scholarly journals Comparison of three diagnostic methods in the diagnosis of cryptosporidiosis and gp60 subtyping of Cryptosporidium parvum in diarrheic calves in Central Anatolia Region of Turkey

2021 ◽  
Vol 5 (2) ◽  
pp. 63-69
Author(s):  
Alparslan Yildirim ◽  
Ferda Sevinc ◽  
Zuhal Onder ◽  
Onder Duzlu ◽  
Ozlem Derinbay Ekici ◽  
...  
Keyword(s):  
Cryptosporidium Parvum ◽  
Nested Pcr ◽  
Diagnostic Methods ◽  
Central Anatolia ◽  
Ssu Rrna ◽  
Sequence Analyses ◽  
Significant Difference ◽  
Pcr Rflp ◽  
Species Specific ◽  
Diarrheic Calves

Abstract The aim of this study was to compare three diagnostic methods for the diagnosis of cryptosporidiosis and to detect subtypes ofCryptosporidium parvum by sequences analyses of gp60 gene in diarrheic calves in several herds in Konya province located in Central Anatolia Region of Turkey. Fecal samples were collected from a total of 194 pre-weaned calves (n=158, ≤15 days old, and n=36, 15 to 40 days old), with diarrhoea. For comparative diagnosis, all samples were examined by modified Ziehl-Neelsen staining of fecal smears for the presence of oocyst, nested PCR-RFLP of SSU rRNA and TaqMan qPCR for the detection of Cryptosporidium DNA. A total of 92 (47.4%) and 104 (53.6%) out of the examined samples were found positive by microscopic examination and molecular tools, respectively. The diagnostic sensitivity and specificity of microscopic identification were determined as 88.5% and 100.0%, respectively compared to molecular assays. Cryptosporidium parvum was the only detected species in all positive samples by species-specific qPCR and nested PCR-RFLP assays. Species identifications were further confirmed by sequence analyses of the SSU rRNA PCR products. There was no statistically significant difference in C. parvum prevalence between early pre-weaned calves and calves older than 15 days. The sequence analyses of the gp60 gene of C. parvum isolates revealed a one subtype IIaA13G2R1 belonging to zoonotic family IIa in diarrheic calves

Prevalence and molecular characterisation of Cryptosporidium spp. in goat kids

2018 ◽  
Author(s):  
A. K. Dixit ◽  
Pooja Dixit ◽  
M.L.V. Rao ◽  
Rohita Gupta ◽  
P. C. Shukla
Keyword(s):  
Cryptosporidium Parvum ◽  
Cryptosporidium Species ◽  
Field Conditions ◽  
Rrna Gene ◽  
Molecular Characterisation ◽  
Ssu Rrna ◽  
Cryptosporidium Spp ◽  
Faecal Samples ◽  
Significant Difference ◽  
Pcr Rflp

Prevalence and molecular characterisation of Cryptosporidium species was done in kids belonging to organised and non-organised goat farms at Jabalpur. The overall prevalence of Cryptosporidium was 14.63%. The prevalence was non-significantly higher in male kids (16.16%) as compared to that of female kids (13.21%). Age wise prevalence was higher in kids up to one month age (16.13%) than that of kids upto 3 months age (13.99%). No significant difference was found in prevalence among different breeds and in kids kept in farm or field conditions. The prevalence was non-significantly higher in non-diarrhoeic kids than diarrhoeic kids. Most of the infections were of one score (76.6%). Molecular characterisation by PCR-RFLP of 18S SSU rRNA gene revealed presence of Cryptosporidium parvum species in positive faecal samples.

scholarly journals INFECÇÃO NATURAL POR Cryptosporidium parvum: RELATO DE CASO (Natural infection with Cryptosporidium parvum: case report)

2020 ◽  
Vol 15 (5) ◽  
Author(s):  
Ana Paula Molinari Candeias ◽  
Karim Cristhine Pase Montagnini ◽  
Liliane Aparecida Oliveira De Paula ◽  
Ana Julia Dal Curtivo Back ◽  
Fransael Franklyn Araújo Da Silva ◽  
...  
Keyword(s):  
Case Report ◽  
Cryptosporidium Parvum ◽  
Nested Pcr ◽  
Natural Infection ◽  
Ssu Rrna ◽  
Cryptosporidium Spp

A diarreia neonatal bovina é uma doença de origem multifatorial e entre os agentes envolvidos está o Cryptosporidium parvum, que ganha destaque por ser o principal protozoário responsável por ocasionar diarreia em bezerros neonatos e possuir elevado potencial zoonótico. O presente trabalho teve como objetivo, descrever um caso de infecção natural por C. parvum em um bovino diagnosticado no Laboratório de Patologia Veterinária (LPV) e no Laboratório de Doenças Parasitárias dos Animais (DOPA) da Universidade Federal do Paraná (UFPR) – Setor Palotina. Foi atendido no Hospital Veterinário da UFPR, um bovino, fêmea, Girolando, com 10 dias de vida, e conforme o histórico, o animal foi adquirido de outra propriedade que realizava a colostragem de forma inadequada, e desde a aquisição, o animal começou a apresentar manifestações clínicas como prostração e diarreia amarelada e fétida. Realizou-se tratamento, porém sem sucesso, e o animal foi a óbito, sendo encaminhado para o LPV para a autópsia. No exame macroscópico, observou-se baixo escore corporal com discreta deposição de tecido adiposo em subcutâneo e mesentério, a mucosa oral e ocular estavam levemente pálidas e no intestino, notou-se leve distensão por conteúdo gasoso e áreas multifocais com leve quantidade de conteúdo avermelhado líquido, por vezes, amarelado e fétido. Na análise histopatológica, notou-se nos no ápice das vilosidades, a presença leve de estruturas multifocais, arredondadas, eosinofílicas, medindo entre 1 a 2µm, compatíveis com Cryptosporidium spp. Parte do conteúdo fecal foi remetido ao DOPA, onde realizou-se a análise microscópica e molecular. Para a análise microscópica, foi confeccionado esfregaço fecal com o conteúdo resultante da centrifugo sedimentação, posteriormente, corado pelo método de Ziehl-Neelsen modificado (ORTOLANI, 2000). A análise confirmou a identificação de oocistos de Cryptosporidium spp. Após a análise microscópica a amostra foi submetida a clarificação, extração de DNA e nested-PCR (nPCR) (XIAO et al. 1999). Para a realização da PCR (Reação em Cadeia pela Polimerase) e nPCR, uma alíquota da amostra foi utilizada para a extração de DNA utilizando o Kit ChargeSwitch® gDNA Mini Tissue (Invitrogen). A região 18 SSU rRNA foi selecionada como sequência alvo para amplificação de DNA, sendo que, o amplicon esperado era de 826-864pb. A reação foi realizada com o volume final de 25μL e o produto amplificado foi submetido à eletroforese em gel de agarose a 1,6%. Com o intuito de identificar a espécie envolvida na infecção, após a amostra apresentar resultado positivo na técnica de nPCR, esta foi encaminhada para o sequenciamento e, após a análise da sequência de dados obtida, foi determinado que Cryptosporidium parvum era a espécie envolvida neste caso. Desta forma, o monitoramento dos animais para a obtenção do diagnóstico precoce é de suma importância para evitar as possíveis perdas na produção animal e gerenciar o risco da transmissão para seres humanos.

scholarly journals A Retrospective Study about the Impact of Switching from Nested PCR to Multiplex Real-Time PCR on the Distribution of the Human Papillomavirus (HPV) Genotypes

Medicina ◽  
2019 ◽  
Vol 55 (8) ◽  
pp. 418 ◽  
Author(s):  
Prete ◽  
Ronga ◽  
Addati ◽  
Magrone ◽  
Abbasciano ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Real Time ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
Diagnostic Methods ◽  
Pcr Assay ◽  
Hpv Prevalence ◽  
Significant Difference ◽  
Hpv Genotypes ◽  
The Impact

Background and objectives: Human papillomavirus (HPV) is the most prevalent etiological agent of viral sexually-transmitted infection. This study retrospectively evaluated the impact of a switch to a real-time PCR assay in the HPV prevalence and genotypes distribution by a quasi-experimental before-and-after approach. Materials and Methods: In total, 1742 samples collected from 1433 patients were analyzed at the UOC Microbiology and Virology of Policlinico of Bari, Italy. HPV DNA detection was performed using initially nested PCR and subsequently multiplex real-time PCR assay. Results: Statistically significant difference in HPV overall prevalence after the introduction of the real-time assay was not detected (48.97% vs. 50.62%). According to different extraction-DNA amplification methods, differences were observed in the prevalence rates of HPV-45, 68, 40, 42, and 43. The lowest prevalence for HPV-45 was observed in the Magna Pure-Real Time PCR group, while HPV-68, 40, 42, and 43 were less observed in the Qiagen-Real Time PCR group. After, a multivariate logistic regression, an increase in the prevalence of HPV-42 (aOR: 4.08, 95% CI: 1.71–9.73) was associated with the multiplex real-time PCR assay. Conclusions: Although this study is a not a direct comparison between two diagnostic methods because it has a sequential structure, it serves to verify the impact of a new molecular assay on HPV distribution. Moreover, the stability of HPV prevalence over time suggests that the population composition and the behavioral variables did not likely change during the observation period. Our study proposes that the introduction of a molecular test for HPV detection may be related to changes of HPV genotypes distribution.

PCR-RFLP Detection od PAI-2 Gene Variants: Prevelence in Ethnic Groups and Disease Relationship in patients Undergoing corony Angiography

1997 ◽  
Vol 77 (05) ◽  
pp. 0955-0958 ◽  
Author(s):  
Carole A Foy ◽  
Peter J Grant
Keyword(s):  
Gene Variants ◽  
Genotype Distribution ◽  
Pima Indians ◽  
Significant Difference ◽  
Pcr Rflp ◽  
History Of ◽  
Caucasian Patients ◽  
Clinical Conditions ◽  
Rflp Assay ◽  
Coronary Atheroma

SummaryPAI-2 is a fibrinolytic inhibitor produced predominantly by monocytes. Most PAI-2 is intracellular making study in clinical conditions difficult. Abnormalities in production may be associated with inflammation and fibrinolysis at sites of tissue damage such as the atherosclerotic plaque.PAI-2 gene variants have been described: variant A consists of Asn120, Asn404 and Ser413 and variant B consists of Asp120, Lys404 and Cys413. We designed a PCR-RFLP assay using primers spanning the region containing Asn/Lys404 and Ser/Cys413. Variant B contains an Mwol restriction site. We analysed 302 Pima Indians and 286 healthy Caucasian volunteers. To investigate relationships between genotype and vascular disease we analysed 333 Caucasian patients undergoing coronary angiography.Gene variant B was more common in the Pimas than in Caucasians (p <0.0001). There was no significant difference in genotype distribution between the volunteers and patients. In the patients there was no association between genotype and either a history of MI or extent of coronary atheroma.

Association of CYP2D6*10 (c. 100 C>T) Genotype with Z-END Concentration in Patients with Breast Cancer Receiving Tamoxifen Therapy in Indonesian Population

2019 ◽  
Vol 19 (8) ◽  
pp. 1198-1206 ◽  
Author(s):  
Yenny ◽  
Sonar S. Panigoro ◽  
Denni J. Purwanto ◽  
Adi Hidayat ◽  
Melva Louisa ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Steady State ◽  
Ultra Performance Liquid Chromatography ◽  
Cross Sectional Study ◽  
Threshold Concentration ◽  
Breast Cancer Patients ◽  
Cross Sectional ◽  
Significant Difference ◽  
Pcr Rflp ◽  
Liquid Chromatography Tandem Mass

Background: Tamoxifen (TAM) is a frequently used hormonal prodrug for patients with breast cancer that needs to be activated by cytochrome P450 2D6 (CYP2D6) into Zusammen-endoxifen (Z-END). Objective: The purpose of the study was to determine the association between CYP2D6*10 (c.100C>T) genotype and attainment of the plasma steady-state Z-END minimal threshold concentration (MTC) in Indonesian women with breast cancer. Methods: A cross-sectional study was performed in 125 ambulatory patients with breast cancer consuming TAM at 20 mg/day for at least 4 months. The frequency distribution of CYP2D6*10 (c.100C>T) genotypes (C/C: wild type; C/T: heterozygous mutant; T/T: homozygous mutant) was detected using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), the results of which were subsequently confirmed by sequencing. The genotypes were categorized into plasma Z- END concentrations of <5.9 ng/mL and ≥5.9 ng/mL, which were measured using ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Results: Percentages of C/C, CT, and T/T genotypes were 22.4%, 29.6%, and 48.8%, respectively. Median (25-75%) Z-END concentrations in C/C, C/T, and T/T genotypes were 9.58 (0.7-6.0), 9.86 (0.7-26.6), and 3.76 (0.9-26.6) ng/mL, respectively. Statistical analysis showed a significant difference in median Z-END concentration between patients with T/T genotype and those with C/C or C/T genotypes (p<0.001). There was a significant association between CYP2D6*10 (c.100C>T) genotypes and attainment of plasma steady-state Z-END MTC (p<0.001). Conclusion: There was a significant association between CYP2D6*10 (c.100C>T) and attainment of plasma steady-state Z-END MTC in Indonesian breast cancer patients receiving TAM at a dose of 20 mg/day.

Polymorphisms in alcohol metabolizing genes ADH2 and ADH3 and susceptibility to pancreatitis in alcoholics

2017 ◽  
Vol 6 (03) ◽  
pp. 5297
Author(s):  
Vedangi Aaren* ◽  
Godi Sudhakar ◽  
Girinadh L.R.S.
Keyword(s):  
Frequency Distribution ◽  
Ethanol Metabolism ◽  
Alcoholic Pancreatitis ◽  
Cytochrome P450 Enzyme ◽  
Increased Risk ◽  
Significant Difference ◽  
Pcr Rflp ◽  
P450 Enzyme ◽  
Acute Alcoholic Pancreatitis ◽  
Acute And Chronic Pancreatitis

In both developed and developing countries, overuse of alcohol is a considered as the major cause of acute and chronic pancreatitis. Prolonged overconsumption of alcohol for 5–10 years typically precedes the initial attack of acute alcoholic pancreatitis. It is observed that only a minority (around 5%) of alcoholics develop pancreatitis. It is now established that the pancreas has the capacity to metabolize ethanol. Previous studies have shown that there are two major pathways of ethanol metabolism, oxidative and non-oxidative. Oxidative ethanol metabolism involves the conversion of ethanol to acetaldehyde, a reaction that is catalysed by aldehyde dehydrogenase (ADH) with contributions from cytochrome P450 enzyme (CYP2E1) and possibly also catalase. Genetic factors regulating alcohol metabolism could predispose in developing alcoholic pancreatitis (AP). We investigated the association of polymorphisms in ADH enzymes with the alcoholic pancreatitis in North coastal Andhra Pradesh. Patients with alcoholic pancreatitis (AP; n = 100), alcoholic controls (AC; n = 100), and healthy controls (HC; n = 100) were included in the study. Blood samples were collected from the subjects in EDTA coated vials. DNA was extracted and genotyping for ADH2 and ADH3 was done by PCR-RFLP (polymerase chain reaction restriction fragment length polymorphism). The products were analysed by gel electrophoresis. The frequency distribution of ADH3*1/*1 genotype was significantly higher in AP group (54%) compared with AC (35%), and HC (42%), and was found to be associated with increased risk of alcoholic pancreatitis. There was no statistically significant difference between the frequency distribution of ADH3*1/*1, ADH3*1/*2, and ADH3*2/*2 genotypes between AC and HC. There was no statistically significant difference between the frequency distribution of ADH2*1/*1, ADH2*1/*2, and ADH2*2/*2 genotypes in AP compared with AC and HC. This study shows that carriers of ADH3*1/*1 individuals consuming alcohol are at higher risk for alcoholic pancreatitis than those with other genotypes such as ADH3*1/*2 and ADH3*2/*2. 

scholarly journals Responses of Bat Communities (Mammalia: Chiroptera) to Forest Loss and Habitat Conversion in Southern Cameroon

2021 ◽  
Vol 14 ◽  
pp. 194008292110103
Author(s):  
Patrick Jules Atagana ◽  
Eric Moïse Bakwo Fils ◽  
Sevilor Kekeunou
Keyword(s):  
Secondary Forest ◽  
Biosphere Reserve ◽  
Abundant Species ◽  
Primary Forest ◽  
Habitat Types ◽  
Habitat Transformation ◽  
Significant Difference ◽  
Southern Cameroon ◽  
Habitat Conversion ◽  
Species Specific

We aimed to assess how bats are affected by habitat transformation by comparing bat assemblages in four habitat types: primary forest, secondary forest, cocoa plantations and human habitations in the Dja Biosphere Reserve of southern Cameroon. Bats were sampled in the four habitat types using mist nets. During 126 nights, a total of 413 bats were captured, belonging to four families, 16 genera and 24 species. Ninety three individuals (17 species) were captured in the primary forest, followed by plantations (105 individuals, 14 species), human habitations (159 individuals, 10 species), and secondary forest (55 individuals, eight species). Megaloglossus woermanni was recorded in all the four habitats, and was the most abundant species (105 individuals). The analysis of bat assemblage between habitat types showed a statistically significant difference in species composition. The distribution of the six most abundant species ( Epomops franqueti, Megaloglossus woermanni, Rousettus aegyptiacus, Dohyrina cyclops, Hipposideros cf. caffer and Hipposideros cf. ruber) was influenced by habitat types. Our results suggest that the decrease in species richness observed in disturbed habitats may be due to habitat perturbations of primary forest habitats. Therefore, it is important to examine the effects of habitat conversion at species level, as responses are often species-specific.

scholarly journals Molecular Detection and Phylogeny of Anaplasmaphagocytophilum and Related Variants in Small Ruminants from Turkey

Animals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 814
Author(s):  
Münir Aktaş ◽  
Sezayi Özübek ◽  
Mehmet Can Uluçeşme
Keyword(s):  
16S Rrna ◽  
Small Ruminants ◽  
Rrna Gene ◽  
Infection Rates ◽  
Sheep And Goats ◽  
Significant Difference ◽  
Pcr Rflp ◽  
First Time ◽  
Japan And China ◽  
Apparently Healthy

Anaplasma phagocytophilum causes tick-borne fever in small ruminants. Recently, novel Anaplasma variants related to A. phagocytophilum have been reported in ruminants from Tunisia, Italy, South Korea, Japan, and China. Based on 16S rRNA and groEL genes and sequencing, we screened the frequency of A. phagocytophilum and related variants in 433 apparently healthy small ruminants in Turkey. Anaplasma spp. overall infection rates were 27.9% (121/433 analyzed samples). The frequency of A. phagocytophilum and A. phagocytophilum-like 1 infections was 1.4% and 26.5%, respectively. No A. phagocytophilum-like 2 was detected in the tested animals. The prevalence of Anaplasma spp. was comparable in species, and no significant difference was detected between sheep and goats, whereas the prevalence significantly increased with tick infestation. Sequencing confirmed PCR-RFLP data and showed the presence of A. phagocytophilum and A. phagocytophilum-like-1 variant in the sampled animals. Phylogeny-based on 16S rRNA gene revealed the A. phagocytophilum-like 1 in a separate clade together with the previous isolates detected in small ruminants and ticks. In this work, A. phagocytophilum-like 1 has been detected for the first time in sheep and goats from Turkey. This finding revealed that the variant should be considered in the diagnosis of caprine and ovine anaplasmosis.

scholarly journals High Frequency of Cryptosporidium hominis Infecting Infants Points to A Potential Anthroponotic Transmission in Maputo, Mozambique

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 293
Author(s):  
Idalécia Cossa-Moiane ◽  
Hermínio Cossa ◽  
Adilson Fernando Loforte Bauhofer ◽  
Jorfélia Chilaúle ◽  
Esperança Lourenço Guimarães ◽  
...  
Keyword(s):  
Public Hospitals ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
P Value ◽  
Cryptosporidium Hominis ◽  
Cryptosporidium Spp ◽  
Pcr Rflp ◽  
Maputo City ◽  
Stool Samples ◽  
Polymerase Chain

Cryptosporidium is one of the most important causes of diarrhea in children less than 2 years of age. In this study, we report the frequency, risk factors and species of Cryptosporidium detected by molecular diagnostic methods in children admitted to two public hospitals in Maputo City, Mozambique. We studied 319 patients under the age of five years who were admitted due to diarrhea between April 2015 and February 2016. Single stool samples were examined for the presence of Cryptosporidium spp. oocysts, microscopically by using a Modified Ziehl–Neelsen (mZN) staining method and by using Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP) technique using 18S ribosomal RNA gene as a target. Overall, 57.7% (184/319) were males, the median age (Interquartile range, IQR) was 11.0 (7–15) months. Cryptosporidium spp. oocysts were detected in 11.0% (35/319) by microscopy and in 35.4% (68/192) using PCR-RFLP. The most affected age group were children older than two years, [adjusted odds ratio (aOR): 5.861; 95% confidence interval (CI): 1.532–22.417; p-value < 0.05]. Children with illiterate caregivers had higher risk of infection (aOR: 1.688; 95% CI: 1.001–2.845; p-value < 0.05). An anthroponotic species C. hominis was found in 93.0% (27/29) of samples. Our findings demonstrated that cryptosporidiosis in children with diarrhea might be caused by anthroponomic transmission.

scholarly journals Sarcocystis species in bovine carcasses from a Belgian abattoir: a cross-sectional study

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Hang Zeng ◽  
Inge Van Damme ◽  
Teresia Wanjiru Kabi ◽  
Barbara Šoba ◽  
Sarah Gabriël
Keyword(s):  
Safety Issue ◽  
Cross Sectional Study ◽  
Occurrence Rate ◽  
Sarcocystis Species ◽  
Cross Sectional ◽  
Significant Difference ◽  
Pcr Rflp ◽  
Prevalent Species ◽  
Potential Food ◽  
Effect Of Age

Abstract Background Sarcocystis species are obligatorily heteroxenous parasites, of which some are zoonotic, representing a public health and economic impact. This study investigated the occurrence of Sarcocystis spp. in cattle sampled from a Belgian slaughterhouse. Methods A total of 200 carcasses were included in the study, sampled during 10 sampling days. The sedimentation method was applied to isolate the sarcocysts from both heart and diaphragm muscles collected from each carcass. Multiplex PCR, PCR–RFLP as well as cox1 gene sequencing techniques were applied serially on collected sarcocysts for species identification. Results Sarcocystis spp. were detected in 64% (128/200; 95% CI 57–71%) of the sampled carcasses. Female dairy cattle presented the highest Sarcocystis occurrence rate (91%) as well as the highest Sarcocystis species diversity compared to female beef and male beef. Sarcocystis spp. were detected more often in the heart muscles than in the diaphragm among female beef (p < 0.001) and dairy carcasses (p = 0.001), while in male carcasses no significant difference was observed (p = 0.763). The effect of age was not significant in male carcasses (p = 0.872), while the odds of finding sarcocysts significantly increased with age (p = 0.003) within both types of female carcasses. S. cruzi was the most prevalent species and was found in 56.5% (113/200) of the carcasses, followed by S. hominis (21.0%, 42/200), S. bovifelis (12.5%, 25/200), S. bovini (2.0%, 4/200), S. hirsuta (1.5%, 3/200) and S. heydorni (0.5%, 1/200). Six different species were detected in the diaphragm, while only two species were recovered from the heart. S. cruzi was the most prevalent species in heart, while in the diaphragm, this was S. hominis. Conclusions The detection of S. hominis in 21% of the sampled carcasses presents a potential food safety issue, and further research is warranted into controlling this infection. Graphic Abstract

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