scholarly journals MixedTheileriainfections in free-ranging buffalo herds: implications for diagnosingTheileria parvainfections in Cape buffalo (Syncerus caffer)

Parasitology ◽  
2011 ◽  
Vol 138 (7) ◽  
pp. 884-895 ◽  
Author(s):  
RONEL PIENAAR ◽  
FRED T. POTGIETER ◽  
ABDALLA A. LATIF ◽  
ORIEL M. M. THEKISOE ◽  
BEN J. MANS
Keyword(s):  
South Africa ◽  
Hypervariable Region ◽  
18S Rrna Gene ◽  
Control Measures ◽  
Locked Nucleic Acid ◽  
Rrna Gene ◽  
Mixed Infections ◽  
Endemic Region ◽  
Signal Suppression

SUMMARYBuffalo-adaptedTheileria parvacauses Corridor disease in cattle. Strict control measures therefore apply to the movement of buffalo in South Africa and include mandatory testing of buffalo for the presence ofT. parva. The official test is a real-time hybridization PCR assay that amplifies the V4 hypervariable region of the 18S rRNA gene ofT. parva, T.sp. (buffalo) andT.sp. (bougasvlei). The effect that mixedT. parvaandT.sp. (buffalo)-like infections have on accurateT. parvadiagnosis was investigated in this study.In vitromixed infection simulations indicated PCR signal suppression at 100 to 1000-foldT.sp. (buffalo) excess at lowT. parvaparasitaemia. Suppression of PCR signal was found in field buffalo with mixed infections. TheT. parva-positive status of these cases was confirmed by selective suppression ofT.sp. (buffalo) amplification using a locked nucleic acid clamp and independent assays based on the p67, p104 andTprgenes. The incidence of mixed infections in the Corridor disease endemic region of South Africa is significant, while the prevalence in buffalo outside the endemic area is currently low. A predicted increase ofT.sp. (buffalo)-like infections can affect future diagnoses where mixed infections occur, prompting the need for improvements in current diagnostics.

Geographic distribution of Theileria sp. (buffalo) and Theileria sp. (bougasvlei) in Cape buffalo (Syncerus caffer) in southern Africa: implications for speciation

Parasitology ◽  
2013 ◽  
Vol 141 (3) ◽  
pp. 411-424 ◽  
Author(s):  
RONEL PIENAAR ◽  
ABDALLA A. LATIF ◽  
ORIEL M. M. THEKISOE ◽  
BEN J. MANS
Keyword(s):  
South Africa ◽  
Real Time ◽  
Southern Africa ◽  
Hybrid Sterility ◽  
Control Measures ◽  
Coi Gene ◽  
Mixed Infections ◽  
Endemic Region ◽  
Significant Discrepancy ◽  
Specific Distribution

SUMMARYStrict control measures apply to movement of buffalo in South Africa including testing for Theileria parva, the causative agent of Corridor disease in cattle. The official test is a real-time hybridization PCR assay that amplifies the 18S rRNA V4 hyper-variable region of T. parva, T. sp. (buffalo) and T. sp. (bougasvlei). Mixed infections with the latter organisms affect diagnostic sensitivity due to PCR suppression. While the incidence of mixed infections in the Corridor disease endemic region of South Africa is significant, little information is available on the specific distribution and prevalence of T. sp. (buffalo) and T. sp. (bougasvlei). Specific real-time PCR assays were developed and a total of 1211 samples known to harbour these parasites were screened. Both parasites are widely distributed in southern Africa and the incidence of mixed infections with T. parva within the endemic region is similar (∼25–50%). However, a significant discrepancy exists in regard to mixed infections of T. sp. (buffalo) and T. sp. (bougasvlei) (∼10%). Evidence for speciation between T. sp. (buffalo) and T. sp. (bougasvlei) is supported by phylogenetic analysis of the COI gene, and their designation as different species. This suggests mutual exclusion of parasites and the possibility of hybrid sterility in cases of mixed infections.

scholarly journals Detection of Protozoan Hosts for Legionella pneumophila in Engineered Water Systems by Using a Biofilm Batch Test

2010 ◽  
Vol 76 (21) ◽  
pp. 7144-7153 ◽  
Author(s):  
Rinske M. Valster ◽  
Bart A. Wullings ◽  
Dick van der Kooij
Keyword(s):  
Legionella Pneumophila ◽  
Biofilm Formation ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Batch Test ◽  
Free Living ◽  
Water Types ◽  
Operational Taxonomic Units ◽  
Hartmannella Vermiformis

ABSTRACT Legionella pneumophila proliferates in aquatic habitats within free-living protozoa, 17 species of which have been identified as hosts by using in vitro experiments. The present study aimed at identifying protozoan hosts for L. pneumophila by using a biofilm batch test (BBT). Samples (600 ml) collected from 21 engineered freshwater systems, with added polyethylene cylinders to promote biofilm formation, were inoculated with L. pneumophila and subsequently incubated at 37°C for 20 days. Growth of L. pneumophila was observed in 16 of 18 water types when the host protozoan Hartmannella vermiformis was added. Twelve of the tested water types supported growth of L. pneumophila or indigenous Legionella anisa without added H. vermiformis. In 12 of 19 BBT flasks H. vermiformis was indicated as a host, based on the ratio between maximum concentrations of L. pneumophila and H. vermiformis, determined with quantitative PCR (Q-PCR), and the composition of clone libraries of partial 18S rRNA gene fragments. Analyses of 609 eukaryotic clones from the BBTs revealed that 68 operational taxonomic units (OTUs) showed the highest similarity to free-living protozoa. Forty percent of the sequences clustering with protozoa showed ≥99.5% similarity to H. vermiformis. None of the other protozoa serving as hosts in in vitro studies were detected in the BBTs. In several tests with growth of L. pneumophila, the protozoa Diphylleia rotans, Echinamoeba thermarum, and Neoparamoeba sp. were identified as candidate hosts. In vitro studies are needed to confirm their role as hosts for L. pneumophila. Unidentified protozoa were implicated as hosts for uncultured Legionella spp. grown in BBT flasks at 15°C.

scholarly journals Genomic Profiling for Piroplasms in Feeding Ixodid Ticks in the Eastern Cape, South Africa

Pathogens ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1061
Author(s):  
Olusesan Adeyemi Adelabu ◽  
Benson Chuks Iweriebor ◽  
Anthony Ifeanyi Okoh ◽  
Larry Chikwelu Obi
Keyword(s):  
South Africa ◽  
Nested Pcr ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Rhipicephalus Appendiculatus ◽  
Ixodid Ticks ◽  
Farm Animals ◽  
Homology Search ◽  
Rrna Gene ◽  
Eastern Cape

Importation of tick-infected animals and the uncontrollable migration of birds and wild animals across borders can lead to geographical expansion and redistribution of ticks and pathogen vectors, thus leading to the emergence and re-emergence of tick-borne diseases in humans and animals. Comparatively, little is known about the occurrence of piroplasms in ixodid ticks in the Eastern Cape, South Africa, thus necessitating this study, which is aimed at detecting piroplasms (Theileria and Babesia) from feeding tick samples collected from cattle, sheep, and goats in selected sites in the Eastern Cape, South Africa. A total of 1200 feeding ixodid ticks collected from farm animals at selected homesteads were first subjected to molecular identification using mitochondrial 12S ribosomal RNA (rRNA) gene by PCR and were further tested for the presence of piroplasms through amplification of the 18S rRNA gene via nested-PCR followed by sequencing of the PCR products. The results indicated that 853 (71.1%) corresponded to the genus Rhipicephalus, 335 (27.9%) corresponded to genus Amblyomma, and 12 (1%) corresponded to genus Haemaphysalis. Amblyomma hebraeum and Rhipicephalus appendiculatus were the most common identified ticks from this study. The 18S rRNA nested-PCR revealed that 44 (3.7%) samples were confirmed positive for Theileria. A homology search for the generated sequences revealed a high percentage identity of 98–98.9% similarity to T. buffeli, T. orientalis, and T. sergenti in the GenBank. Based on the results obtained herein, we conclude that there is a big diversity of Theileria species; therefore, we suggest that this research should cover more geographical areas in order to reveal the true prevalence of this pathogen in the studied area because this will be a great step in the possible prevention of an outbreak that could have devastating effects on livestock production and human health in both the studied areas and South Africa at large.

scholarly journals Short-term responses to ocean acidification: effects on relative abundance of eukaryotic plankton from the tropical Timor Sea

2020 ◽  
Author(s):  
Janina Rahlff ◽  
Sahar Khodami ◽  
Lisa Voskuhl ◽  
Matthew P. Humphreys ◽  
Christian Stolle ◽  
...  
Keyword(s):  
Ocean Acidification ◽  
Community Composition ◽  
Hypervariable Region ◽  
18S Rrna Gene ◽  
Marine Biodiversity ◽  
Rrna Gene ◽  
Tropical Regions ◽  
Timor Sea ◽  
Decreasing Ph ◽  
Species Specific

ABSTRACTAnthropogenic carbon dioxide (CO2) emissions drive climate change and pose one of the major challenges of our century. The effects of increased CO2 in the form of ocean acidification (OA) on the communities of marine planktonic eukaryotes in tropical regions such as the Timor Sea are barely understood. Here, we show the effects of high CO2 (pCO2=1823±161 μatm, pHT=7.46±0.05) versus in situ CO2 (pCO2=504±42 μatm, pHT=7.95±0.04) seawater on the community composition of marine planktonic eukaryotes immediately and after 48 hours of treatment exposure in a shipboard microcosm experiment. Illumina sequencing of the V9 hypervariable region of 18S rRNA (gene) was used to study the eukaryotic community composition. Down-regulation of extracellular carbonic anhydrase occurred faster in the high CO2 treatment. Increased CO2 significantly suppressed the relative abundances of different eukaryotic operational taxonomic units (OTUs), including important primary producers. These effects were consistent between abundant (DNA-based) and active (cDNA-based) taxa after 48 hours, e.g., for the diatoms Trieres chinensis and Stephanopyxis turris. Effects were also very species-specific among different diatoms. Planktonic eukaryotes showed adaptation to the CO2 treatment over time, but many OTUs were adversely affected by decreasing pH. OA effects might fundamentally impact the base of marine biodiversity, suggesting profound outcomes for food web functioning in the future ocean.

scholarly journals High prevalence of Babesia microti in small mammals in Beijing

2020 ◽  
Vol 9 (1) ◽  
Author(s):  
Chun-Yan Wei ◽  
Xiao-Mei Wang ◽  
Zhen-Sheng Wang ◽  
Zhi-Hua Wang ◽  
Zeng-Zhi Guan ◽  
...  
Keyword(s):  
Risk Factors ◽  
Small Mammals ◽  
18S Rrna Gene ◽  
Host Population ◽  
Pathogen Transmission ◽  
Control Measures ◽  
Babesia Microti ◽  
Mammalian Host ◽  
Rrna Gene ◽  
Sequence Alignments

Abstract Background Babesiosis is an emerging tick-borne zoonotic infectious disease. Babesia microti is responsible for most cases of human babesiosis globally. It is important to investigate the prevalence of B. microti in the mammalian host population of a specific region in order to elucidate mechanisms of pathogen transmission and to define geographic areas where humans face the greatest risk of exposure. The aim of this study is to understand the prevalence and genotypes of B. microti in the small mammals that are found in Beijing, China. Methods We trapped small mammals from all of the 16 urban, suburban, and outer suburban districts of Beijing during the years 2014, 2017 and 2018. Genomic DNA was extracted from the heart tissues individually and the Babesia 18S rRNA gene was detected by PCR. The genotypes of B. microti were identified based on sequence alignments and phylogenetic analysis. The morphology of the parasites was observed under light microscopy. The risk factors were analyzed statistically based on both univariate analyses and multivariate logistic regression. Results A total of 1391 small mammals were collected. Positive infection of B. microti was detected in 12.1% (168/1391) of small mammals from 15 out of the 16 districts. Both Kobe-type and U.S.-type B. microti, accounting for 9.5% and 2.7%, respectively, were identified. Classic diverse morphologic forms of B. microti were observed. Specific types of ecological habitats including shrub areas, broad-leaved forest, and cropland were revealed to be risk factors associated with B. microti infection. Conclusions This study demonstrated the wide prevalence of B. microti infection in eight species of small mammals in Beijing, with Kobe-type more prevalent than U.S.-type. This study provides fundamental information for the development of informed prevention and control measures by public health authorities; the data gathered indicates a need for further monitoring of both clinical diseases in individuals presenting with babesiosis-like symptoms, as well as the infection status of ticks in high risk areas.

scholarly journals Species, Sequence Types and Alleles: Dissecting Genetic Variation in Acanthamoeba

Pathogens ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 534
Author(s):  
Paul A. Fuerst ◽  
Gregory C. Booton
Keyword(s):  
Genetic Variation ◽  
18S Rrna ◽  
Hypervariable Region ◽  
18S Rrna Gene ◽  
Sequence Type ◽  
Rrna Gene ◽  
Dna Databases ◽  
Molecular Approaches ◽  
History Of ◽  
Sequence Types

Species designations within Acanthamoeba are problematic because of pleomorphic morphology. Molecular approaches, including DNA sequencing, hinted at a resolution that has yet to be fully achieved. Alternative approaches were required. In 1996, the Byers/Fuerst lab introduced the concept of sequence types. Differences between isolates of Acanthamoeba could be quantitatively assessed by comparing sequences of the nuclear 18S rRNA gene, ultimately producing 22 sequence types, designated T1 through T22. The concept of sequence types helps our understanding of Acanthamoeba evolution. Nevertheless, substantial variation in the 18S rRNA gene differentiates many isolates within each sequence type. Because the majority of isolates with sequences in the international DNA databases have been studied for only a small segment of the gene, designated ASA.S1, genetic variation within this hypervariable region of the 18S rRNA gene has been scrutinized. In 2002, we first categorized variation in this region in a sample of T3 and T4 isolates from Hong Kong, observing ten “alleles” within type T4 and five “alleles” within T3. Subsequently, confusion occurred when different labs applied redundant numerical labels to identify different alleles. A more unified approach was required. We have tabulated alleles occurring in the sequences submitted to the international DNA databases, and determined their frequencies. Over 150 alleles have occurred more than once within 3500+ isolates of sequence type T4. Results from smaller samples of other sequence types (T3, T5, T11 and T15, and supergroup T2/6) have also been obtained. Our results provide new insights into the evolutionary history of Acanthamoeba, further illuminating the degree of genetic separation between significant taxonomic units within the genus, perhaps eventually elucidating what constitutes a species of Acanthamoeba.

Diversity and distribution of nematodes associated with terrestrial slugs in the Western Cape Province of South Africa

2011 ◽  
Vol 86 (2) ◽  
pp. 215-221 ◽  
Author(s):  
J.L. Ross ◽  
E.S. Ivanova ◽  
W.F. Sirgel ◽  
A.P. Malan ◽  
M.J. Wilson
Keyword(s):  
South Africa ◽  
18S Rrna ◽  
Parasitic Nematode ◽  
Gene Sequencing ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Western Cape ◽  
Western Cape Province ◽  
Rrna Gene Sequencing ◽  
Three New Species

AbstractA survey of nematodes associated with native and introduced species of terrestrial slugs was conducted in the Western Cape Province of South Africa, in order to gather new data regarding diversity and distribution. A total of 521 terrestrial slugs were collected from 35 localities throughout the Western Cape. All slugs were dissected and examined for the presence of internal nematodes. Extracted nematodes were identified using a combination of molecular (18S rRNA gene sequencing) and morphological techniques. Nematodes were found parasitizing slugs at 14 of the 35 sites examined, amounting to 40% of sample sites. Of all slugs, 6% were infected with nematodes. A total of seven species of nematode were identified in the province, includingAgfa flexilis,Angiostomasp.,Phasmarhabditissp. SA1,Phasmarhabditissp. SA2,Caenorhabditis elegans,Panagrolaimussp. andRhabditissp. Of these species, four were thought to be parasitic to slugs (A. flexilis, Angiostomasp.,Phasmarhabditissp. SA1 andPhasmarhabditissp. SA2), as opposed to forming necromenic or phoretic associations. Three new species of slug-parasitic nematode were identified during this study (Angiostomasp.,Phasmarhabditissp. SA1 andPhasmarhabditissp. SA2).

Bench-scale experiments for the development of a unified loop-mediated isothermal amplification (LAMP) assay for the in vitro diagnosis of Leishmania species' promastigotes

2013 ◽  
Vol 142 (8) ◽  
pp. 1671-1677 ◽  
Author(s):  
M. KARANI ◽  
I. SOTIRIADOU ◽  
J. PLUTZER ◽  
P. KARANIS
Keyword(s):  
Lamp Assay ◽  
High Sensitivity ◽  
Leishmania Species ◽  
18S Rrna Gene ◽  
Isothermal Amplification ◽  
Rrna Gene ◽  
Bench Scale ◽  
Loop Mediated Isothermal Amplification ◽  
Wide Range

SUMMARYWe developed, in bench-scale experiments, a unified loop-mediated isothermal amplification (LAMP) assay for the detection of cutaneous, mucocutaneous and visceral leishmaniasis using DNA of cultivated promastigotes. Two primer sets for the LAMP assay were designed based on the 18S rRNA gene, and their sensitivity and specificity were tested and compared. Both of them were specific for Leishmania as the DNA of all ten Leishmania species tested was amplified, whereas the DNA of other parasites, including that of Trypanosoma, was not. The detection limit for primer set 1 ranged between 30 pg and 3·6 fg, depending on which Leishmania species tested. Primer set 2 showed high sensitivity, but was less sensitive than primer set 1. Our findings lead to the conclusion that the LAMP assay with primer set 1 is a promising and effective assay for the successful detection of a wide range of Leishmania infections using only a unified multiplex LAMP test.

scholarly journals Design and Evaluation of Illumina MiSeq-Compatible, 18S rRNA Gene-Specific Primers for Improved Characterization of Mixed Phototrophic Communities

2016 ◽  
Vol 82 (19) ◽  
pp. 5878-5891 ◽  
Author(s):  
Ian M. Bradley ◽  
Ameet J. Pinto ◽  
Jeremy S. Guest
Keyword(s):  
Community Structure ◽  
18S Rrna ◽  
Target Genes ◽  
Hypervariable Region ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Sequencing Bias ◽  
Primer Sets

ABSTRACTThe use of high-throughput sequencing technologies with the 16S rRNA gene for characterization of bacterial and archaeal communities has become routine. However, the adoption of sequencing methods for eukaryotes has been slow, despite their significance to natural and engineered systems. There are large variations among the target genes used for amplicon sequencing, and for the 18S rRNA gene, there is no consensus on which hypervariable region provides the most suitable representation of diversity. Additionally, it is unclear how much PCR/sequencing bias affects the depiction of community structure using current primers. The present study amplified the V4 and V8-V9 regions from seven microalgal mock communities as well as eukaryotic communities from freshwater, coastal, and wastewater samples to examine the effect of PCR/sequencing bias on community structure and membership. We found that degeneracies on the 3′ end of the current V4-specific primers impact read length and mean relative abundance. Furthermore, the PCR/sequencing error is markedly higher for GC-rich members than for communities with balanced GC content. Importantly, the V4 region failed to reliably capture 2 of the 12 mock community members, and the V8-V9 hypervariable region more accurately represents mean relative abundance and alpha and beta diversity. Overall, the V4 and V8-V9 regions show similar community representations over freshwater, coastal, and wastewater environments, but specific samples show markedly different communities. These results indicate that multiple primer sets may be advantageous for gaining a more complete understanding of community structure and highlight the importance of including mock communities composed of species of interest.IMPORTANCEThe quantification of error associated with community representation by amplicon sequencing is a critical challenge that is often ignored. When target genes are amplified using currently available primers, differential amplification efficiencies result in inaccurate estimates of community structure. The extent to which amplification bias affects community representation and the accuracy with which different gene targets represent community structure are not known. As a result, there is no consensus on which region provides the most suitable representation of diversity for eukaryotes. This study determined the accuracy with which commonly used 18S rRNA gene primer sets represent community structure and identified particular biases related to PCR amplification and Illumina MiSeq sequencing in order to more accurately study eukaryotic microbial communities.

scholarly journals Metagenetic Analysis for Microbial Characterization of Focaccia Doughs Obtained by Using Two Different Starters: Traditional Baker’s Yeast and a Selected Leuconostoc citreum Strain

Foods ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1189
Author(s):  
Massimo Ferrara ◽  
Angelo Sisto ◽  
Giuseppina Mulè ◽  
Paola Lavermicocca ◽  
Palmira De Bellis
Keyword(s):  
Hypervariable Region ◽  
18S Rrna Gene ◽  
Biochemical Properties ◽  
Baker’S Yeast ◽  
Rrna Gene ◽  
Baker's Yeast ◽  
Hypervariable Regions ◽  
Leuconostoc Citreum ◽  
Significant Difference ◽  
Microbial Environment

Lactic acid bacteria (LAB) decisively influence the technological, nutritional, organoleptic and preservation properties of bakery products. Therefore, their use has long been considered an excellent strategy to improve the characteristics of those goods. The aim of this study was the evaluation of microbial diversity in different doughs used for the production of a typical Apulian flatbread, named focaccia. Leavening of the analyzed doughs was obtained with baker’s yeast or by applying an innovative “yeast-free” protocol based on a liquid sourdough obtained by using Leuconostoc citreum strain C2.27 as a starter. The microbial populations of the doughs were studied by both a culture-dependent approach and metagenetic analyses. The flours used for dough preparation were also subjected to the same analyses. The metagenetic analyses were performed by sequencing the V5–V6 hypervariable regions of the 16S rRNA gene and the V9 hypervariable region of the 18S rRNA gene. The results indicate that these hypervariable regions were suitable for studying the microbiota of doughs, highlighting a significant difference between the microbial community of focaccia dough with baker’s yeast and that of the dough inoculated with the bacterial starter. In particular, the dough made with baker’s yeast contained a microbiota with a high abundance of Proteobacteria (82% of the bacterial population), known to be negatively correlated with the biochemical properties of the doughs, while the Proteobacteria in dough produced with the L. citreum starter were about 43.5% lower than those in flour and dough prepared using baker’s yeast. Moreover, the results show that the L. citreum C2.27 starter was able to dominate the microbial environment and also reveal the absence of the genus Saccharomyces in the dough used for the production of the “yeast-free” focaccia. This result is particularly important because it highlights the suitability of the starter strain for obtaining an innovative “yeast-free” product.

Sign in / Sign up

Export Citation Format

Share Document