scholarly journals Illuminating Microbial Dark Matter in Meromictic Sakinaw Lake

2014 ◽  
Vol 80 (21) ◽  
pp. 6807-6818 ◽  
Author(s):  
Esther A. Gies ◽  
Kishori M. Konwar ◽  
J. Thomas Beatty ◽  
Steven J. Hallam
Keyword(s):  
Single Cell ◽  
Niche Partitioning ◽  
454 Sequencing ◽  
Small Subunit ◽  
Rrna Gene ◽  
Small Subunit Rrna ◽  
Metabolic Potential ◽  
Content Type ◽  
Lake Ecosystems ◽  
Pathway Reconstruction

ABSTRACTDespite recent advances in metagenomic and single-cell genomic sequencing to investigate uncultivated microbial diversity and metabolic potential, fundamental questions related to population structure, interactions, and biogeochemical roles of candidate divisions remain. Numerous molecular surveys suggest that stratified ecosystems manifesting anoxic, sulfidic, and/or methane-rich conditions are enriched in these enigmatic microbes. Here we describe diversity, abundance, and cooccurrence patterns of uncultivated microbial communities inhabiting the permanently stratified waters of meromictic Sakinaw Lake, British Columbia, Canada, using 454 sequencing of the small-subunit rRNA gene with three-domain resolution. Operational taxonomic units (OTUs) were affiliated with 64 phyla, including more than 25 candidate divisions. Pronounced trends in community structure were observed for all three domains with eukaryotic sequences vanishing almost completely below the mixolimnion, followed by a rapid and sustained increase in methanogen-affiliated (∼10%) and unassigned (∼60%) archaeal sequences as well as bacterial OTUs affiliated withChloroflexi(∼22%) and candidate divisions (∼28%). Network analysis revealed highly correlated, depth-dependent cooccurrence patterns betweenChloroflexi, candidate divisions WWE1, OP9/JS1, OP8, and OD1, methanogens, and unassigned archaeal OTUs indicating niche partitioning and putative syntrophic growth modes. Indeed, pathway reconstruction using recently published Sakinaw Lake single-cell genomes affiliated with OP9/JS1 and OP8 revealed complete coverage of the Wood-Ljungdahl pathway with potential to drive syntrophic acetate oxidation to hydrogen and carbon dioxide under methanogenic conditions. Taken together, these observations point to previously unrecognized syntrophic networks in meromictic lake ecosystems with the potential to inform design and operation of anaerobic methanogenic bioreactors.

scholarly journals Regulation of Plasmodium yoelii Oocyst Development by Strain- and Stage-Specific Small-Subunit rRNA

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Yanwei Qi ◽  
Feng Zhu ◽  
Richard T. Eastman ◽  
Young Fu ◽  
Martine Zilversmit ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Small Subunit ◽  
Plasmodium Yoelii ◽  
Rrna Genes ◽  
Parasite Development ◽  
Rrna Gene ◽  
Small Subunit Rrna ◽  
Malaria Parasites ◽  
Content Type ◽  
Small Subunit Rrna Gene

ABSTRACT One unique feature of malaria parasites is the differential transcription of structurally distinct rRNA (rRNA) genes at different developmental stages: the A-type genes are transcribed mainly in asexual stages, whereas the S-type genes are expressed mostly in sexual or mosquito stages. Conclusive functional evidence of different rRNAs in regulating stage-specific parasite development, however, is still absent. Here we performed genetic crosses of Plasmodium yoelii parasites with one parent having an oocyst development defect (ODD) phenotype and another producing normal oocysts to identify the gene(s) contributing to the ODD. The parent with ODD—characterized as having small oocysts and lacking infective sporozoites—was obtained after introduction of a plasmid with a green fluorescent protein gene into the parasite genome and subsequent passages in mice. Quantitative trait locus analysis of genome-wide microsatellite genotypes of 48 progeny from the crosses linked an ~200-kb segment on chromosome 6 containing one of the S-type genes (D-type small subunit rRNA gene [D-ssu]) to the ODD. Fine mapping of the plasmid integration site, gene expression pattern, and gene knockout experiments demonstrated that disruption of the D-ssu gene caused the ODD phenotype. Interestingly, introduction of the D-ssu gene into the same parasite strain (self), but not into a different subspecies, significantly affected or completely ablated oocyst development, suggesting a stage- and subspecies (strain)-specific regulation of oocyst development by D-ssu. This study demonstrates that P. yoelii D-ssu is essential for normal oocyst and sporozoite development and that variation in the D-ssu sequence can have dramatic effects on parasite development. IMPORTANCE Malaria parasites are the only known organisms that express structurally distinct rRNA genes at different developmental stages. The differential expression of these genes suggests that they play unique roles during the complex life cycle of the parasites. Conclusive functional proof of different rRNAs in regulating parasite development, however, is still absent or controversial. Here we functionally demonstrate for the first time that a stage-specifically expressed D-type small-subunit rRNA gene (D-ssu) is essential for oocyst development of the malaria parasite Plasmodium yoelii in the mosquito. This study also shows that variations in D-ssu sequence and/or the timing of transcription may have profound effects on parasite oocyst development. The results show that in addition to protein translation, rRNAs of malaria parasites also regulate parasite development and differentiation in a strain-specific manner, which can be explored for controlling parasite transmission.

scholarly journals Description of Pelistega indica sp. nov., isolated from human gut

2014 ◽  
Vol 64 (Pt_4) ◽  
pp. 1389-1394 ◽  
Author(s):  
Om Prakash ◽  
Hitendra Munot ◽  
Yogesh Nimonkar ◽  
Madhu Sharma ◽  
Shreyas Kumbhare ◽  
...  
Keyword(s):  
Similarity Search ◽  
Type Species ◽  
Sequence Similarity ◽  
Small Subunit ◽  
Rrna Gene ◽  
Small Subunit Rrna ◽  
Healthy Human ◽  
Content Type ◽  
Link Type ◽  
Polyphasic Characterization

A Gram-stain-negative, motile, non-spore-forming, coccoid bacterium was isolated from a stool sample of a healthy human subject and formed cream colour colonies on tryptic soy agar. Almost full-length (1500 bp) small subunit rRNA (16S rRNA) gene sequences were generated and a similarity search was conducted by blast. The results of the similarity search indicated that the bacterium belongs to the class Betaproteobacteria , family Alcaligenaceae . It showed maximum sequence similarity (96.5 %) with Pelistega europaea CCUG 39967T followed by Advenella mimigardefordensis DSM 17166T (96.1 %) and Taylorella asinigenitalis LMG 19572T (95.3 %). The DNA G+C content of strain HM-7T was 42 mol%. Strain HM-7T contained C14 : 0, C16 : 0, C16 : 0 3-OH and C18 : 0 as the dominant fatty acids. Morphological, physiological and biochemical data also indicated that strain HM-7T represents a member of the genus Pelistega , but at the same time distinguished it from Pelistega europaea CCUG 39967T, the only species of the genus with a validly published name. Based on polyphasic characterization we conclude that the bacterium represents a novel species of the genus Pelistega and propose the name Pelistega indica sp. nov., with strain HM-7T ( = MCC 2185T = DSM 27484T) as the type strain of the species.

scholarly journals The Role of Host Phylogeny Varies in Shaping Microbial Diversity in the Hindguts of Lower Termites

2014 ◽  
Vol 81 (3) ◽  
pp. 1059-1070 ◽  
Author(s):  
Vera Tai ◽  
Erick R. James ◽  
Christine A. Nalepa ◽  
Rudolf H. Scheffrahn ◽  
Steve J. Perlman ◽  
...  
Keyword(s):  
Community Composition ◽  
Bacterial Community Composition ◽  
Small Subunit ◽  
Rrna Gene ◽  
Small Subunit Rrna ◽  
Microbial Composition ◽  
Variable Regions ◽  
Content Type ◽  
Symbiotic Associations ◽  
Host Phylogeny

ABSTRACTThe hindguts of lower termites andCryptocercuscockroaches are home to a distinct community of archaea, bacteria, and protists (primarily parabasalids and some oxymonads). Within a host species, the composition of these hindgut communities appears relatively stable, but the evolutionary and ecological factors structuring community composition and stability are poorly understood, as are differential impacts of these factors on protists, bacteria, and archaea. We analyzed the microbial composition of parabasalids and bacteria in the hindguts ofCryptocercus punctulatusand 23 species spanning 4 families of lower termites by pyrosequencing variable regions of the small-subunit rRNA gene. Especially for the parabasalids, these data revealed undiscovered taxa and provided a phylogenetic basis for a more accurate understanding of diversity, diversification, and community composition. The composition of the parabasalid communities was found to be strongly structured by the phylogeny of their hosts, indicating the importance of historical effects, although exceptions were also identified. Particularly, spirotrichonymphids and trichonymphids likely were transferred between host lineages. In contrast, host phylogeny was not sufficient to explain the majority of bacterial community composition, but the compositions of theBacteroidetes,Elusimicrobia,Tenericutes,Spirochaetes, andSynergisteswere structured by host phylogeny perhaps due to their symbiotic associations with protists. All together, historical effects probably resulting from vertical inheritance have had a prominent role in structuring the hindgut communities, especially of the parabasalids, but dispersal and environmental acquisition have played a larger role in community composition than previously expected.

Desulfotomaculum peckii sp. nov., a moderately thermophilic member of the genus Desulfotomaculum , isolated from an upflow anaerobic filter treating abattoir wastewaters

2013 ◽  
Vol 63 (Pt_6) ◽  
pp. 2082-2087 ◽  
Author(s):  
Linda Jabari ◽  
Hana Gannoun ◽  
Jean-Luc Cayol ◽  
Moktar Hamdi ◽  
Bernard Ollivier ◽  
...  
Keyword(s):  
Type Species ◽  
Small Subunit ◽  
Rrna Gene ◽  
Small Subunit Rrna ◽  
Terminal Electron Acceptor ◽  
Content Type ◽  
Moderately Thermophilic ◽  
Sulfate Reducing ◽  
Link Type ◽  
Nitrate And Nitrite

A novel anaerobic thermophilic sulfate-reducing bacterium designated strain LINDBHT1T was isolated from an anaerobic digester treating abattoir wastewaters in Tunisia. Strain LINDBHT1T grew at temperatures between 50 and 65 °C (optimum 55–60 °C), and at pH between 5.9 and 9.2 (optimum pH 6.0–6.8). Strain LINDBHT1T required salt for growth (1–40 g NaCl l−1), with an optimum of 20–30 g l−1. In the presence of sulfate as terminal electron acceptor, strain LINDBHT1T used H2/CO2, propanol, butanol and ethanol as carbon and energy sources but fumarate, formate, lactate and pyruvate were not utilized. Butanol was converted to butyrate, while propanol and ethanol were oxidized to propionate and acetate, respectively. Sulfate, sulfite and thiosulfate were utilized as terminal electron acceptors but elemental sulfur, iron (III), fumarate, nitrate and nitrite were not used. The G+C content of the genomic DNA was 44.4 mol%. Phylogenetic analysis of the small-subunit rRNA gene sequence indicated that strain LINDBHT1T was affiliated to the genus Desulfotomaculum with the type strains of Desulfotomaculum halophilum and Desulfotomaculum alkaliphilum as its closest phylogenetic relatives (about 89 % similarity). This strain represents a novel species of the genus Desulfotomaculum , Desulfotomaculum peckii sp. nov.; the type strain is LINDBHT1T ( = DSM 23769T = JCM 17209T).

scholarly journals Single-cell genomics unveils a canonical origin of the diverse mitochondrial genomes of euglenozoans

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Kristína Záhonová ◽  
Gordon Lax ◽  
Savar D. Sinha ◽  
Guy Leonard ◽  
Thomas A. Richards ◽  
...  
Keyword(s):  
Single Cell ◽  
Small Subunit ◽  
Rrna Genes ◽  
Mitochondrial Genomes ◽  
Trna Genes ◽  
Rrna Gene ◽  
Heterotrophic Flagellates ◽  
Small Subunit Rrna ◽  
Single Chromosome ◽  
Linear Chromosomes

Abstract Background The supergroup Euglenozoa unites heterotrophic flagellates from three major clades, kinetoplastids, diplonemids, and euglenids, each of which exhibits extremely divergent mitochondrial characteristics. Mitochondrial genomes (mtDNAs) of euglenids comprise multiple linear chromosomes carrying single genes, whereas mitochondrial chromosomes are circular non-catenated in diplonemids, but circular and catenated in kinetoplastids. In diplonemids and kinetoplastids, mitochondrial mRNAs require extensive and diverse editing and/or trans-splicing to produce mature transcripts. All known euglenozoan mtDNAs exhibit extremely short mitochondrial small (rns) and large (rnl) subunit rRNA genes, and absence of tRNA genes. How these features evolved from an ancestral bacteria-like circular mitochondrial genome remains unanswered. Results We sequenced and assembled 20 euglenozoan single-cell amplified genomes (SAGs). In our phylogenetic and phylogenomic analyses, three SAGs were placed within kinetoplastids, 14 within diplonemids, one (EU2) within euglenids, and two SAGs with nearly identical small subunit rRNA gene (18S) sequences (EU17/18) branched as either a basal lineage of euglenids, or as a sister to all euglenozoans. Near-complete mitochondrial genomes were identified in EU2 and EU17/18. Surprisingly, both EU2 and EU17/18 mitochondrial contigs contained multiple genes and one tRNA gene. Furthermore, EU17/18 mtDNA possessed several features unique among euglenozoans including full-length rns and rnl genes, six mitoribosomal genes, and nad11, all likely on a single chromosome. Conclusions Our data strongly suggest that EU17/18 is an early-branching euglenozoan with numerous ancestral mitochondrial features. Collectively these data contribute to untangling the early evolution of euglenozoan mitochondria.

scholarly journals Respiratory Microbiota Dynamics following Streptococcus pneumoniae Acquisition in Young and Elderly Mice

2014 ◽  
Vol 82 (4) ◽  
pp. 1725-1731 ◽  
Author(s):  
Cassandra L. Krone ◽  
Giske Biesbroek ◽  
Krzysztof Trzciński ◽  
Elisabeth A. M. Sanders ◽  
Debby Bogaert
Keyword(s):  
Streptococcus Pneumoniae ◽  
Young Adult ◽  
Hypervariable Region ◽  
Small Subunit ◽  
Rrna Gene ◽  
Upper Respiratory Tract ◽  
Small Subunit Rrna ◽  
Content Type ◽  
Portal Of Entry ◽  
The Impact

ABSTRACTThe upper respiratory tract (URT) is a distinct microbial niche of low-density bacterial communities and, also, a portal of entry for many potential pathogens, includingStreptococcus pneumoniae. Thus far, animal models have been used to study the dynamics of and interactions between limited numbers of different species in the URT. Here, we applied a deep sequencing approach to explore, for the first time, the impact ofS. pneumoniaeacquisition on URT microbiota in a mouse model, as well as potential age-dependent effects. Young-adult and elderly mice were inoculated intranasally withS. pneumoniae, and nasal lavage samples were collected for up to 28 days postcolonization. Bacterial DNA extracted from lavage samples was subjected to barcoded pyrosequencing of the V5-to-V7 hypervariable region of the small-subunit rRNA gene. We observed highly diverse microbial profiles, with the presence overall of 15 phyla and approximately 645 operational taxonomic units (OTUs). We noted differences in the composition of microbiota between young and elderly mice, with a significantly higher abundance ofBacteroidetesin the young mice. The introduction ofS. pneumoniaeinto the URT led to a temporary dominance of pneumococci in the microbiota of all mice, accompanied by a significant decrease in microbial diversity. As mice gradually cleared the colonization, the diversity returned to baseline levels. Diversification was accompanied by an early expansion ofBacteroidetes,Staphylococcusspp., andLachnospiraceae. Moreover, theBacteroidetesexpansion was significantly greater in young-adult than in elderly mice. In conclusion, we observed differences in URT microbiota composition between naive young-adult and elderly mice that were associated with differences in pneumococcal clearance over time.

scholarly journals Comparative Single-Cell Genomics of Chloroflexi from the Okinawa Trough Deep-Subsurface Biosphere

2016 ◽  
Vol 82 (10) ◽  
pp. 3000-3008 ◽  
Author(s):  
Heather Fullerton ◽  
Craig L. Moyer
Keyword(s):  
Single Cell ◽  
Okinawa Trough ◽  
Reductive Dehalogenation ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Metabolic Potential ◽  
Deep Subsurface ◽  
Content Type ◽  
Reductive Dehalogenase ◽  
The Okinawa Trough

ABSTRACTChloroflexismall-subunit (SSU) rRNA gene sequences are frequently recovered from subseafloor environments, but the metabolic potential of the phylum is poorly understood. The phylumChloroflexiis represented by isolates with diverse metabolic strategies, including anoxic phototrophy, fermentation, and reductive dehalogenation; therefore, function cannot be attributed to these organisms based solely on phylogeny. Single-cell genomics can provide metabolic insights into uncultured organisms, like the deep-subsurfaceChloroflexi. Nine SSU rRNA gene sequences were identified from single-cell sorts of whole-round core material collected from the Okinawa Trough at Iheya North hydrothermal field as part of Integrated Ocean Drilling Program (IODP) expedition 331 (Deep Hot Biosphere). Previous studies of subsurfaceChloroflexisingle amplified genomes (SAGs) suggested heterotrophic or lithotrophic metabolisms and provided no evidence for growth by reductive dehalogenation. Our nineChloroflexiSAGs (seven of which are from the orderAnaerolineales) indicate that, in addition to genes for the Wood-Ljungdahl pathway, exogenous carbon sources can be actively transported into cells. At least one subunit for pyruvate ferredoxin oxidoreductase was found in four of theChloroflexiSAGs. This protein can provide a link between the Wood-Ljungdahl pathway and other carbon anabolic pathways. Finally, one of the sevenAnaerolinealesSAGs contains a distinct reductive dehalogenase homologous (rdhA) gene.IMPORTANCEThrough the use of single amplified genomes (SAGs), we have extended the metabolic potential of an understudied group of subsurface microbes, theChloroflexi. These microbes are frequently detected in the subsurface biosphere, though their metabolic capabilities have remained elusive. In contrast to previously examinedChloroflexiSAGs, our genomes (several are from the orderAnaerolineales) were recovered from a hydrothermally driven system and therefore provide a unique window into the metabolic potential of this type of habitat. In addition, a reductive dehalogenase gene (rdhA) has been directly linked to marine subsurfaceChloroflexi, suggesting that reductive dehalogenation is not limited to the classDehalococcoidia. This discovery expands the nutrient-cycling and metabolic potential present within the deep subsurface and provides functional gene information relating to this enigmatic group.

Mesotoga infera sp. nov., a mesophilic member of the order Thermotogales , isolated from an underground gas storage aquifer

2013 ◽  
Vol 63 (Pt_8) ◽  
pp. 3003-3008 ◽  
Author(s):  
Wajdi Ben Hania ◽  
Anne Postec ◽  
Thomas Aüllo ◽  
Anthony Ranchou-Peyruse ◽  
Gaël Erauso ◽  
...  
Keyword(s):  
Type Species ◽  
Elemental Sulfur ◽  
Gas Storage ◽  
Small Subunit ◽  
Rrna Gene ◽  
Small Subunit Rrna ◽  
Underground Gas Storage ◽  
Terminal Electron Acceptor ◽  
Content Type ◽  
Link Type

Strain VNs100T, a novel mesophilic, anaerobic, rod-coccoid-shaped bacterium, having a sheath-like outer structure (toga), was isolated from a water sample collected in the area of an underground gas storage aquifer. It was non-motile with cells appearing singly (2–4 µm long×1–2 µm wide), in pairs or as long chains and stained Gram-negative. Strain VNs100T was heterotrophic, able to use arabinose, cellobiose, fructose, galactose, glucose, lactose, lactate, mannose, maltose, raffinose, ribose, sucrose and xylose as energy sources only in the presence of elemental sulfur as terminal electron acceptor. Acetate, CO2 and sulfide were the end products of sugar metabolism. Hydrogen was not detected. Elemental sulfur, but not thiosulfate, sulfate or sulfite, were reduced to sulfide. Strain VNs100T grew at temperatures between 30 and 50 °C (optimum 45 °C), at pH values between 6.2 and 7.9 (optimum 7.3–7.5) and at NaCl concentrations between 0 and 15 g l−1 (optimum 2 g l−1). The DNA G+C content was 47.5 mol%. The main cellular fatty acid was C16 : 0. Phylogenetic analysis of the small subunit rRNA gene sequence indicated that strain VNs100T had as its closest relatives ‘ Mesotoga sulfurireducens’ (97.1 % similarity) and Mesotoga prima (similarity of 97.1 % and 97.7 % with each of its two genes, respectively) within the order Thermotogales . Hybridization between strain VNS100T and ‘M. sulfurireducens’ and between strain VNS100T and M. prima showed 12.9 % and 20.6 % relatedness, respectively. Based on phenotypic, phylogenetic and taxonomic characteristics, strain VNs100T is proposed as a representative of a novel species of the genus Mesotoga in the family Thermotogaceae , order Thermotogales . The name Mesotoga infera sp. nov. is proposed. The type strain is VNs100T ( = DSM 25546T = JCM 18154T).

scholarly journals Culture-Independent Analysis of Aerosol Microbiology in a Metropolitan Subway System

2013 ◽  
Vol 79 (11) ◽  
pp. 3485-3493 ◽  
Author(s):  
Charles E. Robertson ◽  
Laura K. Baumgartner ◽  
J. Kirk Harris ◽  
Kristen L. Peterson ◽  
Mark J. Stevens ◽  
...  
Keyword(s):  
Human Skin ◽  
Bacterial Species ◽  
Environmental Water ◽  
Small Subunit ◽  
Commensal Bacteria ◽  
Health Concern ◽  
Rrna Gene ◽  
Small Subunit Rrna ◽  
Content Type ◽  
Culture Independent

ABSTRACTThe goal of this study was to determine the composition and diversity of microorganisms associated with bioaerosols in a heavily trafficked metropolitan subway environment. We collected bioaerosols by fluid impingement on several New York City subway platforms and associated sites in three sampling sessions over a 1.5-year period. The types and quantities of aerosolized microorganisms were determined by culture-independent phylogenetic analysis of small-subunit rRNA gene sequences by using both Sanger (universal) and pyrosequencing (bacterial) technologies. Overall, the subway bacterial composition was relatively simple; only 26 taxonomic families made up ∼75% of the sequences determined. The microbiology was more or less similar throughout the system and with time and was most similar to outdoor air, consistent with highly efficient air mixing in the system. Identifiable bacterial sequences indicated that the subway aerosol assemblage was composed of a mixture of genera and species characteristic of soil, environmental water, and human skin commensal bacteria. Eukaryotic diversity was mainly fungal, dominated by organisms of types associated with wood rot. Human skin bacterial species (at 99% rRNA sequence identity) included theStaphylococcusspp.Staphylococcus epidermidis(the most abundant and prevalent commensal of the human integument),S. hominis,S. cohnii,S. caprae, andS. haemolyticus, all well-documented human commensal bacteria. We encountered no organisms of public health concern. This study is the most extensive culture-independent survey of subway microbiota so far and puts in place pre-event information required for any bioterrorism surveillance activities or monitoring of the microbiological impact of recent subway flooding events.

scholarly journals Targeted Next-Generation Sequencing and Informatics as an Effective Tool to Establish the Composition of Bovine Piroplasm Populations in Endemic Regions

2020 ◽  
Vol 9 (1) ◽  
pp. 21
Author(s):  
Abdul Ghafar ◽  
Anson V. Koehler ◽  
Ross S. Hall ◽  
Charles G. Gauci ◽  
Robin B. Gasser ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Water Buffalo ◽  
Hypervariable Region ◽  
Small Subunit ◽  
Rrna Gene ◽  
Next Generation ◽  
Small Subunit Rrna ◽  
Tropical Theileriosis ◽  
Ecological Zones ◽  
Generation Sequencing

Protists of the genera Babesia and Theileria (piroplasms) cause some of the most prevalent and debilitating diseases for bovines worldwide. In this study, we established and used a next-generation sequencing-informatic approach to explore the composition of Babesia and Theileria populations in cattle and water buffalo in a country (Pakistan) endemic for these pathogens. We collected individual blood samples from cattle (n = 212) and water buffalo (n = 154), extracted genomic DNAs, PCR-amplified the V4 hypervariable region of 18S small subunit rRNA gene from piroplasms, sequenced amplicons using Illumina technology, and then analysed data using bioinformatic platforms. The results revealed piroplasms in 68.9% (252/366) samples, with overall occurrence being markedly higher in cattle (85.8%) than in water buffaloes (45.5%). Babesia (B.) occultans and Theileria (T.) lestoquardi-like species were recorded for the first time in Pakistan, and, overall, T. annulata was most commonly detected (65.8%) followed by B. bovis (7.1%), B. bigemina (4.4%), and T. orientalis (0.5%), with the genetic variability within B. bovis being pronounced. The occurrence and composition of piroplasm species varied markedly across different agro-ecological zones. The high detection of T. annulata in asymptomatic animals suggested a relatively high level of endemic stability of tropical theileriosis in the bovine population.

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