scholarly journals Molecular Identification of Selected Tick-Borne Protozoan and Bacterial Pathogens in Thoroughbred Racehorses in Cavite, Philippines

Pathogens ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 1318
Author(s):  
Eloiza May Galon ◽  
Adrian Miki Macalanda ◽  
Mary Margarett Garcia ◽  
Chrysler James Ibasco ◽  
Anatolio Garvida ◽  
...  
Keyword(s):  
18S Rrna ◽  
Bacterial Pathogens ◽  
Significant Risk ◽  
The Philippines ◽  
23S Rrna ◽  
Borrelia Burgdorferi Sensu Lato ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Horse Racing ◽  
Thoroughbred Racehorses

Tick-borne diseases (TBDs) considerably impair equine health and productivity. Moreover, TBDs, particularly equine piroplasmosis, impede international movement and trade of equids, which is a vital component of the global horse racing industry. In the Philippines, horse racing is a lucrative industry generating millions of USD annually. However, information on equine TBDs is scarce. This study intended to describe molecularly the equine tick-borne infections in a racehorse park in Cavite, Philippines and identify the risk factors associated with the infections. One hundred twenty-four (n = 124) thoroughbred racehorses were sampled and screened for selected tick-borne protozoan and bacterial pathogens using polymerase chain reaction (PCR) assays. Racehorses were positive for Babesia caballi (12.10%; 15/124), Theileria equi (0.81%; 1/124), Anaplasma phagocytophilum (10.48%; 13/124), Borrelia burgdorferi sensu lato (38.71%; 48/124), A. marginale (0.81%; 1/124), and Coxiella burnetii (0.81%; 1/124). Rickettsia was not detected in the samples. Gender was determined as a significant risk factor for B. caballi infection. Sequencing analysis revealed that seven partial 18S rRNA B. caballi isolates shared 98.63–100% identity with each other and were classified as genotype A. Meanwhile, the sequence obtained from the lone T. equi-positive sample was 99.77% identical to isolates from Spain, Switzerland, China, Saudi Arabia, and South Korea, and was confirmed as genotype E based on the 18S rRNA gene. Eight Anaplasma 16S rRNA partial sequences were highly identical to A. phagocytophilum and A. ovis. Partial sequences of Borrelia 5–23S rRNA were most closely related to Bo. japonica and other Borrelia sp. isolates from various countries. This study reports the first molecular detection of Borrelia and Anaplasma and the identification of B. caballi and T. equi genotypes in racehorses in the Philippines. Findings from this study shall be useful in crafting equine tick and TBD control and prevention programs in the country.

scholarly journals Molecular Characterization of Ciliate Diversity in Stream Biofilms

2008 ◽  
Vol 74 (6) ◽  
pp. 1740-1747 ◽  
Author(s):  
Andrew Dopheide ◽  
Gavin Lear ◽  
Rebecca Stott ◽  
Gillian Lewis
Keyword(s):  
18S Rrna ◽  
Pcr Primers ◽  
18S Rrna Gene ◽  
Sequence Diversity ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Specific Pcr ◽  
Stream Biofilms

ABSTRACT Free-living protozoa are thought to be of fundamental importance in aquatic ecosystems, but there is limited understanding of their diversity and ecological role, particularly in surface-associated communities such as biofilms. Existing eukaryote-specific PCR primers were used to survey 18S rRNA gene sequence diversity in stream biofilms but poorly revealed protozoan diversity, demonstrating a need for protozoan-targeted primers. Group-specific PCR primers targeting 18S rRNA genes of the protozoan phylum Ciliophora were therefore designed and tested using DNA extracted from cultured protozoan isolates. The two most reliable primer combinations were applied to stream biofilm DNA, followed by cloning and sequencing analysis. Of 44 clones derived from primer set 384F/1147R, 86% were of probable ciliate origin, as were 25% of 44 clones detected by primer set 121F/1147R. A further 29% of 121F/1147R-detected clones matched sequences from the closely related phylum Apicomplexa. The highly ciliate-specific primer set 384F/1147R was subsequently used in PCRs on biofilm DNA from four streams exhibiting different levels of human impact, revealing differences in ciliate sequence diversity in samples from each site. Of a total of 240 clones, 73% were of probable ciliate origin; 54 different putative ciliate sequences were detected from throughout seven taxonomic ciliate classes. Sequences from Oligohymenophorea were most commonly detected in all samples, followed by either Spirotrichea or Phyllopharyngea. Restriction fragment length polymorphism profile-based analysis of clones suggested a potentially higher level of diversity than did sequencing. Nevertheless, newly designed PCR primers 384F/1147R were considered to provide an effective molecular basis for characterization of ciliate diversity in stream biofilms.

scholarly journals Prevalence and distribution pattern of Cryptosporidium spp. among pre-weaned diarrheic calves in the Republic of Korea

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0259824
Author(s):  
Dong-Hun Jang ◽  
Hyung-Chul Cho ◽  
Seung-Uk Shin ◽  
Eun-Mi Kim ◽  
Yu-Jin Park ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Infection Rate ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Republic Of Korea ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
High Genetic Diversity ◽  
The Republic ◽  
Diarrheic Calves

Cryptosporidium spp. are protozoan parasites that belong to subphylum apicomplexa and cause diarrhea in humans and animals worldwide. Data on the prevalence of Cryptosporidium spp. and its subtypes among calves in the Republic of Korea (KOR) are sparse. Hence, our study aimed to investigate the prevalence and association between the age of calf and the identified Cryptosporidium spp. and to determine the genotypes/subtypes of Cryptosporidium spp. in pre-weaned calves with diarrhea in the KOR. A total of 460 diarrheic fecal samples were collected from calves aged 1−60 days and screened for Cryptosporidium spp. by the 18S rRNA gene. Species identification was determined using the sequencing analysis of the 18S rRNA gene, and C. parvum-positive samples were subtyped via the sequence analysis of the 60-kDa glycoprotein (gp60) gene. Sequence analysis based on the 18S rRNA gene revealed the presence of three Cryptosporidium spp., namely, C. parvum (n = 72), C. ryanae (n = 12), and C. bovis (n = 2). Co-infection by these species was not observed. The infection rate was the highest in calves aged 11−20 days (26.1%, 95% CI 17.1−35.1), whereas the lowest rate was observed in calves aged 21−30 days (7.7%, 95% CI 0.0−16.1). The prevalence of C. parvum was detected exclusively in calves aged ≤20 days, and the highest infection rate of C. ryanae was seen in calves ≥31 days of age. The occurrence of C. parvum (χ2 = 25.300, P = 0.000) and C. ryanae (χ2 = 18.020, P = 0.001) was significantly associated with the age of the calves. Eleven different subtypes of the IIa family that belonging to C. parvum were recognized via the sequence analyses of the gp60 gene. Except for two (IIaA18G3R1 and IIaA15G2R1) subtypes, nine subtypes were first identified in calves with diarrhea in the KOR. IIaA18G3R1 was the most frequently detected subtype (72.2% of calves), followed by IIaA17G3R1 (5.6%), IIaA15G2R1 (4.2%), IIaA19G4R1 (4.2%), IIaA16G4R1 (2.8%), IIaA17G4R1 (2.8%), IIaA19G3R (2.8%), IIaA14G1R1 (1.4%), IIaA14G3R1 (1.4%), IIaA15G1R1 (1.4%), and IIaA19G1R1 (1.4%) These results suggest that the prevalence of Cryptosporidium spp. is significantly associated with calf age. Furthermore, the findings demonstrate the high genetic diversity of C. parvum and the widespread occurrence of zoonotic C. parvum in pre-weaned calves. Hence, calves are a potential source of zoonotic transmission with considerable public health implications.

scholarly journals Comparative Study of PCR-Based Approaches for the Genetic Characterization of Three Strains of Acidithiobacillus caldus Isolated from Different Sites in China

2013 ◽  
Vol 62 (4) ◽  
pp. 351-358
Author(s):  
Xueling Wu ◽  
Hong Duan ◽  
Hongwei Fan ◽  
Zhenzhen Zhang ◽  
Lili Liu
Keyword(s):  
16S Rrna ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
23S Rrna ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Intergenic Spacers ◽  
Acidithiobacillus Caldus ◽  
Pcr Fingerprinting ◽  
23S Rrna Gene

Comparative study of the genetic characteristics among three Acidithiobacillus caldus strains isolated from different typical environments in China was performed using a combination of molecular methods, namely sequencing analysis of PCR-amplified 16S rRNA genes and 16S-23S rRNA gene intergenic spacers (ITS), repetitive element PCR (rep-PCR), arbitrarily primed PCR (AP-PCR) fingerprinting and random amplified polymorphic DNA (RAPD). Both of the 16S rRNA gene and 16S-23S rRNA gene intergenic spacers sequences of the three strains exhibited small variations, with 99.9-100%, 99.7-100% identity respectively. In contrast, according to the analysis of bacterial diversity based on rep-PCR and AP-PCR fingerprinting, they produced highly discriminatory banding patterns, and the similarity values between them varied from 61.97% to 71.64%. RAPD analysis showed that banding profiles of their genomic DNA exhibited obvious differences from each other with 53.44-75% similarity. These results suggested that in contrast to 16S rRNA genes and 16S-23S rRNA gene intergenic spacers sequencing analysis, rep-PCR, AP-PCR fingerprinting and RAPD analysis possessed higher discriminatory power in identifying these closely related strains. And they could be used as rapid and highly discriminatory typing techniques in studying bacterial diversity, especially in differentiating bacteria within Acidithiobacillus caldus.

Persistent and recurrent urethritis due to macrolide-resistant Mycoplasma genitalium: first reports in Argentina

2021 ◽  
Author(s):  
Gabriela Baldoni ◽  
Gabriela Iribarren ◽  
Claudia Garbasz ◽  
Pablo Striebeck ◽  
Micaela Mayer Wolf ◽  
...  
Keyword(s):  
Male Patient ◽  
Mycoplasma Genitalium ◽  
Purulent Discharge ◽  
23S Rrna ◽  
Rrna Gene ◽  
Reference Laboratory ◽  
Sequencing Analysis ◽  
Adequate Treatment ◽  
23S Rrna Gene ◽  
Clinical Cases

Introduction: Mycoplasma genitalium (MG) is responsible for 15%-20% nongonococcal urethritis in men. In Argentina, the diagnosis is only performed by few laboratories. Single-dose 1 g azithromycin (AZM1D) treatment leads to emergence of macrolide resistance (mutations at 23S rRNA gene, region V, position 2058 or 2059). Recommendations include 5-day AZM (AZM5D) regimen, moxifloxacin as second-line therapy. Doxycycline is only 30% effective. Test of Cure (ToC) is advisable. Objective: The aim of this study was to describe the first two clinical cases of persistent and recurrent urethritis due to macrolide-resistant MG in Argentina. Methods: End point polymerase chain reaction (PCR) for diagnosis and ToC. Sanger sequencing analysis of mutations. Results: Case 1: A 26-year-old male patient with occasional heterosexual contacts and no history of sexually transmitted infections (STIs) complained urethral thick purulent discharge and dysuria (January 2018), with negative microbiological cultures and Chlamydia trachomatis PCR. The patient received ceftriaxone/AZM1D. However, symptoms persisted (April 2018). Later, doxycycline was prescribed for 1 month. Five days after treatment, the sample was referred to the STI national reference laboratory (NRL) and results were found positive for MG. The patient was given AZM5D. As a result, symptoms disappeared, posterior ToC was found negative, and retrospectively, sequencing 23S rRNA gene showed A2058G transition. Case 2: An 18-year-old male patient with stable heterosexual relationship complained of previous gonococcal urethritis and urethral serous exudate with inflammatory reaction (September 2017), with negative microbiological cultures. The patient received ceftriaxone and AZM1D as initial treatment. Later, he was given doxycycline for 10 days. On February 2018, symptoms reappeared and sample referred to the NRL was positive for MG (negative for other STIs). With AZM1D treatment, symptoms disappeared. After 1 month, the symptoms recurred. Results showed a new MG-positive sample (April 2018). AZM5D administration induced 2 weeks symptoms free and recurrence, requiring moxifloxacin treatment. Symptoms disappeared completely. Posterior ToC is negative. Subsequently, sequencing both samples referred to the NRL showed A2059G transition. Conclusion: The clinical cases presented notified the importance of early and accurate diagnosis of MG infections and use of adequate treatment schemes. We emphasized the relevance of monitoring and surveillance prevalence of macrolide-resistant MG in Argentina.

scholarly journals Intestinal Microbiota and Species Diversity ofCampylobacterandHelicobacterspp. in Migrating Shorebirds in Delaware Bay

2014 ◽  
Vol 80 (6) ◽  
pp. 1838-1847 ◽  
Author(s):  
Hodon Ryu ◽  
Kirsten Grond ◽  
Bram Verheijen ◽  
Michael Elk ◽  
Deborah M. Buehler ◽  
...  
Keyword(s):  
16S Rrna ◽  
Delaware Bay ◽  
23S Rrna ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Clone Libraries ◽  
Sequence Identity ◽  
Semipalmated Sandpiper ◽  
Source Markers ◽  
Red Knot

ABSTRACTUsing 16S rRNA gene sequencing analysis, we examined the bacterial diversity and the presence of opportunistic bacterial pathogens (i.e.,CampylobacterandHelicobacter) in red knot (Calidris canutus;n= 40), ruddy turnstone (Arenaria interpres;n= 35), and semipalmated sandpiper (Calidris pusilla;n= 22) fecal samples collected during a migratory stopover in Delaware Bay. Additionally, we studied the occurrence ofCampylobacterspp., enterococci, and waterfowl fecal source markers using quantitative PCR (qPCR) assays. Of 3,889 16S rRNA clone sequences analyzed, the bacterial community was mostly composed ofBacilli(63.5%),Fusobacteria(12.7%),Epsilonproteobacteria(6.5%), andClostridia(5.8%). When epsilonproteobacterium-specific 23S rRNA gene clone libraries (i.e., 1,414 sequences) were analyzed, the sequences were identified asCampylobacter(82.3%) orHelicobacter(17.7%) spp. Specifically, 38.4%, 10.1%, and 26.0% of clone sequences were identified asC. lari(>99% sequence identity) in ruddy turnstone, red knot, and semipalmated sandpiper clone libraries, respectively. Other pathogenic species ofCampylobacter, such asC. jejuniandC. coli, were not detected in excreta of any of the three bird species. MostHelicobacter-like sequences identified were closely related toH. pametensis(>99% sequence identity) andH. anseris(92% sequence identity). qPCR results showed that the occurrence and abundance ofCampylobacterspp. was relatively high compared to those of fecal indicator bacteria, such asEnterococcusspp.,E. faecalis, andCatellicoccus marimammalium. Overall, the results provide insights into the complexity of the shorebird gut microbial community and suggest that these migratory birds are important reservoirs of pathogenicCampylobacterspecies.

scholarly journals Iron encrustations on filamentous algae colonized by <i>Gallionella</i>-related bacteria in a metal-polluted freshwater stream

2015 ◽  
Vol 12 (18) ◽  
pp. 5277-5289 ◽  
Author(s):  
J. F. Mori ◽  
T. R. Neu ◽  
S. Lu ◽  
M. Händel ◽  
K. U. Totsche ◽  
...  
Keyword(s):  
Laser Scanning ◽  
18S Rrna ◽  
Stream Water ◽  
18S Rrna Gene ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Mining District ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Freshwater Algae

Abstract. Filamentous macroscopic algae were observed in slightly acidic to circumneutral (pH 5.9–6.5), metal-rich stream water that leaked out from a former uranium mining district (Ronneburg, Germany). These algae differed in color and morphology and were encrusted with Fe-deposits. To elucidate their potential interaction with Fe(II)-oxidizing bacteria (FeOB), we collected algal samples at three time points during summer 2013 and studied the algae-bacteria-mineral compositions via confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectra, and a 16S and 18S rRNA gene-based bacterial and algae community analysis. Surprisingly, sequencing analysis of 18S rRNA gene regions of green and brown algae revealed high homologies with the freshwater algae Tribonema (99.9–100 %). CLSM imaging indicated a loss of active chloroplasts in the algae cells, which may be responsible for the change in color in

scholarly journals Iron encrustations on filamentous algae colonized by <i>Gallionella</i>-related bacteria in a metal-polluted freshwater stream

2015 ◽  
Vol 12 (10) ◽  
pp. 7705-7737
Author(s):  
J. F. Mori ◽  
T. R. Neu ◽  
S. Lu ◽  
M. Händel ◽  
K. U. Totsche ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Green Algae ◽  
Brown Algae ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Laser Scanning Microscopy ◽  
Gene Copy ◽  
Rrna Gene ◽  
Sequencing Analysis

Abstract. Filamentous macroscopic algae were observed in slightly acidic to circumneutral (pH 5.9~6.5) metal-rich stream water that leaked out in a former uranium-mining district (Ronneburg, Germany). These algae differ in color and morphology and were encrusted with Fe-deposits. To elucidate the potential interaction with Fe(II)-oxidizing bacteria (FeOB), we collected algal samples at three time points during summer 2013 and studied the algae-bacteria-mineral compositions via confocal laser scanning microscopy (CLSM), scanning electronic microscopy, Fourier transform infrared spectra, and a 16S and 18S rRNA gene based bacterial and algae community analysis. Surprisingly, sequencing analysis of 18S rRNA gene regions of green and brown algae revealed high homologies with the yellow-green freshwater algae Tribonema (99.9~100%). CLSM imaging indicates a loss of active chloroplasts in the algae cells, which may be responsible for the change in color in Tribonema. Fe(III)-precipitates on algal cells identified as ferrihydrite and schwertmannite were associated with microbes and extracellular polymeric substances (EPS)-like glycoconjugates. While the green algae were fully encrusted with Fe-precipitates, the brown algae often exhibited discontinuous series of precipitates. This pattern was likely due to the intercalary growth of algal filaments which allowed them to avoid fatal encrustation. 16S rRNA gene targeted studies based on DNA and RNA revealed that Gallionella-related FeOB dominated the bacterial RNA and DNA communities (70–97 and 63–96%, respectively) suggesting their contribution to Fe(II) oxidation. Quantitative PCR revealed higher Gallionella-related 16S rRNA gene copy numbers on the surface of green algae compared to the brown algae. The latter harbored a higher microbial diversity, including some putative predators of algae. Lower photosynthetic activities of the brown algae lead to reduced EPS production which may have enabled predator colonization. The differences observed between green and brown algae suggest that metal-tolerant Tribonema sp. provide suitable microenvironments for microaerophilic Fe-oxidizing bacteria. However, high levels of iron orchres can be fatal to the alga.

scholarly journals Molecular pathways to high-level azithromycin resistance in Neisseria gonorrhoeae

2021 ◽  
Author(s):  
J G E Laumen ◽  
S S Manoharan-Basil ◽  
E Verhoeven ◽  
S Abdellati ◽  
I De Baetselier ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Ribosomal Proteins ◽  
Efflux Pump ◽  
23S Rrna ◽  
Rrna Gene ◽  
23S Rrna Gene ◽  
Evolution Of Resistance ◽  
High Level ◽  
Azithromycin Resistance ◽  
Level Resistance

Abstract Background The prevalence of azithromycin resistance in Neisseria gonorrhoeae is increasing in numerous populations worldwide. Objectives To characterize the genetic pathways leading to high-level azithromycin resistance. Methods A customized morbidostat was used to subject two N. gonorrhoeae reference strains (WHO-F and WHO-X) to dynamically sustained azithromycin pressure. We tracked stepwise evolution of resistance by whole genome sequencing. Results Within 26 days, all cultures evolved high-level azithromycin resistance. Typically, the first step towards resistance was found in transitory mutations in genes rplD, rplV and rpmH (encoding the ribosomal proteins L4, L22 and L34 respectively), followed by mutations in the MtrCDE-encoded efflux pump and the 23S rRNA gene. Low- to high-level resistance was associated with mutations in the ribosomal proteins and MtrCDE efflux pump. However, high-level resistance was consistently associated with mutations in the 23S ribosomal RNA, mainly the well-known A2059G and C2611T mutations, but also at position A2058G. Conclusions This study enabled us to track previously reported mutations and identify novel mutations in ribosomal proteins (L4, L22 and L34) that may play a role in the genesis of azithromycin resistance in N. gonorrhoeae.

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