Molecular Diagnosis and Typing of Cryptosporidium spp. Species in Human Stools with Diarrhea.

OBJECTIVE: This study was conducted to molecularly identify and classify Cryptosporidium spp. in fecal samples (n=150) from patients with diarrhea received at the microbiology laboratory of a private hospital in Denizli. METHODS: In this study, the positivity of Cryptosporidium spp. in fecal samples was investigated using direct microscopy, Kinyoun's acid-fast staining method, and Nested polymerase chain reaction (PCR) techniques. Positive PCR products were sequenced. RESULTS: In the examined fecal samples of patients with diarrhea, no parasites were detected through direct microscopic examination. Using the Kinyoun acid-fast staining method, Cryptosporidium spp. was identified in 2.7% (n=4) of the samples, while Nested PCR detected it in 4.67% (n=7) of the samples. The four positive samples were sequenced using primers that amplify the 18S rRNA gene region. The sequencing results identified the isolates as C. parvum. CONCLUSION: Cryptosporidiosis is an important public health issue as it is a zoonotic disease caused by the Cryptosporidium parasite that can be transmitted from animals to humans. This study focuses on the molecular characterization of Cryptosporidium species detected in human fecal samples, which is significant for understanding which specific strains or species are involved in human infections. According to the findings, it is recommended that control measures be implemented to reduce the risk of exposure to Cryptosporidium in both humans and animals in Türkiye.

Cryptosporidium species and subtypes in Norway: Predominance of C. parvum and emergence of C. mortiferum.

AbstractPCR-based diagnostics has revealed the previously largely unknown Cryptosporidium transmission and infections in high-income countries. This study aimed to determine domestic and imported subtypes of Cryptosporidium species in Norway, evaluate their demographic distribution, and identify potential small outbreaks. Cryptosporidium-positive human faecal samples were obtained from six medical microbiology laboratories between February 2022 and January 2024, together with 22 Cryptosporidium-positive animal samples. Species and subtypes were identified by sequencing PCR products from gp60 and SSU rRNA genes. Most cryptosporidiosis cases occurred during late summer/early autumn, primarily in children and young adults. Of 550 human samples, 359 were successfully characterized molecularly (65%), revealing infection with 10 different Cryptosporidium species. C. parvum occurred in 245 (68%) human isolates with IIa and IId being major allele families, with distinct regional distribution patterns of common subtypes. A kindergarten outbreak with 5 cases was due to C. parvum IIaA14G1R1. C. mortiferum was identified in 33 (9.2%) human cases of which 24 were known to be of domestic origin, making it the second most common species in human autochthonous cases in Norway. All C. mortiferum isolates were of the same genotype; XIVaA20G2T1, including 13 cases from a suspected small outbreak in Trøndelag. C. hominis occurred in 68 typed cases (19%), but mostly in infections acquired abroad, with allele families Ib and If occurring most often. In conclusion, this study of recent Cryptosporidium spp. and subtypes in Norway, highlights the predominance of C. parvum and the emergence of C. mortiferum among autochthonous cases.

Fever of unknown origin, blood and cerebrospinal fluid involvement: a leprosy case report.

Leprosy is a chronic infectious disease that mainly affects the skin and peripheral nerves, it can also invade deeper tissues and organs, including mucous membranes, lymph nodes, testes, eyes, and internal organs. Severe cases can result in deformities and disabilities. We encountered the case of a 39-year-old male with unexplained fever, headache and rash. The patient's lesions were taken for histopathological examination and slit skin smear analysis. Further, the patient was detected of Mycobacterium leprae (M.leprae) nucleic acid sequences in the cerebrospinal fluid (CSF) and plasma, and M.leprae gene targets in the skin lesion tissue and blood. The patient was eventually diagnosed with multibacillary leprosy and type II leprosy reaction. These results suggest the possibility of bacteremia in patients with leprosy to some extent, and observation implies the potential invasion of CSF by M.leprae or its genetic material.

Use of a synthetic oligonucleotide to detect false positives caused by cross-contamination in nested PCR.

Nested PCR is a useful tool for identifying low-abundance target sequences of pathogens and avoiding false negatives. However, it carries an increased risk of cross-contamination, especially with its positive control. Here, we propose using customized synthetic oligonucleotides to detect false positives due to cross-contamination.

Optimizing the nested PCR method for Decapod iridescent virus 1 (DIV1) targeting ATPase gene by reselecting the inner primers.

DIV1 has the characteristics of fast transmission and a broad host range. Its infection leads to a high mortality rate, posing a serious threat to the global crustacean aquaculture industry. In order to increase the accuracy of DIV1 detection and reduce the difficulty of result interpretation, this study modified the original nested PCR method targeting the DIV1 ATPase gene. The internal primers for the nested PCR were redesigned to produce a 338 bp amplification product in the second step PCR, effectively distinguishing the target band from primer dimers. The newly established nested PCR method exhibits strong specificity and high sensitivity, with a detection limit as low as 1.37 × 101 copies/reaction. The developed nested PCR assay provides new technical support for the accurate detection of DIV1 in global crustacean aquaculture.

Comparison of PCR, Nested PCR, and RT-LAMP for Rapid Detection of Feline Calicivirus Infection in Clinical Samples.

Feline calicivirus (FCV) is a highly contagious virus that causes upper respiratory tract disease, commonly known as cat flu. It is widely distributed worldwide and poses a major threat to feline health. Therefore, it is essential to find an efficient and rapid method for detecting FCV. In this study, the colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay, using neutral red as an indicator, was developed and validated to target the ORF2 gene of FCV for the first time. Additionally, the study compared the diagnostic abilities of polymerase chain reaction (PCR), nested PCR, and RT-LAMP assays for detecting FCV in clinical samples. The optimized RT-LAMP amplification was carried out at 56.3 °C. The technique visually detected FCV within 70 min, with a limit of detection of 14.3 × 101 copies/µL, and showed no cross-reactivity with other feline pathogens. Out of 54 oropharyngeal swab samples, 17 tested positive for FCV using both nested PCR and RT-LAMP, while only one tested positive using conventional PCR. The positivity rate was higher with nested PCR and RT-LAMP (31.48%) compared to conventional PCR (1.85%). Consequently, these results demonstrated the effectiveness of the colorimetric RT-LAMP assay developed in this study as an alternative for diagnosing FCV in cats.

Pyrexia of Unknown Origin in a Young Male: Unmasking Melioidosis in a Tuberculosis-Endemic Setting.

A young male, plantation worker from Southeast Asia, presented with a non-productive cough, intermittent high-grade fever with chills, and significant weight loss over two months. Prior investigations were non-contributory, despite various antibiotics, his symptoms persisted. Physical examination and routine investigations, including an extensive microbiological workup for fever were non-contributory. A positron emission tomography-computed tomography (PET-CT) scan performed for pyrexia of unknown origin (PUO) revealed pulmonary consolidation, mediastinal lymphadenopathy, and splenic microabscesses. Material aspirated via endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) from the left interlobar lymph node was positive for Burkholderia pseudomallei on conventional nested polymerase chain reaction (PCR), confirming a diagnosis of melioidosis. Following appropriate antibiotic therapy, there was a complete resolution of symptoms. This case underscores the diagnostic challenges and the need for advanced techniques in identifying melioidosis, which can mimic tuberculosis.

Epidemiological Features of Leptospirosis and Identification of Leptospira wolffii as a Persistently Prevailing Species in North-Central Bangladesh.

Leptospirosis is considered to be the most widespread, yet neglected, re-emerging zoonotic disease caused by infection with a pathogenic species of the genus Leptospira. Although this disease is prevalent in Bangladesh, the recent epidemiological status has not yet been well documented. In this study, we aimed to determine the prevalence of leptospirosis among febrile patients using different diagnostic methods and to characterize the epidemiological features and species of Leptospira in Mymensingh, north-central Bangladesh. Among the blood samples of 186 patients with suspected leptospirosis who met the inclusion criteria, including having a fever for more than 5 days (November 2021-June 2022), 88 samples (47%) were Leptospira-positive according to IgM LAT, IgM ELISA, or nested PCR (positivity rates: 38%, 37%, and 42%, respectively). Nested PCR showed a significantly higher positivity rate (54%) in patients with a short fever (5-10 day) than the other methods did, with lower rates among those with a longer fever. Leptospirosis cases were more common in males (68%), those 16-45 years of age (70%), residents of rural areas (81%), and farmers (41%). In addition to a fever, myalgia and jaundice were found in more than 70% of the patients, while variable symptoms were observed. The 16S rRNA sequencing analysis revealed that the Leptospira species in all the 22 samples tested were L. wolffii, belonging to the pathogenic subclade P2. This study showed the recent epidemiological features of leptospirosis in Bangladesh, indicating the presumptive predominance of L. wolffii since 2019.

Nested PCR effective to detect low viral loads of SARS-CoV-2 in animal samples.

SARS-CoV-2 emerged from an animal source and was then transmitted to humans, causing the COVID-19 pandemic. Since a wide range of animals are susceptible to SARS-CoV-2 infection, the zoonotic potential of SARS-CoV-2 increases with every new animal infected. The molecular gold standard assay for SARS-CoV-2 detection is real-time RT-PCR, where the Ct obtained is proportional to the amount of nucleic acid and can be a semi-quantitative measure of the viral load. However, since the use of real-time RT-PCR assays in animal samples is low due to the high costs, the use of validated nested PCR assays will help to monitor large-scale animal samplings, by reducing the costs of detection. In the present study, 140 samples from dogs and cats (15 SARS-CoV-2-positive samples with Ct values from 27 to 33, and 125 negative samples), previously analyzed by real-time RT-PCR, were analyzed by nested PCR. To increase the number of positive samples to determine the sensitivity of the assay, 40 human samples obtained during COVID-19 diagnosis in 2020 were included. The specificity of the primers was analyzed against samples positive to canine coronavirus (CCV) and feline infectious peritonitis virus (FIPV). To calculate the limit of detection (LoD) of the nested PCR, the viral load was estimated extrapolating the Ct value obtained by real-time RT-PCR. The Ct values obtained were considered as semi-quantitative and were able to distinguish between high, moderate and low viral loads. The Kappa value or "agreement" between assays and reliability of the nested PCR were also determined. Eleven of the animal samples analyzed by nested PCR targeting the N gene were detected as positive, while 129 were detected as negative to the virus, with Ct values ranging between17 and 31.5. All the samples from humans analyzed by nested PCR were positive. These results indicate that the assay has a sensitivity of near 95 % and a specificity of 100 %. No unspecific reactions analyzed by nested PCR were observed with the samples positive to CCV and FIPV. The samples detected as positive to SARS-CoV-2 by nested PCR were those that presented a Ct between17 and 31.5. The LoD of the nested PCR was estimated close to 50 copies/µL of viral load, corresponding with a Ct of 31.5. The Kappa value between assays was excellent (k = 0.829). The results obtained demonstrate that nested PCR is useful to detect SARS-CoV-2 low viral loads at a lower cost than with real-time RT-PCR.

Rapid and sensitive detection of chikungunya virus using one-tube, reverse transcription, semi-nested multi-enzyme isothermal rapid amplification, and lateral flow dipstick assays.

Chikungunya fever is an acute infectious disease caused by chikungunya virus (CHIKV), which is transmitted by Aedes mosquitoes. Simple, rapid, and sensitive detection of CHIKV is critical for its prevention and spread. To address this issue, we combined one-tube, reverse transcription semi-nested, multi-enzyme isothermal rapid amplification, and lateral flow dipstick strips assay to detect CHIKV RNA. The study used a 318-bp gene fragment of CHIKV NSP4 as the target of the assay. This method of amplification takes 30 min for two-step amplification at 39°C. The dilution of amplification products was added to the LFD strip with results visible to the naked eye after 10 min. The method has a sensitivity of 1 copy/µL for the detection of CHIKV RNA, which is 100-fold higher than the conventional reverse transcription-multi-enzyme isothermal rapid amplification and 10-fold higher than the reverse transcription quantitative PCR (RT-qPCR) method. In addition, the method demonstrated good specificity and a better detection rate (85.7%, 18 of 21) than RT-qPCR (80.9%, 17 of 21) in clinically confirmed patient plasma samples. Thus, the rapid CHIKV RNA assay developed in this study will be an important tool for the rapid and accurate screening of patients for chikungunya fever. IMPORTANCE: This study presents a new one-tube, reverse transcription semi-nested, multi-enzyme isothermal rapid amplification assay combined with lateral flow dipstick strips for the detection of CHIKV. This technique significantly improves sensitivity and outperforms RT-qPCR for the detection of CHIKV, especially in samples with low viral loads. It is also significantly faster than conventional RT-qPCR and does not require special equipment or a standard PCR laboratory. The combination of the isothermal amplification technology developed in this study with point-of-care molecular testing offers the potential for rapid, on-site, low-cost molecular diagnosis of CHIKV.

Pathogenic Leptospira interrogans in Jos North Abattoir, Nigeria: Occurrence, serology, and molecular characterization.

Leptospirosis, a neglected zoonotic disease, adversely affects animal, human health, and socioeconomic conditions, particularly in developing countries like Nigeria. This study aimed to determine the occurrence and molecular identification of pathogenic Leptospira spp. among abattoir workers, cattle, and rats in Jos North, Plateau State, Nigeria. Using a cross-sectional study design, a total of 394 samples were collected, including 149 urine samples from abattoir workers, 125 urine samples from cattle bladders, and 120 bladders from trapped rats. Samples were processed and cultured in Ellinghausen McCullough Johnson Harrison (EMJH) medium and examined under a darkfield microscope. Positive cultures were confirmed using the Microscopic Agglutination Test (MAT) and nested Polymerase Chain Reaction (N-PCR) targeted the 16 S rDNA gene. Results revealed a prevalence of 33.76 % for Leptospira spp. across all samples, with the highest occurrence in abattoir workers (13.96 %), followed by rats (13.45 %), and cattle (6.35 %). The MAT showed L. interrogans serovar Hardjo str. Hardjoprajitno as the most prevalent serotype (41.61 %), followed by L. interrogans serovar Icterohaemorrhagiae str. RGA (34.31 %). N-PCR confirmed the presence of pathogenic Leptospira spp., showing bands of 1200 bp. Phylogenetic analysis of the 16 S rDNA gene sequences revealed close similarities to known pathogenic Leptospira strains from Brazil and the USA. The study underscores the significant public health risk posed by leptospirosis in Jos North and highlights the need for improved diagnostic capabilities, increased awareness, and effective control measures to mitigate the disease burden. Enhanced surveillance and preventive strategies are crucial to protect both animal and human health in the region.

N7-Ended Walker PCR: An Efficient Genome-Walking Tool.

As a tool for acquiring uncharacterized genomic DNA adjacent to characterized DNA, genome-walking is integral to bioscience-related research works. Herein, a new genome-walking tool known as N7-ended walker PCR (polymerase chain reaction) is presented. The key aspect for this method is the use of a degenerate walker primer in secondary/tertiary PCR. The 7 nt 5' tail of this primer completely degenerates to N7 relative to its corresponding primary walker primer. The degeneracy reduces the efficiency of annealing this primer to its predecessor site. Clearly, primary nontarget DNA defined by the primary walker primer prefers to form a hairpin structure via the inverted ends rather than hybridizing with the degenerate primer. As a result, N7-ended walker PCR achieves genome-walking by selectively boosting the DNA of interest. The feasibility of the N7-ended walker PCR method was proven by acquiring uncharacterized DNAs flanking several characterized DNAs.

Status of Cassava Witches' Broom Disease in the Philippines and Identification of Potential Pathogens by Metagenomic Analysis.

Cassava witches' broom disease (CWBD) is one of the most devastating diseases of cassava (Manihot esculenta Crantz), and it threatens global production of the crop. In 2017, a phytoplasma, Candidatus Phytoplasma luffae (Ca. P. luffae), was reported in the Philippines, and it has been considered as the causal agent, despite unknown etiology and transmission of CWBD. In this study, the nationwide occurrence of CWBD was assessed, and detection of CWBD's pathogen was attempted using polymerase chain reaction (PCR) and next-generation sequencing (NGS) techniques. The results showed that CWBD has spread and become severe, exhibiting symptoms such as small leaf proliferation, shortened internodes, and vascular necrosis. PCR analysis revealed a low phytoplasma detection rate, possibly due to low titer, uneven distribution, or absence in the CWBD-symptomatic cassava. In addition, NGS techniques confirm the PCR results, revealing the absence or extremely low phytoplasma read counts, but a surprisingly high abundance of fastidious and xylem-limited fungus, Ceratobasidium sp. in CWBD-symptomatic plants. These findings cast doubt over the involvement of phytoplasma in CWBD and instead highlight the potential association of Ceratobasidium sp., strongly supporting the recent findings in mainland Southeast Asia. Further investigations are needed to verify the etiology of CWBD and identify infection mechanisms of Ceratobasidium sp. to develop effective diagnostic and control methods for disease management.

Cryptosporidium Infections in Neonatal Calves on a Dairy Farm.

This study was conducted with the aim of the molecular identification of the protozoan parasite Cryptosporidium spp. in calves in the early stage of their development on a dairy farm in Eastern Slovakia. Twenty-five Holstein and Holstein cross calves were included in the study and monitored from their birth to the fifth week of life (1-5 weeks). Fresh fecal samples were collected from the same group of calves each week, except during the fourth week, and with the exception of Sample 8. All samples were analyzed using the Ziehl-Neelsen staining method and coproantigen was tested using the ELISA test as the screening method. Using the ELISA method, the highest incidence of cryptosporidiosis was observed in the second week of life of the calves, while the antigen was detected in 21 (91.6%) calves. Using the Ziehl-Neelsen staining method, the highest incidence was also observed in the second week, with an incidence rate of 62.5%. Positive isolates confirmed by the ELISA test were molecularly characterized. The species and subtypes of Cryptosporidium in the positive isolates were identified using PCR and the sequence analysis of the small subunit of the ribosomal 18S RNA (ssu rRNA) and the 60 kDa glycoprotein (gp60) genes of the parasite. The sequence analysis of 29 isolates at the 18S rRNA loci confirmed the presence of two species-Cryptosporidium parvum and Cryptosporidium ryanae. Out of 29 isolates, 25 were assigned to the species C. parvum, with the gp60 locus identified as genotype IIaA17G1R1. Among the individual animal groups, calves are the most common reservoirs of the C. parvum zoonotic species. This disease has significant public health implications as contact with livestock and their feces and working with barn manure are major sources of infection, not only for other animals but also for humans.

Innovative Semi-Nested Realtime PCR Assay with Extendable Blocking Probe for Enhanced Analysis of SEPT9 Methylation in Colorectal Cancer.

(1) Background: The detection of methylated SEPT9 (mSEPT9) in plasma is a promising approach to non-invasive colorectal cancer (CRC) screening. Traditional approaches have limitations in sensitivity and cost-effectiveness, particularly in resource-limited settings. (2) Methods: We developed a semi-nested realtime PCR assay utilizing extendable blocking probes (ExBP) to enhance the detection of low-level mSEPT9 based on DNA melting. This assay allows for the discrimination of mSEPT9 in the presence of high concentrations of non-methylated SEPT9 (up to 100,000 times higher). (3) Results: The assay demonstrated a sensitivity of 73.91% and specificity of 80%, showcasing its ability to detect very low levels of methylated DNA effectively. The innovative use of ExBP without costly modified probes simplifies the assay setup and reduces the overall costs, enhancing its applicability in diverse clinical settings. (4) Conclusions: This novel assay significantly improves the detection of mSEPT9, offering a potential advance in CRC screening and monitoring. Its cost-efficiency and high sensitivity make it particularly suitable for the early detection and management of CRC, especially in settings with limited resources. Future studies are encouraged to validate this assay in larger populations to establish its clinical benefits and practical utility.

Rapid Molecular Diagnosis of Sporotrichosis Directly from Biological Samples from a Reference Center in Brazil.

The gold standard diagnosis of sporotrichosis is the isolation of Sporothrix sp. in culture media, but this is a time-consuming test that is susceptible to contamination and can be affected by the fungal load. Molecular methods such as nested PCR are gaining more ground in the management of several infections as they are tools for the rapid and accurate identification of microorganisms from pure cultures or directly from biological samples. This study aimed to apply a nested PCR molecular protocol for the rapid detection of Sporothrix spp. directly from clinical samples. Thirteen samples-six from skin biopsies, five from skin exudates, and two from conjunctival secretions-were obtained from patients diagnosed with sporotrichosis due to S. brasiliensis. Calmodulin gene sequencing identified all the isolates as S. brasiliensis. Nested PCR was able to detect all the Sporothrix sensu lato directly from clinical samples as well as the CBS 120339 reference strain. The nested PCR protocol stands out as a diagnostic alternative, as it allows the identification of Sporothrix spp. directly from clinical samples without the need for fungal isolation.

Novel Semi-Nested Real-Time PCR Assay Leveraging Extendable Blocking Probes for Improved SHOX2 Methylation Analysis in Lung Cancer.

Lung cancer is the leading cause of cancer deaths globally, necessitating effective early detection methods. Traditional diagnostics like low-dose computed tomography (LDCT) often yield high false positive rates. SHOX2 gene methylation has emerged as a promising biomarker. This study aimed to develop and validate a novel semi-nested real-time PCR assay enhancing sensitivity and specificity for detecting SHOX2 methylation using extendable blocking probes (ExBPs). The assay integrates a semi-nested PCR approach with ExBPs, enhancing the detection of low-abundance methylated SHOX2 DNA amidst unmethylated sequences. It was tested on spiked samples with varied methylation levels and on clinical samples from lung cancer patients and individuals with benign lung conditions. The assay detected methylated SHOX2 DNA down to 0.01%. Clinical evaluations confirmed its ability to effectively differentiate between lung cancer patients and those with benign conditions, demonstrating enhanced sensitivity and specificity. The use of ExBPs minimized non-target sequence amplification, crucial for reducing false positives. The novel semi-nested real-time PCR assay offers a cost-effective, highly sensitive, and specific method for detecting SHOX2 methylation, enhancing early lung cancer detection and monitoring, particularly valuable in resource-limited settings.

Establishment and evaluation of a qPCR method for the detection of pfmdr1 mutations in Plasmodium falciparum, the causal agent of fatal malaria.

Drug resistance surveillance is a major integral part of malaria control programs. Molecular methods play a pivotal role in drug resistance detection and related molecular research. This study aimed to develop a rapid and accurate detection method for drug resistance of Plasmodium falciparum (P. falciparum). A quantitative real-time PCR (qPCR) assay has been developed that identifies the mutation at locus A256T in the P.falciparum multi-drug resistance(pfmdr1) gene producing amino acid change at position 86. The results of 198 samples detected by qPCR were consistent with nested PCR and sequencing, giving an accuracy of 94.3%. The sensitivity, specificity, positive and negative predictive value of qPCR were 85.7%, 97.6%, 90.0% and 96.4%, respectively. The results of qPCR are basically consistent with the nested PCR, which is expected to replace the nested PCR as a new molecular biological method for drug resistance detection, providing reliable technical support for global malaria prevention and control.

Analysis of serum proteomic profiles of endangered Siamese and Burmese Eld's deer infected with subclinical Babesia bovis in Thailand.

The endangered Eld's deer is a conserved species in Thailand, where tropical parasitic infections are endemic. Although Eld's deer with babesiosis are generally asymptomatic, they can still harbor the parasite and serve as reservoirs for ticks, spreading the infection to healthy animals within the herd. The present study aimed to investigate potential serum proteome biomarkers of Eld's deer with subclinical Babesia bovis infection. A total of 67 blood samples were collected from captive Siamese and Burmese Eld's deer showing no signs of parasitic infection. The nested polymerase chain reaction (nPCR) of a conserved spherical body protein 2 (sbp-2) gene of B. bovis was utilized to classify Eld's deer groups, with 25.37 % (17/67) testing positive for B. bovis. Additionally, the application of proteomic studies showed that six B. bovis proteins, such as Obg-like ATPase 1 (OLA1) and heat shock protein 90 (HSP90), were significantly upregulated by more than a two-fold change compared with the PCR-negative samples. Of the 55 overexpressed serum proteins in the PCR-positives, alpha 2-HS glycoprotein (AHSG) and immunoglobulin lambda variable 2-8 (IGLV2-8) were notably among the top 10 proteins with the highest area under curve (AUC) values. Hence, they were proposed as potential biomarkers for subclinical B. bovis infection in Eld's deer. Analysis of the protein interaction network revealed interactions between Eld's deer AHSG and B. bovis OLA1 and HSP90, alongside associations with other proteins such as erb-b2 receptor tyrosine kinase 2 (ERBB2) and epidermal growth factor receptor (EGFR). These interactions were involved in the immune system pathway and inflammatory responses. Our findings shed light on subclinical infection of B. bovis in Eld's deer and identify potential biomarkers, contributing to the further effective detection and monitoring of B. bovis infection in this endangered species.

Impact of whole grain highland hull-less barley on the denaturing gradient gel electrophoresis profiles of gut microbial communities in rats fed high-fat diets.

Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) is a traditional non-culture technique that can provide a fingerprint of the microbial community. In the field of gut microbiota analysis, PCR-DGGE still holds potential for development. In the present study, we utilized an improved nested PCR-DGGE approach targeting the V3 region of 16S ribosomal DNA to investigate the impact of whole grain highland hull-less barley (WHLB), a cereal known for its significant hypocholesterolemic effect, on the gut microbiota profiles of high-fat diet rats. Seventy-two male Sprague-Dawley rats were divided into four groups and fed a normal control diet, a high-fat diet, or a high-fat diet supplemented with a low or high dose of WHLB for 4 or 8 weeks. The results revealed that the dominant bands varied among different dose groups and further changed with different treatment times. The compositions of bacterial communities in feces and cecal content were similar, but the dominant bacterial bands differed. After performing double DGGE, extracting the bands, sequencing the DNA, and aligning the sequences, a total of 19 bands were classified under the Firmicutes and Bacteroidetes phyla, while two bands were identified as unclassified uncultured bacteria. The relative abundance of Lactobacillus gasseri, Uncultured Prevotella sp., and Clostridium sp. increased following the administration of WHLB. Illumina-based sequencing was employed to assess the reliability of DGGE, demonstrating its reliability in analyzing the dominant taxonomic composition, although it may have limitations in accurately detecting the alpha diversity of bacterial species. IMPORTANCE: While next-generation sequencing has overshadowed polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), the latter still holds promise for advancing gut microbiota analysis due to its unique advantages. In this study, we used optimized nested PCR-DGGE to investigate the gut microbiota profile of high-fat diet rats after administering whole grain highland hull-less barley. High-throughput sequencing was employed to validate the DGGE results. Our results proved the reliability of PCR-DGGE for analyzing the dominant taxonomic composition while also providing visual evidence of a notable relationship between the composition of cecal and fecal microbial communities, highlighting substantial differences in both richness and abundance.