scholarly journals First report and molecular characterization of Cryptosporidium spp. in humans and animals in Khartoum state, Sudan

2019 ◽  
Vol 12 (1) ◽  
pp. 183-189 ◽  
Author(s):  
Kaltoum Yagoub Adam ◽  
A. A. Ismail ◽  
M. A. Masri ◽  
A. A. Gameel
Keyword(s):  
Nested Pcr ◽  
Mammalian Species ◽  
Pcr Amplification ◽  
Health Concern ◽  
Economic Losses ◽  
Staining Procedure ◽  
Rrna Gene ◽  
Nested Polymerase Chain Reaction ◽  
Cryptosporidium Spp ◽  
Pcr Targeting

Background and Aim: Cryptosporidium is recognized to infect several mammalian species as well as humans, causing substantial economic losses and serious public health concern. Infected animals can be a source of environmental contamination and human infections. In general, the occurrence of Cryptosporidium species in animals and human in Sudan and zoonotic importance is not well documented. This study aimed to identify Cryptosporidium spp. infecting different animal species and humans and to compare between different isolates obtained. Materials and Methods: To provide molecular information about Cryptosporidium in animals and humans, both modified Ziehl-Neelsen (MZN) specific stain and molecular assay were used. Concentration techniques followed by three protocols of DNA extraction were carried out. After microscopic screening of 263 fecal samples (goats [n=197], cattle [n=12], sheep [n=12], and human [n=42]), 61 positive and 30 negative, randomly selected samples were used in nested polymerase chain reaction (PCR) targeting part of the 18S RNA. Results: Nested PCR amplification confirmed 91.8% (56/61) of microscopic-positive samples. 8.2% (5/61) of negative samples by PCR (positive by microscopy) were considered false negatives. Sequencing followed by alignment of the 14 isolates indicated that all samples were identical (100%) and belonged to Cryptosporidium parvum. Conclusion: MZN staining procedure is reliable for the routine diagnosis of Cryptosporidium; cetyltrimethylammonium bromide extraction buffer and nested PCR targeting 18S rRNA gene are reliable and useful in epidemiological studies of this parasite.

scholarly journals Molecular Characterization ofCryptosporidiumspp. in Wild Rodents of Southwestern Iran Using 18s rRNA Gene Nested-PCR-RFLP and Sequencing Techniques

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Jasem Saki ◽  
Masoud Foroutan-Rad ◽  
Reza Asadpouri
Keyword(s):  
Molecular Characterization ◽  
Nested Pcr ◽  
18S Rrna ◽  
Restriction Enzymes ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Wild Rodents ◽  
Nested Polymerase Chain Reaction ◽  
Pcr Rflp ◽  
Southwest Of Iran

Background. Rodents could act as reservoir forCryptosporidiumspp. speciallyC. parvum, a zoonotic agent responsible for human infections. Since there is no information aboutCryptosporidiuminfection in rodents of Ahvaz city, southwest of Iran, hence, this survey was performed to determine the prevalence and molecular characterization ofCryptosporidiumspp. in this region.Materials and Methods. One hundred rodents were trapped from different regions of Ahvaz city. Intestine contents and fecal specimens of rodents were studied using both microscopy examination to identify oocyst and nested-polymerase chain reaction (PCR) technique for 18s rRNA gene detection. Eventually restriction fragment length polymorphism (RFLP) method usingSspIandVspIrestriction enzymes was carried out to genotype the species and then obtained results were sequenced.Results. Three out of 100 samples were diagnosed as positive and overall prevalence ofCryptosporidiumspp. was 3% using both modified Ziehl-Neelsen staining under light microscope and nested-PCR (830 bp) methods. Afterwards, PCR-RFLP was performed on positive samples andC. parvumpattern was identified. Finally PCR-RFLP findings were sequenced and presence ofC. parvumwas confirmed again.Conclusions. Our study showed rodents could be potential reservoir forC. parvum. So an integrated program for control and combat with them should be adopted and continued.

scholarly journals Detection of Pneumocystis in lungs of bats from Brazil by PCR amplification

2009 ◽  
Vol 29 (6) ◽  
pp. 469-473 ◽  
Author(s):  
Edna Maria Cavallini Sanches ◽  
Susi M. Pacheco ◽  
Alison S. Cericatto ◽  
Rosane M. Melo ◽  
Edson Molleta Colodel ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Urban Areas ◽  
Pcr Amplification ◽  
Small Subunit ◽  
Rrna Gene ◽  
Wild Animals ◽  
Tadarida Brasiliensis ◽  
Desmodus Rotundus ◽  
Mato Grosso ◽  
Wide Range

Pneumocystis has been isolated from a wide range of unrelated mammalian hosts, including humans, domestic and wild animals. It has been demonstrated that the genome of Pneumocystis of one host differs markedly from that of other hosts. Also, variation in the chromosome and DNA sequence of Pneumocystis within a single host species has been observed. Since information about the occurrence and nature of infections in wild animals is still limited, the objective of this work was to detect the presence of Pneumocystis sp. in lungs of bats from two states from Brazil by Nested-PCR amplification. The bats, captured in caves and in urban areas, were obtained from the Program of Rabies Control of two States in Brazil, Mato Grosso and Rio Grande do Sul, located in the Mid-Western and Southern regions of the country, respectively. DNAs were extracted from 102 lung tissues and screened for Pneumocystis by nested PCR at the mtLSU rRNA gene and small subunit of mitochondrial ribosomal RNA (mtSSU rRNA). Gene amplification was performed using the mtLSU rRNA, the primer set pAZ102H - pAZ102E and pAZ102X - pAZY, and the mtSSU rRNA primer set pAZ102 10FRI - pAZ102 10R-RI and pAZ102 13RI - pAZ102 14RI. The most frequent bats were Tadarida brasiliensis (25), Desmodus rotundus (20), and Nyctinomops laticaudatus (19). Pneumocystis was more prevalent in the species Nyctinomops laticaudatus (26.3% = 5/19), Tadarida brasiliensis (24% = 6/25), and Desmodus rotundus (20% = 4/20). Besides these species, Pneumocystis also was detected in lungs from Molossus molossus (1/11, 9.1%), Artibeus fimbriatus (1/1, 100%), Sturnira lilium (1/3, 33.3%), Myotis levis (2/3, 66.7%)and Diphylla ecaudata (1/2, 50%). PCR products which could indicate the presence of Pneumocystis (21.56%) were identified in DNA samples obtained from 8 out of 16 classified species from both states (5 bats were not identified). This is the first report of detection of Pneumocystis in bats from Brazil.

scholarly journals Molecular characterization of Cryptosporidium spp. in sheep and goat in Al-Qadisiyah province/ Iraq

2018 ◽  
Vol 41 (2) ◽  
pp. 31-37
Author(s):  
Mansoor Jadaan Ali Alkhaled
Keyword(s):  
Dna Sequencing ◽  
Phylogenetic Tree ◽  
Ribosomal Rna ◽  
18S Rrna ◽  
Molecular Technique ◽  
Rrna Gene ◽  
Analysis Method ◽  
Nested Polymerase Chain Reaction ◽  
Ribosomal Rna Gene ◽  
Cryptosporidium Spp

     The present study was conducted during the period from September 2015 until February 2016. 100 fecal samples were collected from 60 sheep and 40 goats for diagnosis of Cryptosporidium parasite from diverse areas in Al-Qadisiyah province. The study amid to know the genetic characters of Cryptosporidium spp. parasite by using a molecular technique such as the nested polymerase chain reaction and DNA sequencing analyzed by phylogenetic tree to identify the parasite species. This study was done on the sheep and goat at first time in the middle region of Iraq and the identified species were recorded in NCBI-Genbank database. In sheep, the results of positive infected samples was (40%) while, in the goats were (32.5%), the DNA sequencing and phylogenetic analysis method based on small ribosomal RNA gene (18s rRNA) for Cryptosporidium species typing. The results were conducted by Neighbor-Joining phylogenetic tree analysis method and the 18s rRNA gene sequences were confirmed by using NCBI-BLAST data analysis in order to compare with NCBI submitted selected references isolates of (18s ribosomal RNA) gene in Cryptosporidium spp. parasites. Our finding in present study appeared to follow spp. (C. parvum, C. hominis, C. andersoni, C. ubiquitum, C. xiaio and C. suis). These identified species which primary affected sheep and goats as mentioned in previous studies when compared with newly Iraq isolates strains.

scholarly journals Molecular survey ofBartonella henselaeandBartonella clarridgeiaein pet cats across Japan by species-specific nested-PCR

2017 ◽  
Vol 145 (13) ◽  
pp. 2694-2700 ◽  
Author(s):  
S. SATO ◽  
H. KABEYA ◽  
A. NEGISHI ◽  
H. TSUJIMOTO ◽  
K. NISHIGAKI ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Bartonella Henselae ◽  
23S Rrna ◽  
Cat Scratch Disease ◽  
Internal Transcribed Spacer Region ◽  
Nested Polymerase Chain Reaction ◽  
Polymerase Chain ◽  
Species Specific ◽  
Pcr Targeting ◽  
Eastern Japan

SUMMARYCats are known to be the main reservoir forBartonella henselaeandBartonella clarridgeiae, which are the agents of ‘cat-scratch disease’ in humans. In the present study, we investigated the prevalence of the twoBartonellaspecies on 1754 cat bloods collected from all prefectures in Japan during 2007–2008 by a nested-polymerase chain reaction (PCR) targeting the 16S–23S rRNA internal transcribed spacer region. Overall,BartonellaDNA was detected in 4·6% (80/1754) of the cats examined. The nested-PCR showed that 48·8% (39/80) of the positive cats were infected withB. henselaemono-infection, 33·8% (27/80) withB. clarridgeiaemono-infection and 17·5% (14/80) were infected with both species. The prevalence (5·9%; 65/1103) ofBartonellainfection in the western part of Japan was significantly higher than that (2·3%; 15/651) of eastern Japan (P< 0·001). Statistical analysis of the cats examined suggested a significant association betweenBartonellainfection and FeLV infection (OR = 1·9; 95% CI = 1·1–3·4), but not with FIV infection (OR = 1·6; 95% CI = 1·0–2·6).

scholarly journals Molecular Epidemiology of Bovine Babesiosis in Punjab, Pakistan

2021 ◽  
Vol 49 ◽  
Author(s):  
Asif Masih ◽  
Azhar Rafique ◽  
Farhat Jabeen ◽  
Shabana Naz
Keyword(s):  
Risk Factors ◽  
Nested Pcr ◽  
Dairy Industry ◽  
Summer Season ◽  
Babesia Bovis ◽  
Economic Losses ◽  
Molecular Evidence ◽  
Conventional Pcr ◽  
Associated Risk Factors ◽  
Pcr Targeting

Background: Babesiosis is endemic in Pakistan and is one of the most important bovine diseases that causes huge economic losses and high mortality in young animals. This disease is transmitted by a protozoan parasite babesia which belongs to genus Babesia (Apicomplexa: Piroplasmida: Babesiidae). This disease is very much prevalent in summers followed by rainy season because humid environment is favorable for the growth of these parasites. An epidemiological and molecular study was conducted to unveil the prevalence and associated risk factors of Babesia bigemina (B. bigemina) and Babesia bovis (B. bovis) in selected districts i.e., Faisalabad, Toba Tek Singh and Jhang of Punjab, Pakistan.Materials, Methods & Results: A total of 518 (Cattle = 360, Buffalo = 158) blood samples were collected. The samples were analyzed by polymerase chain reaction (PCR) and nested PCR (n-PCR) targeting apocytochrome b-genes (CYTb). Chi-square test for univariate analysis was used to analyze the data. The overall prevalence in summer based upon microscopic analysis was 20.55% (37/180) and 13.92% (11/79) in cattle and buffaloes respectively and in winter was 8.80% (16/180), 5.06% (4/79)) in cattle and buffaloes respectively. The samples were further analyzed through conventional PCR (c-PCR) and nested PCR (nPCR). The overall results of conventional PCR in summer showed that 72 cows and buffaloes were infected with babesiosis. The conventional PCR based results of summer showed that prevalence of babesiosis was 29.44% (53/180) in cows and 24.05% (19/79) buffaloes. The results of cPCR during the winter season showed that 12.77% (23/180) and 13.92% (11/79) buffaloes were positive for babesiosis. The overall results of conventional PCR in winter showed that 34/259 cows and buffaloes were infected with babesiosis. On the other hand, the nested PCR results of summer season showed that the prevalence of babesiosis in cows was 32.22% (58/180) and 29.11% (23/79) in buffaloes. In total, 81 cows and buffaloes were infected with babesiosis during summer season. The nPCR results of winter showed that 15% (27/180) cows and 20.25% (16/79) buffaloes were infected with babesiosis. In total, 43 cows and buffaloes were infected with babesiosis. The results have shown that sensitivity of n-PCR is more as compared to conventional PCR. This study is the first molecular evidence of B. bigemina and B. bovis and its associated risk factors in Punjab province, Pakistan.Discussion: Dairy sector in Pakistan is one of the fastest growing sectors. Despite of remarkable growth, dairy industry is facing many problems one of them is tick borne diseases (TBDs). TBDs are more prevalent in tropical and subtropical areas of the world and leads to huge economic losses to dairy industry in terms of decreased milk, meat and wool production. Babesiosis is characterized by increased fever, decreased production, poor quality wool, anemia, hemoglobinuria, paleness of mucous membrane. The risk factors analysis of summer and winter data revealed that, adult animals were more prone to babesiosis (24.00%) [P = 0.032] and (8.50%) [P = 0.048]. In both seasons (summer and winter), females were more infected with babesiosis (20.19% and 8.17%) [P = 0.049 and P =0.021] as compared to males, high prevalence in females was might be due to that females were reared for longer period of time. Babesiosis was more occurred in non-cemented floor system (26.01% and 13.51%) [P = 0.028 and P = 0.044] in summer and winter, respectively. Disease was found more prevalent in closed housing system in summer and winter (27.27% and 10.93%) [P = 0.043 and P = 0.034] as compared to open housing. Weak animals were more infected with babesiosis (30.84%) [P = 0.045] and (12.80%) [P = 0.042] in summer and winter, as compared to healthy ones. The animals with high tick infestations were more suffered with babesia infection (25.49% and 13.34%) [P = 0.036 and P = 0.003] in both seasons as compared to less tick burden. Keywords: apocytochrome gene, babesiosis, bovine, nPCR, PCR, season.

scholarly journals Frequency and Molecular Identification of Cryptosporidium Species among Immunocompromised Patients Referred to Hos-pitals, Central Iran, 2015-16

2020 ◽  
Author(s):  
Shahrokh IZADI ◽  
Mohammad Ali MOHAGHEGH ◽  
Zahra GHAYOUR-NAJAFABADI ◽  
Mehdi AZAMI ◽  
Farzaneh MIRZAEI ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Nested Pcr ◽  
Organ Transplant ◽  
Cross Sectional Study ◽  
Cryptosporidium Species ◽  
Rrna Gene ◽  
Immunocompromised Patients ◽  
Cross Sectional ◽  
Cryptosporidium Spp ◽  
Hiv Aids

Background: The purpose of this study was to determine the prevalence and genotype of Cryptosporidium spp. in different groups of immunocompromised patients admitted to the referral hospitals in center of Iran during 2015–2016. Methods: This cross-sectional study was conducted on 346 immunocompromised patients (HIV+/AIDS, Lymphoma, Leukemia and organ transplants) in referred hospitals from central parts of Iran including Isfahan, Markazi, Yazd and Chaharmahale Bakhtiari provinces. Stool samples were analyzed for Cryptosporidium species, modified Ziehl–Neelsen staining techniques followed by the semi-nested PCR and DNA sequencing methods. Results: The total rate of Cryptosporidium spp. was 3.46% (12/346) in the patients, however, the prevalence of the parasite, was 4.6% (4/87) in HIV+/AIDS patients, 3.6% (6/168) in patients with blood malignancy and 2.1% (2/91) in organ transplant recipients. The SSU rRNA gene of Cryptosporidium spp. in all microscopic-positive samples was effectively amplified by the semi-nested PCR and DNA sequences, exposed the existence of two Cryptosporidium species, including C. hominis 91.6% (11/12) and C. parvum 8.3% (1/12). Conclusion: The predominance of C. hominis in the present study may be certifies the importance of anthroponotic transmission of cryptosporidiosis in center of Iran.

scholarly journals Immunomagnetic Separation (IMS)-Fluorescent Antibody Detection and IMS-PCR Detection of Seeded Cryptosporidium parvum Oocysts in Natural Waters and Their Limitations

2002 ◽  
Vol 68 (6) ◽  
pp. 2991-2996 ◽  
Author(s):  
Gregory D. Sturbaum ◽  
Patricia T. Klonicki ◽  
Marilyn M. Marshall ◽  
B. Helen Jost ◽  
Brec L. Clay ◽  
...  
Keyword(s):  
Cryptosporidium Parvum ◽  
Water Samples ◽  
Natural Waters ◽  
Fluorescent Antibody ◽  
Pcr Amplification ◽  
Immunomagnetic Separation ◽  
Pcr Detection ◽  
Antibody Detection ◽  
Rrna Gene ◽  
Pcr Targeting

ABSTRACT Detection and enumeration of Cryptosporidium parvum in both treated and untreated waters are important to facilitate prevention of future cryptosporidiosis incidents. Immunomagnetic separation (IMS)-fluorescent antibody (FA) detection and IMS-PCR detection efficiencies were evaluated in two natural waters seeded with nominal seed doses of 5, 10, and 15 oocysts. IMS-FA detected oocysts at concentrations at or below the three nominal oocyst seed doses, illustrating that IMS-FA is sensitive enough to detect low oocyst numbers. However, the species of the oocysts could not be determined with this technique. IMS-PCR, targeting the 18S rRNA gene in this study, yielded positive amplification for 17 of the 18 seeded water samples, and the amplicons were subjected to restriction fragment length polymorphism digestion and DNA sequencing for species identification. Interestingly, the two unseeded, natural water samples were also PCR positive; one amplicon was the same base pair size as the C. parvum amplicon, and the other amplicon was larger. These two amplified products were determined to be derived from DNA of Cryptosporidium muris and a dinoflagellate. These IMS-PCR results illustrate that (i) IMS-PCR is able to detect low oocyst numbers in natural waters, (ii) PCR amplification alone is not confirmatory for detection of target DNA when environmental samples are used, (ii) PCR primers, especially those designed against the rRNA gene region, need to be evaluated for specificity with organisms closely related to the target organism, and (iv) environmental amplicons should be subjected to appropriate species-specific confirmatory techniques.

Detection of filamentous genus Gordonia in foam samples using genus-specific primers combined with PCR – denaturing gradient gel electrophoresis analysis

2007 ◽  
Vol 53 (6) ◽  
pp. 768-774 ◽  
Author(s):  
Fo-Ting Shen ◽  
Hsuan-Ru Huang ◽  
A.B. Arun ◽  
Hui-Ling Lu ◽  
Ta-Chen Lin ◽  
...  
Keyword(s):  
Wastewater Treatment ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Nested Pcr ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Wastewater Treatment Plants ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
Gradient Gel Electrophoresis

A nested-PCR amplification combined with denaturing gradient gel electrophoresis (PCR–DGGE) approach was used to detect and identify Gordonia populations from wastewater treatment plant foam samples. The PCR-amplified region (position 722–1119) by specifically designed primers G699F and G1096R covered the hypervariable region of the Gordonia 16S rRNA gene sequence. This approach successfully distinguished Gordonia species to the interspecies level. The differential ability of PCR–DGGE analysis was effectively used to separate 12 Gordonia species belonging to different 16S rRNA gene-based phylogenetic lineages into 8 groups. Based on this method, the minimum limit of Gordonia detection was 5 × 104CFU·g–1in the seeded soil samples. The PCR–DGGE bands obtained were excised and identified by sequence analysis. Gordonia polyisoprenivorans , Gordonia amicalis , DGGE type II Gordonia species, and an uncertain Gordonia species dominated the activated sludge foam samples. Results of this study indicate that the detection and analyses of genus Gordonia within a complex microbial community could be accomplished using the PCR–DGGE approach to a larger extent, with certain limitations. Detection of diverse Gordonia populations in foam samples from wastewater treatment plants revealed the significant role of Gordonia in biological foaming during wastewater treatment. The nested-PCR amplification and DGGE can be used as a diagnostic tool for the early detection of foaming incidents in wastewater treatment plants using Gordonia as indicator organism.

scholarly journals Detection and molecular characterization of Cryptosporidium spp. in captive canaries (Serinus canaria) using different diagnostic methods

2018 ◽  
Vol 27 (1) ◽  
pp. 60-65 ◽  
Author(s):  
Vinícius da Silva Camargo ◽  
Bruna Nicoleti Santana ◽  
Elis Domingos Ferrari ◽  
Alex Akira Nakamura ◽  
Walter Bertequini Nagata ◽  
...  
Keyword(s):  
Real Time ◽  
Malachite Green ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
18S Rrna ◽  
Diagnostic Methods ◽  
Negative Staining ◽  
Cryptosporidium Spp ◽  
Genotype Iii ◽  
Pcr Targeting

Abstract This study used several diagnostic methods to examine the occurrence of and molecularly characterize Cryptosporidium spp. in captive canaries (Serinus canaria) in southern and southeastern Brazil. A total of 498 fecal samples were purified by centrifugal-flotation using Sheather's solution. Cryptosporidium spp. diagnosis was performed using three diagnostic methods: malachite green negative staining, nested PCR targeting the 18S rRNA gene, followed by sequencing the amplified fragments, and duplex real-time PCR targeting the 18S rRNA specific to detect Cryptosporidium galli and Cryptosporidium avian genotype III. The overall positivity for Cryptosporidium spp. (total samples positive in at least one protocol) from the microscopic analysis, nested PCR and duplex real-time PCR protocol results was 13.3% (66/498). The positivity rates were 2.0% (10/498) and 4.6% (23/498) for Cryptosporidium spp. by microscopy and nested PCR, respectively. Sequencing of 20 samples amplified by nested PCR identified C. galli (3.0%; 15/498), Cryptosporidium avian genotype I (0.8%; 4/498) and Cryptosporidium avium (0.2%; 1/498). Duplex real-time PCR revealed a positivity of 7.8% (39/498) for C. galli and 2.4% (12/498) for avian genotype III. Malachite green negative staining differed significantly from nested PCR in detecting Cryptosporidium spp. Duplex real-time PCR was more sensitive than nested PCR/sequencing for detecting gastric Cryptosporidium in canaries.

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