Caloric restriction has been found to extend the lifespan of many organisms including mammals and other vertebrates. With lifespans exceeding months to years, age-related experiments involving fish and mammals can be overtly costly, both in terms of time and funding. The freshwater crustacean, Daphnia, has a relatively short lifespan (∼50 to 100 days), which makes it a cost-effective alternative animal model for longevity and aging studies. Besides age-specific mortality, there are a suite of physiological responses connected to \"healthspan\" that can be tracked as these animals age including growth, reproduction, and metabolic rates. These responses can be complemented by assessment of molecular and cellular processes connected to aging and health. Lifespan and metabolism of this model organism is responsive to long studied modulators of aging, such as rearing temperature and nutritional manipulation, but also pharmacological agents that target aging, e.g., rapamycin, which adds to its usefulness as a model organism. Here we describe how to culture Daphnia for aging experiments including maintaining laboratory populations of Daphnia mothers, growing algal food, and manipulating nutrition of these animals. In addition, we provide methods for tracking common physiological and longevity responses of Daphnia. This protocol provides researchers planning to use this model organism with methods to establish and maintain Daphnia populations and to standardize their experimental approaches. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culturing algae for Daphnia food Basic Protocol 2: General methods for culturing Daphnia Basic Protocol 3: Standardizing and controlling nutrition for experimental Daphnia Basic Protocol 4: Monitoring Daphnia lifespan Basic Protocol 5: Evaluating Daphnia health: Heart rate and respiration, body mass and growth rates, and reproduction.

Tuberculosis (TB) is among the most predominant infectious illnesses in developing areas around the globe. As stated by the World Health Organization (WHO), the number of instances of drug-resistant tuberculosis (DR-TB) has increased lately. This case report describes the effective diagnosis and customized treatment for primary extra-pulmonary multidrug-resistant tubercular pleural effusion, a disease which is difficult to identify due to relatively low bacterial count as well as frequently negative staining on Ziehl Neelsen (ZN) for acid-fast bacilli (AFB). The bacteria causing multidrug-resistant tuberculosis (MDR-TB) is resistant to a minimum of two drugs, isoniazid and rifampicin, the most effective TB medications. We are going to present the case of a 60-year-old male who complained of breathlessness, cough, and loss of weight for one month and chest pain and fever for 12 days. The patient\'s pleural fluid examination was carried out, which showed exudative fluid (according to Light\'s criteria) with adenosine deaminase (ADA) positive. Cartridge-based nucleic acid amplification test (CBNAAT) and line probe assays (LPAs) were carried out, which suggested mycobacterium tuberculosis (MTB) with rifampicin and isoniazid resistance. The patient was started an oral regimen with bedaquiline in accordance with WHO standards, leading to significant improvement. This case reveals that to promptly diagnose and treat DR-TB, pleural effusions, and pleural biopsies need to be exposed early to investigations such as Xpert (MTB)/resistance to rifampicin assay, culturing, and genotype drug sensitivity testing (DST).

Protists are key players in the biosphere. Here, we provide a perspective on integrating protist culturing with omics approaches, imaging, and high-throughput single-cell manipulation strategies, concluding with actions required for a successful return of the golden age of protist culturing.

Optimising diagnostic methods in shelters so that they are as economical as possible for the shelter is especially important because shelters often have a significant lack of funds and so usually do not carry out preventive screening of cats. Dermatophyte fungi spread quickly and can infect shelter staff. The aim of our work was to identify the occurrence of Microsporum canis in shelter cats. It aimed to determine the prevalence of M. canis in cats at the selected shelter and compare the efficiency of detection using a Wood\'s lamp and culturing on Sabouraud\'s agar. All cats present in the shelter at the time of the study (n = 70) were examined with Wood\'s lamp and hair sampling followed by subsequent culturing on Sabouraud\'s agar. Identification of fungi was based on microscopic proof of macroconidia and microconidia. The prevalence of M. canis by diagnosis on Sabouraud\'s agar was 64.29% of cats, with the help of Wood\'s lamp 48.57% of cats showed positive fluorescence. The sensitivity of the Wood lamp examination was 71% and the specificity was 92%. Our study suggests that Wood\'s lamp could be used by trained shelter personnel for the first examination of cats at reception and could significantly reduce the risk of spreading M. canis in shelters.

OBJECTIVE: Emerging evidence suggests that microbiome-targeted approaches may provide a novel opportunity to reduce the incidence of reproductive failures in cattle. To develop such microbiome-based strategies, one of the first logical steps is to identify reproductive microbiome features related to fertility and to isolate the fertility-associated microbial species for developing a future bacterial consortium that could be administered before breeding to enhance pregnancy outcomes. Here, we characterized the vaginal and uterine microbiota in beef cattle that became pregnant or remained open via artificial insemination and identified microbiota features associated with fertility. We compared similarities between vaginal and uterine microbiota and between heifers and cows. Using culturing, we provided new insights into the culturable fraction of the vaginal and uterine microbiota and their antimicrobial resistance. Overall, our findings will serve as an important basis for future research aimed at harnessing the vaginal and uterine microbiome for improved cattle fertility.

Human noroviruses are one of the leading causes of acute gastroenteritis worldwide. Based on quantitative microbial risk assessments, norovirus contributes the greatest infectious risk of any pathogen from exposure to sewage-contaminated water; however, these estimates have been based upon molecular (i.e., RNA-based) data as human norovirus has remained largely unculturable in the laboratory. Current approaches to assess the environmental fate of noroviruses rely on the use of culturable surrogate viruses and molecular methods. Human intestinal enteroids (HIEs) are an emerging cell culture system capable of amplifying viable norovirus. Here, we applied the HIE assay to assess both viable norovirus and norovirus RNA persistence in surface, tap, and deionized water microcosms. Viable norovirus decreased to below the detection limit in tap and deionized water microcosms and was measured in a single replicate in the surface water microcosm at study conclusion (28 days). Conversely, the norovirus RNA signal remained constant over the duration of the study, even when viable norovirus was below the limit of detection. Our findings demonstrate the disconnect between current environmental norovirus detection via molecular methods and viability as assessed through the HIE assay. These results imply that molecular norovirus monitoring is not inherently representative of infectious norovirus.

In this study, we evaluated the seminal and fecal microbiota in yearling beef bulls fed a common diet to achieve moderate (1.13 kg/day) or high (1.80 kg/day) rates of weight gain. Semen samples were collected on days 0 and 112 of dietary intervention (n = 19/group) as well as postbreeding (n = 6/group) using electroejaculation, and the microbiota was assessed using 16S rRNA gene sequencing, quantitative PCR (qPCR), and culturing. The fecal microbiota was also evaluated, and its similarity with seminal microbiota was assessed. A subset of seminal bacterial isolates (n = 33) was screened for resistance against 28 antibiotics. A complex and dynamic microbiota was detected in bovine semen, and the community structure was affected by sampling time (R2 = 0.16, P < 0.001). Microbial richness increased significantly from day 0 to day 112, and diversity increased after breeding (P > 0.05). Seminal microbiota remained unaffected by the differential rates of gain, and its overall composition was distinct from fecal microbiota, with only 6% of the taxa shared between them. A total of 364 isolates from 49 different genera were recovered under aerobic and anaerobic culturing. Among these seminal isolates were pathogenic species and those resistant to several antibiotics. Overall, our results suggest that bovine semen harbors a rich and complex microbiota which changes over time and during the breeding season but appears to be resilient to differential gains achieved via a common diet. Seminal microbiota is distinct from the fecal microbiota and harbors potentially pathogenic and antibiotic-resistant bacterial species. IMPORTANCE Increasing evidence from human and other animal species supports the existence of a commensal microbiota in semen and that this seminal microbiota may influence not only sperm quality and fertility but also female reproduction. Seminal microbiota in bulls and its evolution and factors shaping this community, however, remain largely underexplored. In this study, we characterized the seminal microbiota of yearling beef bulls and its response to the bull age, different weight gains, and mating activity. We compared bacterial composition between seminal and fecal microbiota and evaluated the diversity of culturable seminal bacteria and their antimicrobial resistance. Our results obtained from sequencing, culturing, and antibiotic susceptibility testing provide novel information on the taxonomic composition, evolution, and factors shaping the seminal microbiota of yearling beef bulls. This information will serve as an important basis for further understanding of the seminal microbiome and its involvement in reproductive health and fertility in cattle.

Functional characterization of the microbiome\'s influence on host physiology has been dominated by a few characteristic example strains that have been studied in detail. However, the extensive development of methods for high-throughput bacterial isolation and culture over the past decade is enabling functional characterization of the broader microbiota that may impact human health. Characterizing the understudied majority of human microbes and expanding our functional understanding of the diversity of the gut microbiota could enable new insights into diseases with unknown etiology, provide disease-predictive microbiome signatures, and advance microbial therapeutics. We summarize high-throughput culture-dependent platforms for characterizing bacterial strain function and host-interactions. We elaborate on the importance of these technologies in facilitating mechanistic studies of previously unexplored microbes, highlight new opportunities for large-scale in vitro screens of host-relevant microbial functions, and discuss the potential translational applications for microbiome science.

Collectively, we have been reviewers for microbial ecology, genetics and genomics studies that include environmental DNA (eDNA), microbiome studies, and whole bacterial genome biology for Microbial Ecology and other journals for about three decades. Here, we wish to point out trends and point to areas of study that readers, especially those moving into the next generation of microbial ecology research, might learn and consider. In this communication, we are not saying the work currently being accomplished in microbial ecology and restoration biology is inadequate. What we are saying is that a significant milestone in microbial ecology has been reached, and approaches that may have been overlooked or were unable to be completed before should be reconsidered in moving forward into a new more ecological era where restoration of the ecological trajectory of systems has become critical. It is our hope that this introduction, along with the papers that make up this special issue, will address the sense of immediacy and focus needed to move into the next generation of microbial ecology study.

Pure cultures of chicken intestinal microbial species may still be crucial and imperative to expound on the function of gut microbiota, and also contribute to the development of potential probiotics and novel bioactive metabolites from gut microbiota. In this study, we isolated and identified 507 chicken intestinal bacterial isolates, including 89 previously uncultured isolates. Among these, a total of 63 Lactobacillus strains, belonging to L. vaginalis, L. crispatus, L. gallinarum, L. reuteri, L. salivarius, and L. saerimneri, exhibited antibacterial activity against S. Pullorum. Acid tolerance tests showed Limosilactobacillus reuteri strain YPG14 (L. reuteri strain YPG14) has a particularly strong tolerance to acid. We further characterized other probiotic properties of L. reuteri strain YPG14. In simulated intestinal fluid, the growth of L. reuteri strain YPG14 remained stable after incubation for 4 h. The auto-aggregation test showed the auto-aggregation percentage of L. reuteri strain YPG14 was recorded as 15.0  ±  0.38%, 48.3  ±  2.51%, and 75.1  ±  4.44% at 3, 12, and 24 h, respectively. In addition, the mucin binding assay showed L. reuteri strain YPG14 exhibited 12.07 ±  0.02% adhesion to mucin. Antibiotic sensitivity testing showed that L. reuteri strain YPG14 was sensitive to the majority of the tested antibiotics. The anti-Salmonella Pullorum (S. Pullorum) infection effect in vivo revealed that the consumption of L. reuteri strain YPG14 could significantly improve body weight loss and survival rate of chicks infected by S. Pullorum; reduce the loads of S. Pullorum in the jejunum, liver, spleen, and feces; and alleviate the jejunum villi morphological structure damage, crypt loss, and inflammatory cell infiltration caused by S. Pullorum. Overall, this study may help us to understand the diversity of chicken intestinal microflora and provide some insights for potential probiotic development from gut microbiota and may find application in the poultry industry.