
Author(s):  
H. Ball ◽  
D. Finlay ◽  
G. Reilly
Keyword(s):  
Sandwich Elisa ◽  
Mycoplasma Bovis ◽  
Nasal Swabs
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