scholarly journals Serological and Molecular Evidence of Brucella Species in the Rapidly Growing Pig Sector in Kenya

2020 ◽  
Author(s):  
James Miser Akoko ◽  
Roger Pelle ◽  
Velma Kivali ◽  
Esther Schelling ◽  
Gabriel Shirima ◽  
...  
Keyword(s):  
Real Time ◽  
Epidemiological Data ◽  
Molecular Techniques ◽  
Molecular Evidence ◽  
Rt Pcr ◽  
Conventional Pcr ◽  
Brucella Infection ◽  
Growing Pig ◽  
Pcr Targeting

Abstract Background: Brucellosis is an emerging yet neglected zoonosis that has been reported in Kenya. Epidemiological data on brucellosis in ruminants is readily accessible; however, reports on brucellosis in pigs remain limited. This study sought to detect Brucella infection in pig serum by both serological and molecular techniques. Serum from 700 pigs randomly collected at a centralized abattoir in Nairobi region, Kenya were screened in parallel, using both Rose Bengal Test (RBT) and competitive Enzyme-Linked Immuno-sorbent Assay (cELISA) for antibodies against Brucella spp. All sera positive by RBT and 16 randomly selected negative samples were further tested using conventional PCR targeting bcsp31 geneand real-time PCR (RT-PCR) assays targeting IS711 and bcsp31 genes. Results: A prevalence of 0.57% (n = 4/700) was estimated using RBT; none of these samples was positive on cELISA. All RBT positive sera were also positive by both PCRs, while two sero-negative samples also tested positive on RT-PCR (n = 6/20). Brucella abortus was detected in four out of the six PCR positive samples through a real-time multiplex PCR. Conclusion: The detection of antibodies against Brucella spp. and DNA in serum from slaughterhouse pigs confirm the presence of Brucella in pigs. Therefore, investigation of the epidemiology and role of pigs in the transmission of brucellosis in Kenya is needed. Further targeted studies would be useful to systematically quantify and identify the spp. of Brucella in pigs.

scholarly journals Serological and molecular evidence of Brucella species in the rapidly growing pig sector in Kenya

2020 ◽  
Author(s):  
James Miser Akoko ◽  
Roger Pelle ◽  
Velma Kivali ◽  
Esther Schelling ◽  
Gabriel Shirima ◽  
...  
Keyword(s):  
Real Time ◽  
Epidemiological Data ◽  
Molecular Techniques ◽  
Molecular Evidence ◽  
Brucella Species ◽  
Rt Pcr ◽  
Conventional Pcr ◽  
Growing Pig ◽  
Pcr Targeting

Abstract Background: Brucellosis is an emerging yet neglected zoonosis that has been reported in Kenya. Epidemiological data on brucellosis in ruminants is readily accessible; however, reports on brucellosis in pigs remain limited. This study sought to detect Brucella infection in pig serum by both serological and molecular techniques. Serum from 700 pigs randomly collected at a centralized abattoir in Nairobi region, Kenya were screened in parallel, using both Rose Bengal Test (RBT) and competitive Enzyme-Linked Immuno-sorbent Assay (cELISA) for antibodies against Brucella spp. All sera positive by RBT and 16 randomly selected negative samples were further tested using conventional PCR targeting bcsp31 gene and real-time PCR (RT-PCR) assays targeting IS711 and bcsp31 genes.Results: A prevalence of 0.57% (n = 4/700) was estimated using RBT; none of these samples was positive on cELISA. All RBT positive sera were also positive by both PCRs, while two sero-negative samples also tested positive on RT-PCR (n = 6/20). Brucella abortus was detected in four out of the six PCR positive samples through a real-time multiplex PCR.Conclusion: The detection of antibodies against Brucella spp. and DNA in serum from slaughterhouse pigs confirm the presence of Brucella in pigs. Therefore, investigation of the epidemiology and role of pigs in the transmission of brucellosis in Kenya is needed. Further targeted studies would be useful to systematically quantify and identify the spp. of Brucella in pigs.

scholarly journals Serological and molecular evidence of Brucella species in the rapidly growing pig sector in Kenya

2020 ◽  
Author(s):  
James Miser Akoko ◽  
Roger Pelle ◽  
Velma Kivali ◽  
Esther Schelling ◽  
Gabriel Shirima ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Epidemiological Data ◽  
Molecular Techniques ◽  
Molecular Evidence ◽  
Rt Pcr ◽  
Growing Pig ◽  
Pcr Targeting ◽  
The Rose

Abstract BackgroundBrucellosis is an emerging, yet neglected zoonosis that has been reported in Kenya. Epidemiological data on brucellosis in ruminants is readily accessible; however, reports on brucellosis in pigs remain limited. This study sought to detect Brucella infection in pig serum by both serological and molecular techniques. Serum from 700 pigs randomly collected at a centralized abattoir in Nairobi, Kenya were screened in parallel, using the Rose Bengal Test (RBT) and competitive Enzyme-Linked Immuno-sorbent Assay (cELISA) for antibodies against Brucella spp.. All sera positive by RBT and 16 randomly selected negative samples were further tested using conventional PCR targeting bcsp31 gene and real-time PCR (RT-PCR) assays targeting IS711 and bcsp31 genes.ResultsA prevalence of 0.57% (n=4/700) was estimated using RBT. All RBT positive sera were also positive by both PCRs, while two sero-negative samples also tested positive on RT-PCR (n = 6/20). Brucella abortus was detected in four out of the six PCR positive samples through a multiple real-time PCR. ConclusionThe detection of antibodies against Brucella spp. and DNA in serum from slaughterhouse pigs indicate the presence of Brucella in pigs. Therefore, investigation of the epidemiology and role of pigs in the transmission of brucellosis in Kenya is needed. Further targeted studies would be useful to systematically quantify and identify the spp. of Brucella in pigs.

scholarly journals Molecular detection of cattle Sarcocystis spp. in North-West Italy highlights their association with bovine eosinophilic myositis

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Selene Rubiola ◽  
Tiziana Civera ◽  
Felice Panebianco ◽  
Davide Vercellino ◽  
Francesco Chiesa
Keyword(s):  
18S Rdna ◽  
Inflammatory Myopathy ◽  
Molecular Techniques ◽  
Diaphragm Muscle ◽  
Economic Losses ◽  
Intermediate Hosts ◽  
Slaughter Cattle ◽  
Significant Difference ◽  
Pcr Targeting

Abstract Background Cattle are intermediate hosts of six Sarcocystis species, among which Sarcocystis hominis and Sarcocystis heydorni can infect humans through the consumption of raw or undercooked meat. In addition to the zoonotic potential, there is increasing interest in these protozoa because of the evidence supporting the role of Sarcocystis spp. in the occurrence of bovine eosinophilic myositis (BEM), a specific inflammatory myopathy which leads to carcass condemnation and considerable economic losses. Actually, all the prevalence studies carried out on cattle in Italy have been based on either morphological or 18S rDNA-based molecular techniques, most likely leading to misidentification of closely related species. Therefore, there is a strong need for new data on the prevalence of the different Sarcocystis spp. in cattle in Italy and their association with bovine eosinophilic myositis. Methods To reach our aim, individual striated muscle samples from BEM condemned carcasses (N = 54) and diaphragm muscle samples from randomly sampled carcasses (N = 59) were obtained from Northwest Italy slaughterhouses. Genomic DNA was extracted and analyzed by multiplex-PCR targeting 18S rDNA and cox1 genes. PCR products amplified using the genus-specific primer set in absence of the specific fragment for S. hirsuta, S. cruzi, S. hominis or S. bovifelis were sequenced to achieve species identification. Results Sarcocystis DNA was detected in 67.8% of the samples from slaughter cattle and in 90.7% of the samples from BEM condemned carcasses. S. cruzi was identified as the most prevalent species in slaughter cattle (61%), followed by S. bovifelis (10.2%), S. hominis (8.5%) and S. hirsuta (1.7%). Notably, among the different Sarcocystis spp. detected, the presence of S. bovifelis and S. hominis was significantly higher in samples isolated from BEM condemned carcasses (46.3% and 40.7% respectively), while there was no statistically significant difference between the presence of S. cruzi or S. hirsuta in BEM condemned carcasses (42.6% and 1.8%, respectively) and randomly sampled carcasses. Furthermore, DNA sequence analysis revealed the presence of a putative new species in two carcasses. Conclusions Our study contributes to updating the data on the prevalence of the different Sarcocystis spp. in cattle in Italy, highlighting the presence of three Sarcocystis spp., S. cruzi, S. hominis and S. bovifelis, in BEM lesions and allowing us to speculate on the possible role of S. hominis and S. bovifelis as the major sarcosporidian species involved in bovine eosinophilic myositis. Graphic Abstract

scholarly journals First Report of Hepatitis E Virus in Shellfish in Southeast Italy

2020 ◽  
Vol 11 (1) ◽  
pp. 43
Author(s):  
Gianfranco La Bella ◽  
Maria Grazia Basanisi ◽  
Gaia Nobili ◽  
Valentina Terio ◽  
Elisabetta Suffredini ◽  
...  
Keyword(s):  
Real Time ◽  
Hepatitis E Virus ◽  
Potential Role ◽  
Hepatitis E ◽  
Developed Countries ◽  
Rt Pcr ◽  
Viral Pathogens ◽  
Apulia Region ◽  
Causative Agents

Hepatitis E virus (HEV) represents one of the principal causative agents of hepatitis globally. Among the five HEV genotypes affecting humans, genotypes 3 and 4 are zoonotic and are the main source of hepatitis E in developed countries. HEV has been detected in several foods. The present work investigated the presence of this virus in shellfish sold at retail in the Apulia region of Italy. The presence of HEV RNA was assessed by real-time RT-PCR in 225 shellfish samples collected during 2018. Overall, two (0.89%) of these samples tested positive for HEV RNA. To our knowledge, this is the first notification of the detection of HEV in mussels sold at retail in the Apulia region. These data highlight the potential role of shellfish as a vehicle for the transmission of viral pathogens.

scholarly journals Quantitative assessment of CYP11B1 and CYP11B2 expression in aldosterone-producing adenomas

2002 ◽  
pp. 795-802 ◽  
Author(s):  
F Fallo ◽  
V Pezzi ◽  
L Barzon ◽  
P Mulatero ◽  
F Veglio ◽  
...  
Keyword(s):  
Real Time ◽  
Secretory Activity ◽  
Zona Glomerulosa ◽  
Zona Fasciculata ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Pathophysiological Role ◽  
Aldosterone Production

BACKGROUND: The presence and pathophysiological role of CYP11B1 (11beta-hydroxylase) gene in the zona glomerulosa of human adrenal cortex is still controversial. METHODS: In order to specifically quantify CYP11B1, CYP11B2 (aldosterone synthase) and CYP17(17alpha-hydroxylase) mRNA levels, we developed a real-time RT-PCR assay and examined the expression in a series of adrenal tIssues, including six normal adrenals from patients adrenalectomized for renal cancer and twelve aldosterone-producing adenomas (APA) from patients with primary aldosteronism. RESULTS: CYP11B1 mRNA levels were clearly detected in normal adrenals, which comprised both zona glomerulosa and fasciculata/reticularis cells, but were also measured at a lower range (P<0.05) in APA. The levels of CYP11B2 mRNA were lower (P<0.005) in normal adrenals than in APA. CYP17 mRNAlevels were similar in normal adrenals and in APA. In patients with APA, CYP11B2 and CYP11B1 mRNA levels were not correlated either with basal aldosterone or with the change from basal aldosterone in response to posture or to dexamethasone. No correlation between CYP11B1 mRNA or CYP11B2 mRNA and the percentage of zona fasciculata-like cells was observed in APA. CONCLUSIONS: Real-time RT-PCR can be reliably used to quantify CYP11B1 and CYP11B2 mRNA levels in adrenal tIssues. Expression of CYP11B1 in hyperfunctioning zona glomerulosa suggests an additional formation of corticosterone via 11beta-hydroxylase, providing further substrate for aldosterone biosynthesis. CYP11B1 and CYP11B2 mRNA levels in APA are not related to the in vivo secretory activity of glomerulosa cells, where post-transcriptional factors might ultimately regulate aldosterone production.

Clinical diagnosis of early dengue infection by novel one-step multiplex real-time RT-PCR targeting NS1 gene

2015 ◽  
Vol 65 ◽  
pp. 11-19 ◽  
Author(s):  
Je-Hyoung Kim ◽  
Chom-Kyu Chong ◽  
Mangalam Sinniah ◽  
Jeyaindran Sinnadurai ◽  
Hyun-Ok Song ◽  
...  
Keyword(s):  
Real Time ◽  
Clinical Diagnosis ◽  
Dengue Infection ◽  
Rt Pcr ◽  
One Step ◽  
Ns1 Gene ◽  
Pcr Targeting

scholarly journals Sensitive SYBR Green—Real Time PCR for the Detection and Quantitation of Avian Rotavirus A

2018 ◽  
Vol 6 (1) ◽  
pp. 2 ◽  
Author(s):  
David De la Torre ◽  
Claudete Astolfi-Ferreira ◽  
Ruy Chacon ◽  
Antonio Piantino Ferreira
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Sybr Green ◽  
Molecular Techniques ◽  
Economic Losses ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Rotavirus A ◽  
Avian Nephritis Virus ◽  
Polymerase Chain

Avian rotavirus A (ARtV-A) is a virus that affects young birds, causing acute diarrhea and economic losses in the poultry industry worldwide. The techniques used for the diagnosis of ARtV-A include electron microscopy, isolation in cell culture, and serology, as well as molecular techniques, such as the reverse transcription-polymerase chain reaction (RT-PCR). The objective of this work was to standardize a real-time RT-polymerase chain reaction (RT-qPCR) using SYBR Green chemistry for the rapid detection and quantification of ARtV-A from bird tissues and materials fixed on FTA cards on the basis of the nucleotide sequence of segment 6 (S6), which codes for the structural VP6 protein of ARtV-A. The results show the efficient amplification of the proposed target, with a limit of detection (LoD) of one copy gene (CG) per microliter of cDNA and a limit of quantification (LoQ) of 10 CGs per microliter. The efficiency of the primers was determined to be 95.66% using a standard curve, with an R2 value of 0.999 and a slope of −3.43. The specificity was determined using samples coinfected with ARtV-A, the chicken parvovirus, the chicken astrovirus, and the avian nephritis virus as positive controls and commercially available vaccines of the infectious bronchitis virus, infectious bursa disease virus, avian reovirus and healthy organs as negative controls. This technique, which lacks nonspecific PCR products and dimers, demonstrated greater sensitivity and specificity than conventional RT-PCR, and it reduced the analysis time by more than 50%.

scholarly journals A survey of deformed wing virus and acute bee paralysis virus in honey bee colonies from serbia using real-time RT-PCR

2014 ◽  
Vol 64 (1) ◽  
pp. 81-92 ◽  
Author(s):  
Predrag Simeunović ◽  
Jevrosima Stevanović ◽  
Dejan Vidanović ◽  
Jakov Nišavić ◽  
Dejan Radović ◽  
...  
Keyword(s):  
Real Time ◽  
Honey Bee ◽  
Sampling Location ◽  
Rt Pcr ◽  
Deformed Wing Virus ◽  
Bee Colonies ◽  
Acute Bee Paralysis Virus ◽  
Paralysis Virus ◽  
Bee Viruses

Abstract In this study 55 honey bee colonies from different Serbian regions were monitored for the presence of Deformed Wing Virus (DWV) and Acute Bee Paralysis Virus (ABPV) using TaqMan-based real-time RT-PCR assay. The results revealed the presence of DWV in each sampling location, and ABPV in 10 out of 11 apiaries. High frequency of DWV (76.4%) and ABPV (61.8%) positive samples in asymptomatic colonies can be the consequence of inefficient and postponed Varroa treatment concerning the role of this mite in the transmission and activation of honey bee viruses. The real-time RTPCR technique described in this paper is proved to be the most reliable method for this kind of investigation.

scholarly journals Lyophilized Matrix Containing Ready-to-Use Primers and Probe Solutions for Standardization of Real-Time PCR and RT-qPCR Diagnostics

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 7
Author(s):  
Remi N Charrel ◽  
Laurence Thirion
Keyword(s):  
Real Time ◽  
Molecular Techniques ◽  
Cold Chain ◽  
Rt Pcr ◽  
Emerging Viruses ◽  
Valley Fever ◽  
Term Stability ◽  
E Gene ◽  
Long Term Stability

Real-time molecular techniques have become the reference methods for the direct diagnosis of pathogens. The reduction of steps is a key factor in order to decrease the risk of human errors resulting in invalid series and delayed results. We describe here a process involving the preparation of oligonucleotide primers and a hydrolysis probe in a single tube at predefined optimized concentrations that are stabilized via lyophilization (Lyoph-P&P). Lyoph-P&P was compared to the classic protocol using extemporaneously prepared liquid reagents, assaying (i) sensitivity, (ii) long-term stability at 4 °C, and (iii) long-term stability at 37 °C, mimicking transportation without a cold chain. Two previously published molecular assays were selected for this study. They target two emerging viruses that are listed on the blueprint of the WHO to be considered for preparedness and response actions: chikungunya virus (CHIKV) and Rift Valley fever phlebovirus (RVFV). The results of our study demonstrate that (i) Lyoph-P&P is stable for at least four days at 37 °C, supporting shipping without the need of a cold chain, (ii) Lyoph-P&P rehydrated solution is stable at 4 °C for at least two weeks, (iii) the sensitivity observed with Lyoph-P&P is at least equal to, and often better than, that observed with liquid formulation, and (iv) the validation of results observed with low-copy specimens is rendered easier by higher fluorescence levels. In conclusion, Lyoph-P&P holds several advantages over extemporaneously prepared liquid formulations and merits consideration as a novel real-time molecular assay for implementation into a laboratory with routine diagnostic activity. Since the meeting, this concept has been applied to the COVID-19 situation: two diagnostic assays (E gene and RdRp) have been developed and can be ordered on the European Virus Archive catalog (https://www.european-virus-archive.com/detection-kit/lyophilized-primers-and-probe-rt-pcr-2019-ncov-e-gene; https://www.european-virus-archive.com/detection-kit/lyophilized-primers-and-probe-rt-pcr-sars-cov-2-rdrp-gene).

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