Nested PCR Method Research Articles

A one step nested PCR method for detection of Peronospora sparsa, the downy mildew pathogen, in boysenberry (Rubus ursinus)

Downy mildew is a major disease of boysenberries in New Zealand, caused by Peronospora sparsa. Most boysenberry plant material, including tissue culture propagated plants are systemically infected and this pathogen also presents as a latent infection. The current nested PCR method to detect latent infection of P. sparsa in asymptomatic boysenberry plants is time consuming as it employs two separate PCR, with potential contamination producing false positive and/or false negative results. To overcome these issues a one step nested PCR method was developed. The method was optimised for primer concentrations, PCR cycle number and DNA concentration. DNA was extracted using a CTAB method. The one step nested PCR method could detect latent infection of P. sparsa in both dormant and active plant growth at 0.4 pg genomic DNA. The most reliable detection was achieved from crown or root tissues. For surety of the infection status, replicate plant tissues should be assessed by PCR as inconsistency between the one step nested PCR and fluorescence microscopy indicated that P. sparsa colonisation is discontinuous through the plant. This method can be recommended for screening P. sparsa latent infection in boysenberry mother plants and daughter plants in nurseries, due to high sensitivity, improved throughput, low cost, and low contamination risk.

Identification of Fusobacterium nucleatum in primary and secondary endodontic infections and its association with clinical features by using two different methods.

Fusobacterium nucleatum is an important oral pathogen involved in endodontic infections. This study aimed to assess the frequency of Fusobacterium nucleatum in primary and secondary endodontic infections and its associations with the clinical features in a Brazilian population by using both culture and nested PCR methods. A total of 100 microbial samples from patients with primary (n=50) and secondary endodontic infections (n=50) were analyzed by using culture and nested PCR methods. Strict anaerobic techniques were used for culture and identification of F. nucleatum. The DNA extracted from the samples was analyzed for the presence of target species by using species-specific primers. Culture and nested PCR methods detected F. nucleatum, respectively, in 11/100 and 82/100 root canals. F. nucleatum was isolated by culture from 10/50 (20%) root canals with primary infections and from 1/50 (2%) root canal with secondary/persistent infections. Nested PCR detected F. nucleatum in 42/50 (84%) root canals with primary infections and in 40/50 (80%) root canals with secondary/persistent endodontic infections. F. nucleatum was associated with spontaneous pain, tenderness to percussion, pain on palpation, swelling, tooth mobility, wet root canals, hemorrhagic exudate, tooth decay, inadequate restoration, and poor endodontic filling. F. nucleatum was found in more cases of primary endodontic infections than in cases of secondary/persistent ones. A higher prevalence of F. nucleatum was detected by using the nested PCR method than by using culture. The presence of F. nucleatum in the root canals was associated with several clinical features. The high prevalence of F. nucleatum in the root canals detected by molecular methods, and its association with several clinical features reveals the importance of these species in the development of apical pathologies and reinforces the need of an endodontic treatment directed to bacterial elimination.

Detection of Mycobacterium bovis in environmental samples using nested PCR

The detection of Mycobacterium bovis (M. bovis) in environmental samples with precision is imperative to control bovine tuberculosis (bTB) infections at the herd level, as residual M. bovis remains one of the major causes of recurring infections. In this study, a nested PCR method for the detection of M. bovis in environmental samples was applied to identify potential environmental reservoirs of the bacterium. A set of 200 environmental samples (167 fecal samples and 33 water samples) from 39 herds with a history of bTB outbreak was analyzed using a nested PCR method to detect residual M. bovis. Amplicon libraries of the IS6110 target gene fragment were amplified from M. bovis DNA using two established primer sets. A positive nested PCR result was observed in 69.5% of fecal samples and 66.7% of water samples, thus showing that residual M. bovis was present in the environmental samples of bTB-positive herds in a high proportion. This study is the first to demonstrate high levels of M. bovis DNA in environmental samples and to show that environmental reservoirs of this pathogen contribute to recurring outbreaks of bTB. Environmental monitoring of herds in which bTB outbreaks have occurred with high sensitivity and specificity is expected to help prevent the recurrence of potential bTB disease and improve the herd environment.

ELISA versus Nested PCR for Detection of Hepatitis B Co-infection in HIV-infected People in Bandung Indonesia

The number of Human Immunodeficiency Virus infected patients in Bandung is increasing. HIV patients are often suffering from Hepatitis B Virusco-infection. The co-infection status must be detected using the accurate method. This study aims to compare the accuracy of Enzyme-Linked Immunosorbent Assay and nested Polymerase Chain Reaction in detecting HIV-HBV co-infection in Bandung. The research method used is experimental. The sample used in this study was 50 people. All samples had met the inclusion criteria. The presence of hepatitis B surface antigen in the serum was detected using a qualitative ELISA Wantai kit, while sHBsAg gene in the whole blood was amplified using 2 pairs of the specific primary. The results obtained from both methods were analyzed to determine sensitivity, specificity, and accuracy values. The result of Hepatitis B detection using ELISA method showed that 15 samples (30%) were positive, 1 sample (2%) was a false positive, and 3 samples (6%) were the false negative. Moreover, the nested PCR method showed that 18 samples (36%) were positive without false positive or false negative result. These results showed that ELISA has a sensitivity value of 83.33%, specificity of 96.87%, and the accuracy of 92%, while Nested PCR has a value of 100% for all three parameters. Therefore, it can be concluded that the Nested PCR method was more accurate than ELISA to detect Hepatitis B co-infection in HIV patients in Bandung.The Nested PCR can be recommended for detecting the other co-infection cases.
 
 Keywords: accuracy, HBsAg, co-infection, sensitivity, specificity

Seasonal dynamics of Anaplasma spp. in goats in warm-temperate zone of China

Anaplasma are tick-borne obligate intracellular bacteria that can endanger human and animal health, and until now, there have been few reports on the seasonal dynamics of Anaplasma species in China. In this study, a total of 491 goat blood samples were collected in spring (n = 124), summer (n = 135), autumn (n = 110), and winter (n = 122) from Shaanxi provinces. Single and mixed infections of Anaplasma spp. from warm-temperate regions of China were analyzed according to seasons using a nested PCR method. Positive samples were sequenced to observe the molecular and phylogenetic characteristics of the Anaplasma species, and we determined the co-infection rates of Anaplasma spp. for each season. A molecular survey of Anaplasma phagocytophilum, A. bovis, A. ovis, and A. capra in goats showed average prevalences of 71.6 % (maximum 86.7 % in summer and minimum 48.4 % in winter), 62.2 % (minimum 38.7 % in spring and maximum 94.1 % in summer), 25.5 % (minimum 0% in summer and maximum 51.6 % in spring), and 26.6 % (minimum 8.2 % in winter and maximum 55.6 % in summer), respectively. In the phylogenetic analysis, A. phagocytophilum and A. capra occupied two separate groups, Chinese A. bovis and foreign isolates appeared to be geographically isolated, and all A. ovis isolates were in the same branch as the previously described sequences. The survey indicated that goats in warm-temperate regions of China are frequently exposed to Anaplasma spp. all year round, and thus prevention and treatment efforts for anaplasmosis in the region should be strengthened.

DETECTION AND IDENTIFICATION OF YELLOW MOSAIC STUNT DISEASE ON Petunia sp. USING NESTED PCR METHOD

Detection and identification of yellow mosaic stunt disease on Petunia sp. using nested PCR method. Yellow mosaic stuntdisease was found at some nurseries of Petunia in Sleman, Yogyakarta, also in Muntilan and Magelang Central Java. Thedisease was very important due to its ability reducing the quality and quantity of Petunia seedlings. The causal agent of thedisease may be carried over to imported seeds and necessary to identify as a basic information for developing controlstrategies. This research was done by mechanical transmission on indicator plants. The observation of the causal agents wasconducted using electron microscope with quick dipping method and the molecular detection was done using nested PCRwith TobRT up1-TobRT do2 as the external primers and TobN up3-TobN do4 as the internal primers. Mechanical inoculationshowed chlorosis symptoms that developed into local spot on Chenopodium amaranticolor as well as mosaic and veinbanding on Nicotiana benthamiana. The observation using electron microscope showed rod-shaped virus particles sizedapproximately 300 nm and by PCR method produced around 568 bp and 400 bp DNA band. Based on the sequence analysis,the disease was caused by Rehmania mosaic virus. This type of Tobamovirus has 96% similarity with ReMV-Japan. ReMV, aplant pathogen which was a member of Tobamovirus that has never been reported in Indonesia. This research was the firstreport of ReMV in Indonesia infecting Petunia as ornamental plant.

Prevalence and phylogenetic analysis of HTLV-1 in blood donors in Golestan Province, in the Northeast of Iran

The human T-lymphotropic virus type 1 (HTLV-1) can cause ATL or TSP. This study evaluates the prevalence of HTLV-1 infection in blood donors in Golestan province. The study was conducted among 4226 blood donors and ELISA test was performed for the initial HTLV-1 screening. Reactive samples were confirmed by Western blot and Electrochemiluminescence tests. Then recalling donors with reactive results was done and genomic DNA from the new sample was extracted and tested using the Nested PCR method and phylogenetic analysis was performed. At first, 8 samples were reactive with ELISA test and 4 samples were confirmed with western blot, Electrochemiluminescence and Nested PCR tests.The sequences of isolates was related to the HTLV-1 virus and subtype a (cosmopolitan) subgroup A.The prevalence of HTLV-1 virus in Golestan province was about 0.09 %.The genotype of virus isolates had a common ancestor with isolates of the Khorasan region.

Identification of bovine leukemia virus in raw milk samples in North-West of Iran.

Bovine leukemia virus (BLV) is one of the most important carcinogenic viruses genetically related to the human T-cell lymphotropic viruses (HTLV-1 and HTLV-2). The virus infects type B lymphocytes and creates lymph glands tumors. Recently, the association between the presence of this virus and breast cancer has been addressed in humans. Here, we studied the prevalence of BLV in the samples of raw milk of native Iranian and Iranian-foreign cows in traditional, semi-industrial and industrial dairy farms in rural and urban areas of Zanjan province. Raw milk samples of cows were collected manually in sterile tubes. The samples were tested by nested-PCR method. Forty samples (9.93%) out of 403 samples showed BLV contamination. In this study, nested-PCR was successfully applied to determine the level of contamination in raw milk samples from cows infected with BLV. Furthermore, a relatively high rate of BLV infection was found in dairy cows in Zanjan province, northwestern of Iran.

Species-level identification of trypanosomes infecting Australian wildlife by High-Resolution Melting - Real Time Quantitative Polymerase Chain Reaction (HRM-qPCR)

Conventional nested PCR and Sanger sequencing methods are currently the gold standards for detecting trypanosomes in wildlife. However, these techniques are time-consuming and can often overlook mixed infections. True trypanosome prevalence can thus be underrepresented. Here, we designed an 18S rDNA-based real-time quantitative PCR (qPCR) assay coupled with High-Resolution Melting Analysis (HRMA) to detect and discriminate three Trypanosoma species (T. copemani, T. noyesi, and T. vegrandis) commonly infecting Australian marsupials. A total of 68 genetically characterised samples from blood and tissue were used to validate the High-Resolution Melting - Real Time Quantitative Polymerase Chain Reaction (HRM-qPCR) assay. A further 87 marsupial samples consisting of blood, tissue and in vitro cultures derived from wildlife blood samples, were screened for the first time using this assay, and species identity confirmed using conventional PCR and Sanger sequencing. All three Trypanosoma species were successfully detected in pure cultures using the HRM-qPCR assay, and in samples containing mixed trypanosome infections. Of the 87 marsupial samples screened using the HRM-qPCR assay, 93.1% were positive for trypanosomes, and 8.0% contained more than one trypanosome species. In addition to the three targeted Trypanosoma species, this assay was also able to detect and identify other native and exotic trypanosomes. The turnaround time for this assay, from sample preparation to obtaining results, was less than 2 h, with a detection limit of 10 copies of the amplicon in a reaction for each of the targeted trypanosome species. This more rapid and sensitive diagnostic tool provides a high throughput platform for the detection, identification and quantification of trypanosome infections. It will also improve understanding of host diversity and parasite relationships and facilitate conservation management decisions.

One-Tube Nested Real-Time PCR Assay for Rapid Screening of Porcine Cytomegalovirus in Clinical Samples.

Porcine cytomegalovirus (PCMV) is a pathogen that must be removed from pigs for use as organ donors in xenotransplantation. Recently, it has been found that when donor pigs are infected with PCMV, a pig-to-non-human-primate xenotransplantation lower transplant survival by 2–3 times. Therefore, highly sensitive methods are needed to maintain designated pathogen free (DPF) pig and screen for xenografts. The purpose of this study was to evaluate the performance of commercially available method with one-tube nested real-time PCR assay to quickly detect PCMV infection in clinical samples and compare the results with those of sequence analysis. Molecular diagnostic methods were used to evaluate 127 samples, including tissues and blood samples from pigs suspected of PCMV infection. The detection rate for positive PCMV was 38.6% (n = 49), 23.6% (n = 30), and 12.6% (n = 16) in one-tube nested real-time PCR, nested PCR, and conventional PCR methods, respectively. All PCMV-positive samples in conventional PCR or nested PCR methods were also positive in the one-tube nested real-time PCR assay. All the PCR products in the three methods were checked for amplification of PCMV gene by PCR and subsequent direct sequencing. The results of one-tube nested real-time PCR were found to be consistent with those of sequence analysis for all the samples and showed good agreement (κ = 1). Our study found that the one-tube nested real-time PCR assay is more sensitive than the other two methods. This assay required approximately 1.5 h for completion. Therefore, we concluded that one-tube nested real-time PCR assay is a fast and reliable method for the characterizing pathogen responsible for PCMV infection.

Molecular Monitoring and Phylogenetic Analysis of Betanodavirus in Groupers (Epinephelus spp.) and Asian Sea bass (Lates calcarifer) of Iranian Northern Waters of the Persian Gulf.

The emergence of diseases has caused much health and economic damage. Viral Nervous Necrosis (VNN) is considered as one of the most important threats to aquatic ecosystems. VNN can cause severe mortality and economic loss in fish farms. The high water temperatures in southern Iran and the observed incidences of fish mortality in the Persian Gulf led to the hypothesis of the possible emergence of VNN. Therefore, this study aimed to monitor two species of fish susceptible to VNN using PCR, and Nested PCR methods and comparing the sensitivity of these methods to the identification of Betanodavirus infection in apparently healthy and symptomatic fish. About 850 Grouper (Epinephelus spp.) and Asian Sea bass (Lates calcarifer) fish of the Persian Gulf were collected randomly and examined. Molecular methods were used to identify NNV in visibly healthy and symptomatic fish of the Persian Gulf of Iran. The results of the PCR showed no positive cases, but the Nested PCR revealed some positive results. Then, the phylogenetic analysis of the virus sequence was performed. The nucleotide sequence of Nested PCR products revealed a 98-100% homology with Red Spotted Grouper Viral Nervous Necrosis (RGNNV). This is the first report on VNN tracing and detection as well as phylogenetic analysis of the virus from the Persian Gulf of Iran. Therefore, considering the importance of emerging viral diseases and the irreparable damage they cause, continuous monitoring and epidemiological studies of VNN were recommended by authorized organizations.

A phytoplasma survey reveals the presence of ‘CandidatusPhytoplasma fragariae’ inUlmusspp. andAcer pseudoplatanusin Belgium

AbstractA Belgium‐wide survey to determine the presence of ‘CandidatusPhytoplasma ulmi’ elm trees was performed on nearly 600 elm trees. The randomly sampled elms were screened for the presence of phytoplasmas using a standard generic nested PCR method. In five symptomless elm trees, phytoplasmas were detected. Multiple gene sequence analysis (16S rRNA, tuf, rplV‐rpsC‐rplP), virtual RFLP of the 16S rRNA gene fragment and phylogenetic analysis showed that the phytoplasma strains detected in all five elm trees could be classified into the 16SrXII‐E subgroup (‘CandidatusPhytoplasma fragariae’‐related phytoplasma). In addition, a maple tree (Acer pseudoplatanus) located in close proximity to one of these elm trees was also determined to be infected with ‘Ca. P. fragariae’.UlmusandAcerare new host plant genera for this phytoplasma. Further studies are needed to elucidate the epidemiological impact of these findings.

Representative Genotyping, Recombination and Evolutionary Dynamics Analysis of TSA56 Gene Segment of Orientia tsutsugamushi.

Scrub typhus is a zoonotic disease caused by Orientia tsutsugamushi (O. tsutsugamushi). Orientia tsutsugamushi has various genotypes and more new strains with difference in sequences increasingly appeared. Whether the accurateness of one special nested PCR method which amplifies segment instead of entire open reading frame (ORF) sequence meets the current work of identifying new strains and classifying genotypes remains to be confirmed. And the origins and evolution of this organism have not been thoroughly elucidated. Accordingly, in this study, segments and the entire ORF of the 56-kDa type-specific antigen (TSA56) gene of O. tsutsugamushi were collected, including 209 clinically isolated strains in Guangzhou, China from 2012 to 2016 and 139 reference strains worldwide. By performing phylogenetic analysis, we proved that the accurateness of the particular PCR method which almost met detection need. This re-grouping result showed that segments perfectly represented and identified strains of Karp, Boryong, Gilliam, TA763, Kawasaki and part of Kato genotype, and this accuracy is not restricted by region and time. Sequence diversification of Shimokoshi and some Kato strains made their genotyping need to consider entire ORF sequences, but their weak recognition might not be due to recombination. The frequent genetic recombination and high point mutations contributed to genetic diversification of the TSA56 gene. Major overlapping regions of most recombination events occurred between strains of the same genotype, especially Karp and Kato genotype. And cross-genotype overlapping events occurred between Karp and Boryong/Gilliam/TA763/Kato, Kato and Kawasaki/Gilliam/TA686, Boryong and TA686, and Gilliam and Kawasaki. But Segment has quite low recombination frequency and stable mutation trend from 1943 to 2016. So segment is a relatively conserved part of the TSA56 ORF as for its stable trend of genetic diversity, and it may anchor and represent the entire TSA56 ORF gene. And genetic diversity is rejected as one potential reason for the increased incidence of scrub typhus. But an occasional recombination event created an unrecognized genotype which might be due to the breakage of VD II and AD II. Additionally, strains in Guangzhou were homologous and Karp genotype was detected as a dominant.

Evaluation of the expression level of Sine oculis homeobox homolog 1 in cervical cancer tissue in comparison with healthy adjacent tissue

Background and Aim: This study aimed to investigate the expression level of Sine oculis homeobox homolog 1(SIX1) in normal and cancerous cervical tissues. Methods: Real-time PCR was used to evaluate SIX1 expression level in fresh surgically obtained cervical biopsies. Also, cervical specimens were tested for HPV DNA detection by MY09/11, GP5+/6+ nested PCR method. Results: The expression level of SIX1 mRNA in Cervical cancer was markedly higher than in normal cervical tissue (p<0.0001). Moreover, HPV DNA was detected in 100% of cases and 21.05% of controls (P<0.0001). Conclusions: This finding indicates that increased SIX1 expression might be of relevance to the pathogenesis of cervical cancer. Perhaps, studies to find agents that specifically target SIX1, develop novel anti-cancer and anti-metastasis drugs. This idea has to be investigated in future clinical studies.

Development and application of a nested polymerase chain reaction method for the detection of genetically modified soybean in Chinese traditional fermented soy food-sufu

A nested PCR method has been developed to distinguish traditional soybean from transgenic Roundup Ready soybeans (RRS). Comparing with the standard PCR method, the nested PCR method increases the sensitivity by 200 to 1000-fold. One copy of RRS genome can be detected by the nested PCR. Sufu, a traditional fermented soybean curd, is a very important Chinese condiment and it is a highly processed food. We have used the nested PCR to detect seven time points of sufu throughout the aging period. The nested PCR method showed positive results for sufu made up of 100% RRS at all seven time points from 0th day to 180th day. This work describes the development and application of a sensitive and specific detection method for sufu made from RRS using a nested PCR system.

Investigation of hemotropic Mycoplasma spp. genotypes in client-owned cats in Thailand

The genetic information for three feline hemoplasmas is limited in Southeast Asia. According to the limited genetic data, this study modified a nested-PCR method targeting the 16S rRNA gene by designing a novel primary forward degenerate primer. Two hundred and thirty-one archived DNA extracts from the blood of client-owned cats with a variety of diseases were used. The modified nested PCR detected feline hemoplasma DNA in 64 of 231 (27.7 %) samples. Sanger DNA sequencing, BLAST, and phylogenetic analyses revealed nine nucleotide sequences of Mycoplasma haemofelis (Mhf) (3.9 %, 9/231), fifty-three nucleotide sequences of Candidatus Mycoplasma haemominutum (CMhm) (22.94 %, 53/231) and two nucleotide sequences of Candidatus Mycoplasma turicensis (CMtc) (0.86 %, 2/231). The phylogenetic analysis demonstrated separate genotypes of 30 DNA sequences of Thai CMhm. In addition, this analysis elucidated distinct genotypes of CMhm in Thai fishing cats (Prionailurus viverrinus). The domestic cat and Thai fishing cat groups were the two major groups separating Thai CMhm genotypes based on the 16S rRNA. One CMhm sequence in Thai fishing cats was also present in domestic cat CMhm genotypes. This result suggests that transmission of CMhm between domestic cats and Thai fishing cats has likely occurred. One Mhf sequence had low genetic identity (82 % similarity). The phylogenetic analysis confirmed that this sequence was still very closely related to Mhf reference sequences. This Mhf-like genotype could be a candidate novel Mhf genotype. This new genetic information for feline hemotropic Mycoplasma provides valuable information for future feline-related clinical studies.

Detection Cell-Free DNA (cfDNA) Using Nested-PCR as a Diagnosis Tool for Human Fascioliasis Infection

Background: We aimed to detect Fasciola specific deoxyribonucleic acid (DNA) by nested-PCR assay on human stool and urine samples and compare the results with the respective ELISA diagnostic assay. Methods: Overall, 206 clinically suspected cases of fascioliasis were enrolled in the study. Blood samples were collected from all the patients, and serum samples were isolated. ELISA assay, using Fasciola somatic antigen (SA), was carried out to detect anti Fasciola antibodies for the collected sera. DNA was randomly extracted from 25 stool and 10 urine samples of seropositive individuals and was evaluated by conventional PCR and nested PCR methods. The nested-PCR results were confirmed by sequencing the 430 bp region of ribosomal ITSI gene. Stool and urine samples from patients with different parasitic diseases and 25 stool samples from healthy individuals served as controls. Urine samples were collected from 10 healthy controls as well. Results: Fascioliasis was detected by ELISA in 24.8% of the individuals. Of these, 25 seropositive patients were randomly assigned to the study. Fasciola DNA was identified in the stool samples of 96% of seropositive patients by nested PCR but ova of Fasciola was detected by parasitology methods in only 20% of seropositive cases. Fasciola DNA was identified in 90% of the urine samples by nested PCR. No cross-reactions were observed with other parasites. Conclusion: Detection of cfDNA in stool and urine samples has high accuracy and thus can be used for the diagnosis of Fasciola infection in human.

Are antimicrobial resistance genes key targets to detect genetically modified microorganisms in fermentation products?

As genetically modified microorganisms (GMM), commonly used by the food and feed industry to produce additives, enzymes and flavourings, are frequently harbouring antimicrobial resistance (AMR) genes as selection markers, health and environmental concerns were consequently raised. For this reason, the interest of the competent authorities to control such microbial fermentation products has strongly increased, especially since several recent accidental contaminations of unauthorized GMM, or associated recombinant DNA, in bacterial fermentation products intended for the European food and feed chain. However, no global screening strategy is currently available in enforcement laboratories to assess the presence of GMM harbouring AMR genes and/or the presence of full-length AMR genes. Moreover, the confidentiality of the related GMM dossiers strongly hampers the development of methods to perform such control. To overcome this issue, an analysis of related publicly available patents was performed in this study to identify all reported AMR genes. On this basis, the aminoglycoside adenyltransferase (aadD) gene, conferring a resistance to both kanamycin and neomycin, was identified as a key target to cover a large spectrum of GM bacteria. A real-time PCR method to screen for its potential presence as well as a nested-PCR method associated with a sequencing analysis to assess its full-length were developed to target this aadD gene. The performance of these new methods were successfully evaluated in terms of specificity, sensitivity and applicability, allowing their easy implementation in enforcement laboratories. Moreover, the integration of these newly developed methods to our very recently proposed strategy, initially targeting GMM carrying a chloramphenicol resistance gene, allows to drastically increase the detection spectrum of GM bacteria producing fermentation food and feed products. The data generated by the proposed strategy represents therefore a crucial support for the competent authorities, especially to evaluate potential risks for the food and feed safety.

Prevalence of C. burnetii DNA in sheep and goats milk in the northwest of Iran

Q fever is a common zoonotic disease with worldwide distribution. The causative agent of Q fever is Coxiella burnetii, a gram-negative and polymorphic rod bacterium. Sheep and goats are the primary reservoirs of this disease, although a variety of animal species can be infected. The main route of Q fever transmission from animals to humans is the inhalation of contaminated aerosols with C. burnetii. The bacterium is excreted in milk of infected animals and therefore; the consumption of unpasteurized milk and dairy products might be a route of coxiella burnetii transmission from animals to humans. The present study was conducted to determine the prevalence of C. burnetii in milk samples collected from sheep and goats in west Azerbaijan province, Iran. During 2018, a total number of 420 milk samples were collected from sheep (n = 210) and goats (n = 210) of different regions of the province. All milk samples were subjected to DNA extraction and examined by a highly and specific nested-PCR method. The results showed that 51 (12.1%) (95% CI: 9.3%–15.6%) examined samples [sheep; n = 16 (7.6%) and goat; n = 35 (16.6%)] were positive for C. burnetii. The prevalence of C. burnetii in goat milk samples was significantly higher than sheep milk samples (P < 0.05). The shedding of C. burnetii in milk was significantly higher in summer (25%) (P < 0.05, 95% CI: 17.7%–34%) than the other seasons. It was concluded that sheep and goat populations in west Azerbaijan play an important role in the epidemiology of Q fever.

Prevalence of JC and BK viruses in Patients with Colorectal Cancer: A Systematic Review and Meta- Analysis.

Introduction:Polyomaviruses including BK virus (BKV) and JC virus (JCV) are widespread in human and have been associated with colorectal cancer (CRC) in some studies. The aim of present systematic review and meta-analysis article is to calculate the pooled prevalence of BKV and JCV in patients with CRC and assessing their association with this malignancy. Materials and Methods:Domestic databases and Sciences Direct, PubMed, ProQuest, Web of Sciences and Scopus were searched for relevant articles up to 2nd June 2019Two independent reviewers extracted the related data from eligible articles. The pooled prevalence and pooled odds ratio (POR) and their 95% confidence interval (95% CI) were calculated using “metaprop” and “metan” commands in Stata 14. Where I2 statistics were >50%, the random effect model was used. Results:From 1461 relevant studies, 24 articles were eligible and included in the qualitative while 19 articles included in quantitative analysis. The pooled prevalence based on diagnostic methods varies from 29% using immunohistochemistry to 52% using nested-PCR method. The likelihood of being infected with JCV was significantly higher in CRC patients compared to healthy (POR: 4.41, 95% CI: 2.13 – 9.13) controls, normal adjacent mucosa (POR: 2.79, 95% CI: 1.3-5.9) and colorectal adenoma (POR: 3.1, 95% CI: 1.5-6.5) but was not significant when non-CRC patients used as control group. Conclusion:The prevalence of JCV in colorectal patients was substantially variable by different methods and targets. The significant association between JCV and CRC that was observed in the present study is not indicative of causation and should be studied more in large-scale prospective designs.