16S rRNA Gene Research Articles

Biscuits for the gut: A symphony of FODMAPs and dietary fibre in gut microbiome for irritable bowel syndrome (IBS) management

Emerging evidence suggests gut microbiome alterations significantly influence irritable bowel syndrome (IBS) symptoms with fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) implicated in triggering symptoms.This study investigated the impact of low and high FODMAP biscuit models on gut microbiota including: Biscuit Flour Control (BiFC), Low-FODMAP Control (LFC), Low-FODMAP High-Fibre Prototype (LFP), and Wholemeal Flour Control (WMC). Biscuits were subjected to in vitro digestion which was followed by in vitro colonic fermentation. 16S rRNA gene sequencing and fatty acid analyses were performed at the end of fermentation.Alpha diversity analysis revealed samples fermented with LFP induced higher diversity compared to samples fermented with BiFC and LFC. The relative abundance of Escherichia-Shigella was significantly lower in LFP fermentates than BiFC fermentates. LEfSe analysis showed LFC fermentates were characterized by higher levels of Citrobacter and Ligilactobacillus. Significantly reduced acetate and butyrate levels were found in LFP fermentates compared to LFC fermentates. WMC fermentates had higher acetate and propionate levels compared to other biscuit fermentates.In conclusion, incorporating low-FODMAP, high-fibre food prototypes may enhance gut microbial diversity and reduce potentially pathogenic bacteria such as Escherichia-Shigella. These findings highlight the potential of personalized dietary interventions, such as the manipulation of biscuit formulations, in modulating gut microbiota and managing IBS symptoms. Industrial relevanceThe low FODMAP diet has been developed as an effective method to moderate the symptoms of IBS patients by reducing bloating and gas in the colon. Our results revealed that the low-FODMAP, high-fibre biscuit model proved more effective than the low-FODMAP biscuit for generating a gut microbiota that could contribute to easing IBS symptoms. Future research should focus on improving this formulation and adapting it to treat the different forms of IBS. This study also demonstrates the efficacy of using colon fermentation models to devise food formulations for gut microbiome-associated diseases.

Dynamic changes in microbial communities and volatile compounds in kombucha fermentation using Flos sophorae and Elm fruits, compared to Black and Green Tea

The dynamic changes in physicochemical properties, microbial communities, and volatile compounds in kombucha made from Flos sophorae (FLSK) and Elm fruit (EFK) were compared to those of black tea (BTK) and green tea (GTK) over a 12-day fermentation period. The results revealed that overall flavonoid and polyphenol content, as well as antioxidant activity, increased initially and then decreased, accompanied by a steady reduction in pH within the fermentation broths investigated. Notably, the GTK exhibited stronger antioxidant activity than the other fermentation broths. Furthermore, 16S rRNA gene sequencing revealed that Komagataeibacter rhaeticus, Komagataeibacter saccharivorans, and Acidovorax wautersii were the dominating microbial species in the fermentation broths under this study. Komagataeibacter rhaeticus initially reduced and then increased throughout the FLSK fermentation, whereas Komagataeibacter saccharivorans increased from day 0 to day 6, and remain stable by day 12 during the EFK fermentation. Comparative analysis revealed that Komagataeibacter rhaeticus was more abundant in the FLSK and GTK than in the EFK and BTK, whereas Komagataeibacter saccharivorans showed a higher abundance in the EFK relative to the other fermentation broths. Gas chromatography-mass spectrometry identified acetic acid, linalool, ethanol, and ethyl acetate as the major volatile chemicals that rose significantly in fermentation mixtures of the examined substrates. The FLSK had a much higher linalool concentration than the other fermentation broths, although the EFK and GTK had higher ethanol content. Correlation study found that Komagataeibacter rhaeticus was negatively related with alcohol compounds, but Komagataeibacter saccharivorans was positively associated with a diverse spectrum of acids, alcohols, and esters. The study found changes in bioactive chemicals as well as interactions between bacterial populations and volatile compounds throughout fermentation in the substrates investigated.

Ticks and tick-borne pathogens in ruminant farms of Peninsular Malaysia: First molecular evidence of Borrelia theileri in Rhipicephalus microplus (Acari: Ixodidae).

To shed light on the importance of tick-borne diseases, especially in farm animals that often contact with farm workers, this study aimed to identify ticks and tick-borne pathogens in ruminants in Malaysia. Accordingly, specimen collection was conducted across Peninsular Malaysia yielded a total of 1241 ticks collected from 674 farm ruminants. Among these, four tick species were identified, with Rhipicephalus microplus being the most prevalent, constituting 99.03 % of the total tick population. Analysis of 130 tick pools revealed three positives for Borrelia. BLAST analyses of the flaB and 16S rRNA genes revealed high similarities to Borrelia theileri, ranging from 98.78 to 100 % for flaB and 99.23–99.45 % for 16S rRNA. These results align with the phylogenetic trees, where sequences from both genes clustered together with B. theileri, further supporting this identification. No Rickettsia and Bartonella bacteria were detected. This study represents the first occurrence of B. theileri in R. microplus in Malaysia.

Viability discrimination of bacterial microbiomes in home kitchen dish sponges using propidium monoazide treatment.

Dish sponges are known to support the proliferation of human bacterial pathogens, yet they are commonly used by consumers. Exposure to foodborne pathogens via sponge use may lead to illness, a serious concern among susceptible populations. The extent of exposure risks from sponge use has been limited by constraints associated with culture-independent or dependent methods for bacterial community characterization. Therefore, five used dish sponges were characterized to evaluate the presence of viable bacterial foodborne pathogens using the novel application of propidium monoazide (PMA) treatment and targeted 16S rRNA gene amplicon sequencing. Select pathogen viability was confirmed using targeted selective enrichment. The taxonomic abundance profiles of total and viable sponge microbiomes did not vary significantly. The numbers of unique bacterial species (p = 0.0465) and foodborne pathogens (p = 0.0102) identified were significantly lower in viable sponge microbiomes. Twenty unique bacterial foodborne pathogens were detected across total and viable sponge microbiomes, and three to six viable foodborne pathogens were identified in each sponge. Escherichia coli and Staphylococcus aureus were identified in each viable sponge microbiome, and viable E. coli were recovered from two sponges via targeted selective enrichment. These findings suggest that sponge-associated bacterial communities are primarily viable and contain multiple viable bacterial foodborne pathogens.

Z-ligustilide alleviates atherosclerosis by reconstructing gut microbiota and sustaining gut barrier integrity through activation of cannabinoid receptor 2

BackgroundZ-Ligustilide (ZL) is an essential phthalide found in Ligusticum chuanxiong Hort, a commonly used traditional Chinese medicine for treating atherosclerosis (AS) clinically. ZL has been shown to be effective in treating AS. However, the underlying mechanism of ZL against AS and its potential targets remain elusive. PurposeThe purpose of this research was to assess the influence of ZL on AS and explore the role of the gut microbiome in mediating this effect. MethodsA well-established AS mouse model, apolipoprotein E deficient (ApoE-/-) mice was used to examine the effects of ZL on AS, inflammation, and the intestinal barrier. To analyze the changes in gut microbial community, we employed the 16S rRNA gene sequencing. Antibiotic cocktail and fecal microbiota transplantation (FMT) were employed to clarify the contribution of the gut microbiota to the anti-AS effects of ZL. The mechanism through which ZL provided protective effects on AS and the intestinal barrier was explored by untargeted metabolomics, as well as by validating the involvement of cannabinoid receptor 2 (CB2R) in mice and Caco-2 cells. ResultsOral administration of ZL inhibited the development of atherosclerotic lesions, improved plaque stability, inhibited the increase in serum and atherosclerotic inflammation, and improved intestinal barrier function. Fecal bacteria from ZL-treated mice induced similar beneficial effects on AS and the intestinal barrier. We used 16S RNA gene sequencing to reveal a significant increase in Rikenella abundance in both ZL-treated mice and ZL-FMT mice, which was associated with the beneficial effects of ZL. Further function prediction analysis of the gut microbiota and CB2R antagonist intervention experiment in mice and Caco-2 cells showed that the activation of CB2R resulted in the enhancement of the intestinal barrier by ZL. Furthermore, the analysis of metabolomic profiling revealed the enrichment of capsaicin upon ZL treatment, which induced the activation of CB2R in human colon epithelial cells. ConclusionOur study is the first to demonstrate that oral treatment with ZL has the potential to alleviate AS by reducing inflammation levels and enhancing the intestinal barrier function. This mechanism relies on the gut microbiota in a CB2R-dependent manner, suggesting promising strategies and ideas for managing AS. This study provides insights into a novel mechanism for treating AS with ZL.

Retinol metabolism signaling participates in microbiota-regulated fat deposition in obese mice

Obesity is a global pandemic threatening public health, excess fat accumulation and overweight are its characteristics. In this study, the interplay between gut microbiota and retinol metabolism in modulating fat accumulation was verified. We observed gut microbiota depletion reduced the body weight (P<0.05) and the ratios of white adipose tissues (WATs) to body weight (P<0.05) in high-fat diet (HFD) fed-mice. Both the hepatic metabolomics and transcriptomics analyses confirmed that gut microbiota modulated fat accumulation in obese mice. Besides, the kyoto encyclopedia of genes and genomes (KEGG) analysis and protein-protein interaction (PPI) network of RNA-seq results indicated that retinol metabolism signaling may be involved in the microbiota-regulated fat deposition. Furthermore, activated retinol metabolism signaling by all-trans retinoic acid (atRA) supplementation reduced body weight (P<0.05) and WAT accumulation in obese mice. On the other hand, 16S rRNA gene sequencing of the ileal microbiota suggested that atRA supplementation, in turn, increased the microbial diversity and induced the growth of beneficial bacteria including Parabacteroides, Bacteroides, Clostridium_XVIII, Bifidobacterium, Enterococcus, Bacillus, Leuconostoc, and Lactobacillus in obese mice. Spearman correlation showed that atRA decreased the bacteria (Parvibacter, Asaccharobacter, Romboutsia, and Clostridium_IV) that were positively associated with body and WAT weights, whereas increased the bacteria (Lactobacillus) that were negatively associated with body and WAT weights. Together, this study reveals the interaction between the gut microbiota and retinol metabolism signaling in regulating adipose accumulation and obesity. It is expected of this finding to provide new insights to prevent and develop therapeutic measures of obesity-related metabolic syndrome.

Altered intestinal microbiota induced by high-fat diets affect cognition differently in mice

The role of the gut microbiota in the association between high-fat diet and cognition is not clear. We hypothesized that a high-fat diet may influence cognition by altering the intestinal microbiota. Faecal microbiota isolated from male C57BL/6J mice feeding on various high-fat diets and a control basic diet were transplanted to antibiotic treated recipient mice. The measurement of weight and plasma lipids, novel object recognition test, 16S rRNA gene sequencing of faeces and haematoxylin-eosin staining of the hippocampal cornu ammonis 1 (CA1) and cornu ammonis 3 (CA3) areas were performed for all mice. Compared with those in the control and n-3 polyunsaturated fatty acid (n-3 PUFA) groups, donor obese mice fed with diets high in long-chain saturated fatty acids (LCSFAs), n-6 polyunsaturated fatty acids (n-6 PUFAs) and trans fatty acids (TFAs) exhibited significant cognitive impairment (Ps < 0.05). There were fewer neurons in the hippocampal area in the n-6 PUFA group than in the n-3 PUFA group (P < 0.05). Similar effect on cognition and neurons in hippocampal area in corresponding recipient mice were revealed after faecal microbiota transplantation (FMT). In addition, the composition of intestinal microbiota differed among recipient mice after FMT from donor mice. According to these results, it was concluded that diets rich in LCSFAs, n-6 PUFAs, and TFAs may lead to cognitive impairment by damaging the structure of the hippocampus through influencing the intestinal microbiota in mice, while a diet high in n-3 PUFAs may exhibit a beneficial effect.

Comparative Analyses of Lycodon rufozonatus and Lycodon rosozonatus Gut Microbiota in Different Regions.

The interactions between hosts and the gut microbiota are intricate and can significantly affect the ecology and evolution of both parties. Various host traits, including taxonomy, diet, social behaviour, and external factors such as prey availability and the local environment, all play an important role in shaping composition and diversity of the gut microbiogta. In this study, we explored the impact of intestinal microorganisms on the host in adapting to their respective ecological niches in two species of snakes. We collected feces from Lycodon rufozonatus and Lycodon rosozonatus from different geographical locations and used 16S rRNA gene sequencing technology to sequence the v3-v4 region. The results revealed that there was no significant difference in the alpha diversity of intestinal microorganisms between L. rufozonatus and L. rosozonatus. The gut microbiota of all individuals comprised four main phyla: Pseudomonadota, Bacteroidota, Bacillota, and Actinomycetota. At the genus level, the genus Salmonella dominated the enterobacterial microbiota in the samples from Hainan, while there was no obvious dominant genus in the enterobacterial microbiota of the samples from the other four localities. Comparative analysis of enzyme families annotated to the gut microbiota between L. rufozonatus and L. rosozonatus from the four sampling regions by CAZy carbohydrate annotation revealed that nine enzyme families differed significantly in terms of glycoside hydrolases (GHs). In addition, we compared the composition of gut microbial communities between L. rufozonatus and L. rosozonatus and investigated the impact of the differences on their functions. Our results will provide insights into the coevolution of host and gut microbes.

Bifidobacterium fermentum sp. nov. and Bifidobacterium aquikefiricola sp. nov., isolated from water kefir.

Four strains, representing two novel Bifidobacterium species, were isolated from water kefir, a fermented beverage. 16S rRNA gene analysis suggested that the novel species share high identities (98.82-98.89%) with Bifidobacterium aquikefiri LMG 28769T. Complete genomes were assembled with a short- and long-read hybrid sequencing approach. In agreement with the 16S rRNA gene analysis, phylogenetics with 117 marker genes places the novel species closest to B. aquikefiri LMG 28769T as well. The isolates have average nucleotide identity (ANI) scores ranging from 81.46 to 84.84% and digital DNA-DNA hybridization (dDDH) scores from 23.9 to 38.5% with the closest related species, as well as ANI scores between the proposed new species of 80.50%, indicating that the isolates represent two novel species. Matrix-assisted laser desorption/ionization-time of flight chemotaxonomic analysis supported the gene-based taxonomic placement. We propose the names Bifidobacterium fermentum sp. nov. and Bifidobacterium aquikefiricola sp. nov. for these novel species within the Bifidobacterium genus. The proposed type strain B. fermentum WK012_4_13T (= LMG 33104T = DSM 116073T; GenBank accession number GCF_041080835.1) has a genome size of 2.43 Mbp, with a G+C content of 56.00 mol%. The proposed type strain for B. aquikefiricola WK041_4_12T (= LMG 33105T = DSM 116074T; GenBank accession number GCF_041080795.1) has a genome size of 2.36 Mbp and a G+C content of 53.94 mol%. B. fermentum cells are Gram-positive staining, non-motile, non-spore-forming, fructose-6-phosphate phosphoketolase (F6PPK)-positive, catalase- and oxidase-negative and bacillary club shaped. B. aquikefiricola cells are Gram-positive staining, non-motile, non-spore-forming, F6PPK-positive, catalase- and oxidase-negative and square rod shaped.

Captivity increased the abundance of high-risk antibiotic resistance genes in the giant panda gut microbiome

Captivity is a key strategy for protecting endangered species, but research has primarily focused on artificial breeding and reintroduction to bolster wild populations, often overlooking the environmental and health risks associated with antibiotic resistance genes (ARGs). Here, we conducted a comprehensive analysis of the microbiome and ARG profiles in the gut of wild giant pandas across five representative populations, as well as one captive population, utilizing 16S rRNA gene sequencing and High-Throughput Quantitative PCR. Our findings revealed that both geographic location and captivity significantly influenced the gut microbial community and ARG composition in the gut of giant pandas. Additionally, we identified core microbiomes with essential ecological functions, particularly those related to food utilization, were identified in the giant panda gut across different regions. The gut ARGs in giant pandas exhibited a broad range of subtypes, with multidrug resistance genes being the most prevalent. Notably, the captive population harbored the highest abundance of high-risk ARGs, especially those conferring tetracycline resistance. High-risk multidrug ARGs (e.g., tolC, mepA, and mdtA) were found to be strongly correlated with the potential pathogens, such as Escherichia_Shigellina and Pseudomonas. Furthermore, bamboo-associated ARGs and mobile genetic elements (MGEs) contributed significantly to the ARG abundance in the giant panda gut, indicating that diet plays a crucial role in shaping gut resistome. Collectively, our study provides a detailed mapping of giant panda gut microbiomes and ARG distribution, offering valuable insights for conservation efforts and advancing our understanding of ARG dynamics in giant panda populations.

Provolone del Monaco PDO cheese: Lactic microflora, biogenic amines and volatilome characterization

One commercial production run of Provolone del Monaco − a long-ripened pasta filata cheese − was followed up to the end of ripening for a total of 20 samples. 371 LAB isolates were subject to genetic characterization followed by 16S rRNA gene sequencing. The dominant species were Lacticaseibacillus casei/paracasei (19.4 %), Streptococcus macedonicus (19.1 %) and Enterococcus faecalis (13.2 %). Strains were screened for features of technological interest or safety relevance. Tyramine-producing cultures were quite common, above all within enterococci. By MALDI TOF Mass Spectrometry, one Lactococcus lactis and one Enterococcus faecium strain proved to be bacteriocin producers.Four further cheese wheels from the same production run at 623 days of ripening were evaluated for volatile organic compounds, biogenic amines, and bacterial community by metagenomic sequencing. Three individual wheel samples shared a rather similar microbiome with Lactobacillus delbrueckii and Streptococcus thermophilus as the most represented species, while the fourth wheel appeared wholly different being dominated by Lentilactobacillus buchneri and St. infantarius. Additionally, this sample had the greatest content of biogenic amines and a different VOCs composition.Given the variance seen among cheese wheels processed and ripened under the same conditions, the search for adjunct cultures in the production of this cheese seems to be of utmost importance.

National-scale distribution of protists associated with sorghum leaves and roots.

Protists, as integral constituents of the plant microbiome, are posited to confer substantial benefits to plant health and performance. Despite their significance, protists have received considerably less attention compared to other constituents of the plant microbiome, such as bacteria and fungi. To investigate the diversity and community structure of protists in sorghum leaves and roots, we employed amplicon sequencing of the eukaryotic 18S rRNA gene in 563 leaf and root samples collected from 57 locations across China. We found significant differences in the diversity and community structure of protists in sorghum leaves and roots. The leaf was taxonomically dominated by Evosea, Cercozoa and Ciliophora, while the root was dominated by Endomyxa, Cercozoa and Oomycota. The functional taxa of protists exhibited notable differences between leaves and roots, with the former being predominantly occupied by consumers and the latter by parasites. The community composition of protists in the leaf was predominantly influenced by mean annual precipitation, whereas soil pH played a more significant role in the root. The present study identified the most abundant and distributed protists in sorghum leaves and roots and elucidated the underlying factors that govern their community structure. The present study offers a novel perspective on the factors that shape plant-associated protist communities and their potential roles in enhancing the functionality of plant ecosystems.

Refurbishing the marine parasitoid order Pirsoniales with newly (re)described marine and freshwater free-living predators.

Pirsoniales is a stramenopile order composed of marine parasitoids of diatoms with unique life cycle. Until recently, a single genus, Pirsonia, uniting six species, was known. The recent identification of new free-living eukaryotrophic Pirsoniales Pirsonia chemainus, Feodosia pseudopoda, and Koktebelia satura changed our understanding of this group as exclusively parasitic. However, their cell ultrastructure and feeding preferences were not fully studied due to the death of the cultures. In this study, we re-isolated some of these Pirsoniales and established six new strains exhibiting predatory behavior, including a first freshwater representative. This allowed us to describe five new genera and species, as well as to emend the diagnosis of the order Pirsoniales. The 18S rRNA gene phylogenetic analysis revealed the position of new strains within Pirsoniales and their relationships with parasitoid relatives and environmental sequence lineages. Feeding experiments on novel Pirsoniales strains using diverse algal prey showed that they were not able to form trophosomes and auxosomes. The ability of cell aggregation in Pirsoniales was observed for the first time. One of the studied strains contained intracellular gammaproteobacteria distantly related to Coxiella. Ultrastructural analyses revealed a more complex cytoskeleton structure in Pirsoniales than previously thought and supported the monophyly of Bigyromonadea and Pseudofungi.

Molecular Typing of Cryptosporidium in Cattle in Federal Capital Territory, Nigeria

Cryptosporidium is an enteric pathogen with worldwide distribution in humans and animals with cattle as their reservoirs. Molecular tools have been very useful in determining the epidemiology of this zoonotic pathogen of public health importance. Despite the importance of this pathogen to humans and animals, there is limited published information on the risk factors of its epidemiology in the Federal Capital Territory of Nigeria. Positive faecal samples of 400 cattle in 46 farms located in 6 area councils of the FCT that were earlier screened for Cryptosporidium on light microscopy were genotyped by nested PCR using primers that targeted 18S ssUrRNA gene and followed by RFLP. The PCR-RFLP products were further sub genotyped by nested PCR and GP 60 KDa gene amplification. The nucleotides of the amplicons were subsequently sequenced. The overall PCR detection rate was 5.5%. Analysis of the 18S rRNA gene of the PCR-RFLP fragments revealed Cryptosporidium ryanae (18.3%), C. andersoni (8.5%) and C. parvum (4.2%). Analysis of the nucleotide sequence of the GP 60KDa gene of all the C. parvum detected showed all to be 100% subtype family IIa and 100% sub genotype IIaA18G3R1. In conclusion, this study has established the presence of Cryptosporidium in cattle in the FCT. The detection of the zoonotic C. parvum, its subtype IIa and its allele IIaA18G3R1 is an indication of source of zoonotic transmission of the parasite in the FCT, Nigeria.

Detection of airborne wild waterbird-derived DNA demonstrates potential for transmission of avian influenza virus via air inlets into poultry houses, the Netherlands, 2021 to 2022.

BackgroundOutbreaks of highly pathogenic avian influenza (HPAI) on poultry farms and in wild birds worldwide persists despite intensified control measures. It causes unprecedented mortality in bird populations and is increasingly affecting mammalian species. Better understanding of HPAI introduction pathways into farms are needed for targeted disease prevention and control. The relevance of airborne transmission has been suggested but research involving air sampling is limited and unequivocal evidence on transmission routes is lacking.AimWe aimed to investigate whether HPAI virus from wild birds can enter poultry houses through air inlets by characterising host materials through eukaryote DNA sequencing.MethodsWe collected particulate matter samples in and around three HPAI-affected poultry farms which were cleared and decontaminated before sampling. Indoor measurements (n = 61) were taken directly in the airflow entering through air inlets, while outdoor air samples (n = 60) were collected around the poultry house. Positive controls were obtained from a bird rehabilitation shelter. We performed metabarcoding on environmental DNA by deep sequencing 18S rRNA gene amplicons.ResultsWe detected waterbird DNA in air inside all three, and outside of two, poultry farms. Sequences annotated at species level included swans and tufted ducks. Waterbird DNA was present in all indoor and outdoor air samples from the bird shelter.ConclusionAirborne matter derived from contaminated wild birds can potentially introduce HPAI virus to poultry houses through air inlets. The eDNA metabarcoding could assess breaches in biosecurity for HPAI virus and other pathogens potentially transmitted through air via detection of their hosts.

Molecular Epidemiology and Phylogeny of Theileria ovis and Theileria lestoquardi in Sheep and Goats from Southern Punjab, Pakistan.

Background: Theileria spp. are responsible for ovine and caprine theileriosis, leading to significant morbidity and mortality in small ruminants.The present study aims to investigate Theileria spp. infections in small ruminants from Southern Punjab in Pakistan, and genetic characterize revealed Theileria spp. isolates. Methods: A total of 93 sheep and 107 goats were sampled between May and August 2022. Blood smears were examined microscopically, and PCR amplification targeting the 18S rRNA gene was performed to detect Theileria spp. Additionally, specific PCR assays targeting 18S rRNA and ms1 partial sequences were used to identify Theileria ovis and T. lestoquardi, respectively. Results: The prevalence of Theileria spp. was significantly higher using PCR (13.5%) compared to microscopic screening (5%). Sheep showed a higher prevalence rate (19.4%) compared to goats (8.4%) (p = 0.024). Young sheep aged ≤ 1 year were more commonly infected with Theileria spp. (41%) compared to older sheep (p = 0.006). The prevalence of Theileria spp. was higher in sheep-only herds (37.3%) compared to goat-only herds (18%) or mixed-species herds (8.1%) (p = 0.015). The prevalence rates of T. ovis and T. lestoquardi were 9% and 2.5%, respectively, with four animals (2 goats and 2 sheep) showing co-infection. Phylogenetic analysis revealed that our T. ovis 18S rRNA sequence clustered with previously reported sequences from sheep in Turkey, China, Spain, and goats in Tanzania. The obtained T. lestoquardi ms1 partial sequence formed a distinct cluster from other T. lestoquardi isolates in Pakistan and neighboring countries. Conclusion: Theileria spp. co-circulation in Pakistani small ruminants, particularly the presence of T. ovis and T. lestoquardi, highlights the need for attention from animal health decision-makers due to their financial and health impacts.

Microscopy and molecular survey of Hepatozoon spp. in domestic cats and their ticks: First report of H. silvestris from Türkiye

Hepatozoonosis is a tick-borne infection caused by protozoan species of the Hepatozoon genus in the family Hepatozoidae. This study aimed to determine the presence and prevalence of Hepatozoon spp. in domestic cats and their ticks, to characterize the genotypes, to reveal phylogenetic relationships, and to assess the risk factors associated with infection. Blood samples were collected in EDTA tubes from 311 cats that were brought to the Veterinary Faculty, Animal Hospital for treatment or routine control purposes. Smears were prepared from the buffy coat and examined microscopically for the presence of Hepatozoon spp. gamonts. Ticks were collected during the clinical examination of the cats and the species were identified. Genomic DNA obtained from buffy coats and ticks were analyzed using PCR analyses targeting the 18S rRNA gene region of Hepatozoon spp. The results showed that Hepatozoon spp. gamonts in neutrophils were determined in five samples as 1.6 % (5/311). A total of 58 (18.64 %, 58/311) were positive for the PCR analyses. Twelve ticks were collected from the cats, and all were identified as Rhipicephalus turanicus. Hepatozoon spp. were found in seven (58.3 %) ticks by molecular analyses. Sequence analysis of the 18S rRNA gene region of 58 positive isolates revealed the presence of H. felis (n = 25, 43.1 %) and H. silvestris (n = 33, 56.9 %) species in cats and H. felis in Rh. turanicus (n = 7). This study also presents the first report of H. silvestris in domestic cats from Türkiye. Phylogenetic analyses showed that our H. felis isolates clustered in H. felis genotype I and our H. silvestris were stated in the same cluster with Italian isolates.

Occurrence of Phytoplasmas Belonging to 16SrII Group Associated with Cyanthillium cinereum Witches'-Broom Diseases in China.

Cyanthillium cinereum, which belongs to the family of Asteraceae, is an annual or perennial herbaceous plant with significant medicinal uses for treating colds and fever. During September to November of 2020, C. cinereum showing symptoms of witches'-broom were found in economic forests distributed in Ding'an, Hainan Province of China, with 20% incidence. The symptoms of the plant were consistent with infections by 'Candidatus Phytoplasma' species. To identify the pathogen, five symptomatic and three asymptomatic C. cinereum samples were collected. Total DNAs were extracted using 0.10 g fresh leaf tissues of symptomatic and asymptomatic C. cinereum through a CTAB DNA extraction method according to Doyle and Doyle (1990). PCR amplification were performed employing the primer pairs of R16mF2/R16mR1 (Gundersen and Lee, 1996) and secAfor1/secArev3 (Hodgetts et al., 2008) specific for the conserved gene fragments of 16S rRNA and secA from phytoplasma. The PCR products were purified and sequenced through Biotechnology (Shanghai) Co., Ltd. (Guangzhou, China), and the obtained sequences were deposited in GenBank. The phytoplasmal 16S rRNA and secA gene fragments obtained in the study were all identical with the length of 1325 bp (GenBank accession: PP098738) and 741 bp (PP072217), respectively. The phytoplasma strain was described as CcWB-hnda. A BLAST search based on 16S rRNA genes indicated that CcWB-hnda strain was identical to phytoplasmas belonging to 16SrII group like peanut witches'-broom phytoplasma strain T48 (OR239773) and 'Ca. Phytoplasma aurantifolia' strain TB2022 (CP120449). Virtual RFLP profiles based on 16S rRNA gene fragments obtained by iPhyClassifier (Zhao et al., 2009) showed that CcWB-hnda strain was a member of 16SrII-A subgroup with 1.00 similarity coefficient to the reference phytoplasma strain (L33765). A BLAST search based on secA genes indicated that CcWB-hnda had 100% sequence identity with phytoplasmas belonging to 16SrII group such as 'Ca. Phytoplasma aurantifolia' isolate TB2022 (CP120449), Vigna unguiculata witches'-broom phytoplasma (OR661282) and Emilia sonchifolia witches'-broom phytoplasma (MW353710). Phylogenetic analysis based on 16S rRNA and secA genes by MEGA 7.0 employing Neighbor-Joining method with 1000 bootstrap value (Kumar et al., 2016; Felsenstein, 1985) demonstrated that CcWB-hnda was clustered into one clade with the phytoplasmas belonging to 16SrII group, with 98% and 100% bootstrap value respectively. To our knowledge, this is the first report of C. cinereum infected by phytoplasmas belonging to 16SrII-A subgroup in China. Identification of the vector insects of the pathogens is necessary in future, revealing the epidemiology of the related diseases. Phytoplasmas belonging to same 16Sr group or subgroup can infect different plants and spread through them in nature. The finding in this study will be beneficial to epidemic monitoring and early warning of C. cinereum witches'-broom disease and the related plant diseases caused by the phytoplasmas belonging to 16SrII group.

Detection of Virulence Genes Hla (α-hemolysis), CrtN (staphyloxanthin) for Staphylococcus aureus Isolated from Patients with Otitis and Rhinitis

Staphylococcus aureus is a bacterium that is capable of causing several types of infections acquired in hospitals and other healthcare facilities. The presence of S. aureus in the nasal cavity is significant for both the spread and development of infection. The goal of this study is to detect some virulence genes hla and CrtN of S. aureus. Thirty samples were collected from patients with otitis, and nose and throat infection from Al-Ramadi Teaching Hospital and external Labs for the period December 2021 to February 2022. Oti¬tis and Rhinitis swabs were taken from the patients and cultured on Mannitol salt agar and blood agar then gram stain and biochemical test were done. DNA was extracted from these isolates, and PCR was used to detect 16s rRNA, hla, and CrtN genes. The results showed after the phenotypic, microscopic, and biochem¬ical test, 20 isolates were referred to as S. aureus, which is approximately 66.6% of the total samples. The infection rate among youth and children was higher than that of the elderly and children in particular. Most bacterial infections of the pharynx, nose, and throat are the cause of S. aureus about 90%. DNA extract from 20 positive samples for S. aureus, the diagnosis of the samples was confirmed by 16s rRNA gene. The PCR results showed 18 samples carrying the hla gene by 90% of the total samples, while only 14 samples were carrying the CrtN gene at 80%.

Higher Microbial Abundance and Diversity in Bronchus-Associated Lymphoid Tissue (BALT) Lymphomas than in Non-Cancerous Lung Tissues.

It is well known that the majority of the extranodal marginal zone lymphomas of mucosa-associated lymphoid tissues (MALT lymphomas) are associated with microbiota, e.g., gastric MALT lymphoma with Helicobacter pylori. In general, they are very sensitive to low-dose radiotherapy and chemotherapeutic agents. The microbiota profile is not clearly elucidated in bronchus-associated lymphoid tissue (BALT) lymphoma, a rare type of MALT lymphoma in the lung. Thus, this study aimed to clarify the intratumor microbiome in BALT lymphoma using the third-generation NGS method. DNAs were extracted from 12 formalin-fixed paraffin-embedded (FFPE) tumor tissues obtained from BALT lymphoma patients diagnosed between 1990 and 2016. 16S rRNA gene was amplified by polymerase chain reaction. Amplicons were sequenced using a Nanopore platform. Next-generation sequencing analysis was performed to assess microbial profiles. For comparison, FFPE specimens from nine non-cancerous lung tissues were also analyzed. Specific bacterial families including Burkholderiaceae, Bacillaceae, and Microbacteriaceae were associated with BALT lymphoma by a linear discriminant analysis effect size approach. Although the number of specimens was limited, BALT lymphomas exhibited significantly higher microbial abundance and diversity with distinct microbial composition patterns and correlation networks than non-cancerous lung tissues. This study provides the first insight into intratumor microbiome in BALT lymphoma using the third-generation NGS method. A distinct microbial composition suggests the presence of a unique tumor microenvironment of BALT lymphoma.