Search Results (11,010)

Efficient RNA isolation from filamentous fungi is crucial for gene expression studies, but it poses significant technical challenges due to the robust cell walls and susceptibility of RNA to degradation by ribonucleases. This study presents the effectiveness of two RNA isolation protocols for four species of filamentous fungi: Penicillium crustosum, Penicillium rubens, Penicillium griseofulvum, and Aspergillus fumigatus. Both protocols utilized Fenzol Plus for cell lysis but varied in the mechanical disruption methods: bead-beating versus manual vortexing. The results show that the bead-beater method (Protocol 1) yielded significantly higher RNA quantities, with better purity and integrity, as demonstrated by higher A260/A280 and A260/A230 ratios. RNA concentrations ranged from 30 to 96 µg/g of dry biomass in Penicillium species and up to 52 µg/g in A. fumigatus. The use of chloroform in Protocol 1 also enhanced RNA purity, effectively separating contaminants such as DNA, proteins, and polysaccharides. This optimized protocol is highly efficient and can be applied in routine laboratories handling large numbers of fungal samples, making it a robust method for downstream applications such as cDNA synthesis and transcriptome analysis. Full article

Tularemia is an acute infectious disease classified as a natural focal infection, requiring continuous monitoring of both human and animal morbidity, as well as tracking of pathogen circulation in natural reservoirs and vectors. These efforts are essential for a comprehensive prevention and containment strategy. The causative agent, Francisella tularensis, comprises three subspecies—tularensis, holarctica, and mediasiatica—which differ in their geographic distribution and virulence. The ability to directly detect the pathogen and differentiate between subspecies has enhanced diagnostics and allowed a more accurate identification of circulation areas. Real-time PCR protocols for identification of F. tularensis subspecies tularensis and holarctica have been developed, utilizing specific primers and probes that target unique genomic regions. In this study, we present the development of a new real-time PCR assay for the detection of Francisella spp. and differentiation of F. tularensis subsp. mediasiatica. The specificity of the assay was tested on DNA from 86 bacterial species across 31 families unrelated to Francisella spp., as well as on DNA collections of F. tularensis subsp. mediasiatica and F. tularensis subsp. holarctica. The limit of detection (LOD95%) for real-time PCR in detecting Francisella spp. was 0.297 fg (0.145 genomic equivalents, GE) for holarctica DNA and 0.733 fg (0.358 GE) for mediasiatica DNA. The LOD95% for subspecies differential identification of mediasiatica was 8.156 fg (3.979, GE). The high sensitivity and specificity of these developed protocols enable direct detection of pathogens in biological and environmental samples, thereby improving the efficiency of tularemia surveillance in Kazakhstan. Full article

Coinfections by multiple pathogens, including viruses and bacteria, have severely impacted tilapia aquaculture globally. This study evaluated the impacts of dietary supplementation on red hybrid tilapia (Oreochromis spp.) coinfected with Tilapia lake virus (TiLV) and Aeromonas hydrophila. Fish were divided into three groups: a control group on a normal diet, and two experimental groups received diets supplemented with strategy A, an organic acid blend combined with a lyso-phospholipid-based digestive enhancer, and strategy B, an organic acid blend combined with natural immunostimulants and nutrients. Following exposure to both pathogens, the fish supplemented with strategies A and B showed lower cumulative mortality rates of 50.0% and 41.7%, respectively, compared to 76.3% in the control group. Notably, fish fed with strategy B-supplemented diet displayed a stronger immune response, with a lower expression of il-8, mx, and rsad2, and showed less pathological changes in the liver, spleen, and intestines, suggesting enhanced resistance to coinfection. In contrast, fish receiving strategy A did not exhibit significant changes in the immune-related gene expression or pathogen load, but demonstrate less pathological alterations, indicating intestinal protection. These findings highlight the potential of feed additives, particularly strategy B, to reduce the impact of virus-bacterial coinfections and improve outcomes in tilapia farming. Full article

Recurring outbreaks of cyclosporiasis linked to fresh produce demonstrate the need to develop interventions to reduce C. cayetanensis in irrigation water. C. cayetanensis is resistant to commonly used irrigation water treatments, such as chemical sanitizers, making removal of oocysts by filtration the most suitable intervention. This study evaluated the reduction of Eimeria tenella and E. acervulina, as surrogates for C. cayetanensis, in water using filters packed with sand alone or mixtures of sand and zero-valent iron (ZVI). Water inoculated with Eimeria spp. oocysts was filtered through laboratory-scale (PVC column) and field-scale (swimming pool filter) filters packed with either 100% sand or 50% ZVI/50% sand (v/v). Filtered and backflush water was examined microscopically for oocysts. Laboratory-scale filters with 50% ZVI significantly (p < 0.05) reduced 99.9% of E. tenella oocysts compared to 55.3% with filters containing 100% sand. At the field-scale level, 50% ZVI filters significantly (p < 0.05) reduced 70.5% of E. acervulina oocysts compared to 54.5% by 100% sand filters. Filters were backflushed to examine the recovery of these parasites during routine filter-media cleaning procedures. Backflush recovery of oocysts ranged from 4.42–16.7%. The addition of ZVI significantly improved the reduction of Eimeria spp. oocysts at both filter scales. and should be further investigated as a potential irrigation water intervention to reduce C. cayetanensis. Full article

Background/Objectives: Pandoraea species are Gram-negative, aerobic, rod-shaped bacteria that belong to the Burkholderiaceae family and the Betaproteobacteria class. Despite their rare occurrence in the general population, they have been increasingly observed as the causes of infection in immunocompromised individuals or patients with severe comorbidities. The present review seeks to examine all documented cases of Pandoraea spp. infections in humans, focusing on data related to epidemiology, microbiology, antimicrobial susceptibility, treatment options, and mortality rates. Methods: A systematic review was conducted through a literature search of the PubMed/MedLine and Scopus databases. This review is subjected to certain limitations regarding the data accuracy or pathogen identification molecular techniques applied in the studies. Results: In total, 29 studies provided information on 43 patients with Pandoraea spp. infections. The mean age of the patients was 42 years, and 58% were male. Cystic fibrosis was these patients’ most prevalent risk factor (39.5%). The most frequently reported types of infection were lower respiratory tract infections (74.41%) and bacteremia (30.23%), followed by infective endocarditis, pancreatitis, upper respiratory tract infection, and osteomyelitis (4.65%). P. apista was the most regularly isolated species (37.2%), while antimicrobial resistance was lower for carbapenems, especially for imipenem (17.14%). The most commonly administered antibiotics included carbapenems (82%), cephalosporins, and trimethoprim/sulfamethoxazole (35.89%). The infection outcome primarily depended on the type of infection; mortality rates were high (30.23%) and particularly elevated for bloodstream infections. The protocol for this review was registered in Prospero (ID: CRD42024579385). Conclusions: Due to Pandoraea’s unique antimicrobial resistance pattern and capacity to induce severe infection, clinicians should include it when making a differential diagnosis, especially in patients with severe comorbidities and immunodeficiency. Full article

Approximately 30 distinct Mycoplasma species have been isolated from cattle, but only a few are pathogenic and can cause serious respiratory diseases. Consequently, this study aimed to identify Mycoplasma spp. infections in cattle with bovine respiratory disease (BRD), considering factors such as animal demographics, concurrent infections with other pathogens, post-mortem clinical findings and histological examinations, and seasonality. A total of 326 samples were collected from 322 cattle that had died from BRD in Northwestern Italy. A total of 54 animals (16.8%) tested positive for Mycoplasma spp., and Mycoplasma bovis (n = 22, 40.7%) and Mycoplasma dispar (n = 13, 24.1%) were the most frequently detected species among the examined cattle. Among positive cattle, those aged five months or younger were approximately five times more likely to be infected by Mycoplasma dispar than by Mycoplasma bovis compared to those older than five months (proportional incidence ratio: 5.1, 95% CI 1.2–21.2). The main bacterial pathogens identified in cattle exhibiting co-infection was Pasteurella multocida, whereas the main viral pathogens were BRSV and BoHV-1. Histopathological investigations predominantly revealed catarrhal bronchopneumonia or purulent catarrhal bronchopneumonia among the examined cattle. Finally, Mycoplasma hyopharyngis, a species isolated from the pharyngeal and nasal cavities of pigs so far, was detected for the first time in the pneumonic lung of a bovine infected with BRD. Further investigations are necessary to thoroughly characterize its host range and pathogenic potential. Full article

In predictive microbiology, both primary and secondary models are widely used to estimate microbial growth, often applied through two-step or one-step modelling approaches. This study focused on developing a tool to predict the growth of Pseudomonas spp., a prominent bacterial genus in food spoilage, by applying machine learning regression models, including Support Vector Regression (SVR), Random Forest Regression (RFR) and Gaussian Process Regression (GPR). The key environmental factors—temperature, water activity, and pH—served as predictor variables to model the growth of Pseudomonas spp. in culture media. To assess model performance, these machine learning approaches were compared with traditional models, namely the Gompertz, Logistic, Baranyi, and Huang models, using statistical indicators such as the adjusted coefficient of determination (R²_adj) and root mean square error (RMSE). Machine learning models provided superior accuracy over traditional approaches, with R²_adj values from 0.834 to 0.959 and RMSE values between 0.005 and 0.010, showcasing their ability to handle complex growth patterns more effectively. GPR emerged as the most accurate model for both training and testing datasets. In external validation, additional statistical indices (bias factor, B_f: 0.998 to 1.047; accuracy factor, A_f: 1.100 to 1.167) further supported GPR as a reliable alternative for microbial growth prediction. This machine learning-driven approach bypasses the need for the secondary modelling step required in traditional methods, highlighting its potential as a robust tool in predictive microbiology. Full article

Freshwater ecosystems in Lithuania are threatened by the introduction of invasive fish species including Neogobius fluviatilis, N. melanostomus, Perccottus glenii, and Pseudorasbora parva. Data on helminth parasites of these fishes have not been comprehensively studied, with only two reports on parasites of N. melanostomus from the Curonian Lagoon and Baltic Sea, Lithuania. We examined 278 fish individuals representing 4 invasive species from 13 waterbodies. Using morphological and molecular analyses, we identified 29 helminth taxa representing 15 digenean trematodes, 6 nematodes, 4 cestodes, 2 acanthocephalans, and 2 monogeneans. With 18 species, N. fluviatilis had the highest helminth diversity, followed by N. melanostomus (11 species) and Ps. parva (8 species). Perccottus glenii was found to be free from helminth infection. The availability of historical information on the native fish parasites in Lithuania allowed us to conclude that out of the 29 recorded species, invasive fish serve as hosts for 22 local fish helminth species, while 7 helminth species have been reported exclusively in invasive fish. Based on newly obtained and previously published data, a total of 34 helminth species parasitise invasive fish in Lithuania, of which 30 use these fish as intermediate or paratenic hosts. Full article

White rot fungi, especially representatives of the genus Trametes spp. (Polyporaceae), are effective destructors of various xenobiotics, including oestrogens (phenol-like steroids), which are now widespread in the environment and pose a serious threat to the health of humans, animals and aquatic organisms. In this work, the ability of the white rot fungus Trametes hirsuta LE-BIN 072 to transform oestrone (E1) and 17β-oestradiol (E2), the main endocrine disruptors, was shown. More than 90% of the initial E1 and E2 were removed by the fungus during the first 24 h of transformation. The transformation process proceeded predominantly in the direction of the initial substrates’ detoxification, with the radical oxidative coupling of E1 and E2 as well as their metabolites and the formation of less toxic dimers in various combinations. A number of minor metabolites, in particular, less toxic estriol (E3), were identified by HPLC-MS. The formation of E1 from E2 and vice versa were shown. The exoproteome of the white rot fungus during the transformation of oestrogens was studied in detail for the first time. The contribution of ligninolytic peroxidases (MnP5, MnP7 and VP2) to the process of the extracellular detoxification of oestrogens and their possible metabolites is highlighted. Thus, the studied strain appears to be a promising mycodetoxicant of phenol-like steroids in aquatic environments. Full article

Genital Mycoplasmas are implicated in adverse pregnancy outcomes and the development of infertility. However, the role of Mycoplasma fermentans in these outcomes has not been adequately studied; therefore, its participation in these sufferings requires further investigation. This study aimed to evaluate the prevalence of M. fermentans in pregnant and non-pregnant women. End-point PCR was used to analyze two hundred and twenty-eight endocervical samples for M. hominis, M. genitalium, M. fermentans, M. pirum, Ureaplasma urealyticum, and U. parvum diagnoses. The prevalence of Mycoplasma spp. was as follows: U. parvum was found in 83 samples (36.4%), U. urealyticum in 39 instances (17.1%), M. hominis in 36 (15.7%), M. fermentans in 32 (14%), M. genitalium in 15 (6.6%), and M. pirum in 0 samples. No association was found between the Mycoplasma spp. and some infertility conditions or adverse pregnancy. However, M. fermentans and M. hominis were found to be associated with bacterial vaginosis (RR = 3.4 CI 95% 1.85–6.3, p < 0.005). In conclusion, M. fermentans and M. hominis were isolated more often in women with bacterial vaginosis, which suggests that these bacteria could contribute to the development of this pathology. Full article

Lactococcus spp. has emerged as a pathogen that is affecting global aquaculture, with L. garvieae, L. petauri, and L. formosensis causing piscine lactococcosis. While antimicrobials are commonly used to treat diseases in aquaculture, reports of antimicrobial resistance in fish isolates are increasing. However, little is known about the susceptibility patterns of Lactococcus spp. strains isolated from native fish species in Brazil. This study aimed to assess the antimicrobial susceptibility of these strains and establish a provisional epidemiological cutoff value for L. garvieae using the normalized resistance interpretation approach. A total of 47 isolates were tested: 17 L. garvieae, 24 L. petauri, and 6 L. formosensis. The isolates were classified as wild-type (WT) or non-wild-type (NWT) based on inhibition zone diameters. Isolates classified as NWT for three or more antimicrobial classes were considered multidrug-resistant, and the multiple antibiotic resistance (MAR) index was calculated. The results revealed heterogeneity in antimicrobial resistance profiles, with higher resistance to trimethoprim/sulfamethoxazole and norfloxacin. Resistance to other antimicrobials, including florfenicol and oxytetracycline (approved for use in Brazil), varied according to the bacterial species. Lactococcus petauri (87.5%) and L. formosensis (66.7%) showed the highest multidrug resistance, compared to L. garvieae (11.7%), along with higher MAR index values. These findings suggest that multidrug-resistant strains could pose future challenges in the production of native species, underscoring the need for ongoing monitoring of antimicrobial resistance and responsible use of antimicrobials in aquaculture. Full article

Long-chain polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) have been shown to be effective in enhancing the quality of ruminant products, including meat and milk. However, under these dietary conditions, the de novo lipogenesis could be influenced, too. On the other hand, even if the forage-to-concentrate ratio (F:C) is also a key factor affecting lipid metabolism in small ruminants, there is scarce information about its interaction with dietary PUFA. This study investigates the potential of the F:C ratio as a lever to manipulate lipid metabolism in dairy goats under high dietary PUFA supplementation. For this purpose, twenty-two crossbred dairy goats [Alpine × Local (Greek) breeds] (BW = 50.6 ± 6.1 kg) at early lactation (70 ± 10 days in milk) during the age of 3–4 years old, were separated into two homogeneous subgroups (n = 11). In the first phase, each goat was fed 20 g Schizochytrium spp./day followed by either a high-forage (20 HF) or a high-grain (20 HG) diet, while in the second phase, each goat was fed 40 g Schizochytrium spp./day followed once again either a high-forage (40 HF) or a high-grain (40 HG) diet. The F:C ratio of a high-forage and high-grain diet was 60:40 and 40:60, respectively. Tail fat tissue samples were collected by biopsy on the 42nd day of each experimental phase (last day). Significant decreases (p < 0.05) in the gene expression of ACACA, CBR2, COX4I1, ELOVL5, ELOVL7, LEP, and SCD were presented in goats fed 40 g compared to those fed 20 g Schizochytrium spp., while the gene expression of ACACA, AGPAT2, AGPAT3, ELOVL5, ELOVL6, EPHX2, FASN, and SCD was decreased in high grain compared to high-forage diets. This study also indicated that with the aim to enrich goat products with PUFA by increasing their levels in the diet, lipid metabolism is negatively affected. However, a diet with higher forage inclusion can partially attenuate this condition. Full article

Wisteria vein mosaic virus (WVMV, Potyvirus wisteriae), a virus belonging to the genus Potyvirus, is responsible for Wisteria vein mosaic disease (WMD), a severe disease that affects Wisteria, a genus of garden plants acclaimed worldwide. Although probably originating in the Far East, WVMV infection was first reported in the US, and subsequently in numerous countries. Following the first molecular detection of an Italian isolate, WVMV Bari, its full-length genome was achieved using NGS barcoding technology. A PhyML phylogenetic analysis, supported by clustering algorithm validation, identified a clear separation between two phylogroups. One major clade comprised WVMV strains isolated from Wisteria spp. A second clade grouped three highly divergent strains, at the borderline species threshold, all found in non-wisteria hosts. Relying on a Relative Time Dated Tips (RTDT) molecular clock, the first emergence of WVMV clades has been traced back to around the 17th century. A network inference analysis confirmed the sharp separation between the two host-related phylogroups, also highlighting the presence of potential intermediate variants. Inter-population genetic parameters revealed a very high genetic differentiation in both populations, which was made reliable by statistically significant permutation tests. The migrant number (Nm) and fixation index (F_ST) evidenced a restricted gene flow and strong population structures. According to the dN/dS ratio and negative neutrality tests, it was derived that purifying selection at the expense of non-silent variants is underway within WVMV populations. Targeting WVMV evolutionary traits, the present effort raised interesting questions about the underestimated potential of this culpably neglected species to spread in economically relevant crops. The main intention of our study is, therefore, to propose an evolution-based analysis approach that serves as a case study to investigate how other potyviruses or newly emerging viruses may spread. Full article

Wild animals are recognized as significant reservoirs for various zoonotic pathogens, including antibiotic-resistant bacteria. This study aimed to investigate the presence of Staphylococcus spp. strains in fallow deer (Dama dama) inhabiting a natural preserve in Central Italy and to examine the phenotypic and genotypic antimicrobial resistance and the presence of some virulence genes among the isolates. During July and December 2022, nasal swabs were collected from 175 fallow deer, which were then analyzed through bacteriological cultures. In total, 176 Staphylococcus spp. strains were isolated and subsequently identified using MALDI-TOF mass spectrometry. S. aureus was the most abundant species with 66 (37.5%) strains, followed by S. hyicus, 34 (19.31%) strains, S. sciuri, 32 (18.18%) strains, S. chromogenes, 27 (15.34%) strains, S. xylosus, 11 (6.25%) strains, S. warneri, 5 (2.84%) strains, and S. devriesei, 1 (0.56%) strain. Antimicrobial susceptibility was assessed for each isolate via the agar disk diffusion method, testing a panel of 13 molecules belonging to 9 antimicrobial classes. The highest resistance rates were detected for penicillin (29.55%), rifampicin (22.73%), and amikacin (20.45%). Notably, intermediate susceptibility was observed for erythromycin (61.93%), enrofloxacin (28.41%), and ceftiofur (21.02%). Conversely, the strains exhibited particularly high susceptibility to amoxicillin/clavulanic acid (99.43%), cefoxitin (97.73%), and vancomycin (96.02%). Based on the results, 32 (18.18%) isolates were classified as multidrug-resistant (MDR). Two strains of S. chromogenes and one strain of S. xylosus, both resistant to penicillin, tested positive for the blaZ gene. No methicillin-resistant strains were found, and none of the isolates harbored genes associated with enterotoxin and toxic shock syndrome toxin production. This study highlights the potential role of wildlife, particularly fallow deer, as reservoirs of antibiotic-resistant Staphylococcus spp. strains. Such findings underscore the importance of monitoring wildlife for antimicrobial resistance, which could have implications for public health and veterinary medicine. Full article

Many bacteria, including methicillin-resistant staphylococci and opportunistic pathogens such as Acinetobacter (A.) species, in particular members of the A. calcoaceticus—A. baumannii (Acb) complex, are known to survive in environmental settings. This increases the risk of bacterial spreading and transmission to animals and humans, especially in institutions with a high animal population density. This study aimed to identify the presence of multidrug-resistant (MDR) staphylococci and Acinetobacter species in dog daycare facilities (DDFs). The surfaces of 16 DDFs were sampled and information about cleaning practices was obtained. After microbial identification, the detected isolates were investigated by multiplex PCR for antimicrobial resistance genes. In 200 location samples, 38 staphylococci and 109 Acinetobacter spp. isolates were identified. Methicillin-resistant genes were confirmed in 18 staphylococci isolates from four DDFs, including Staphylococcus (S.) equorum, S. saprophyticus, S. cohnii, S. lentus, and S. haemolyticus. Eight A. radioresistens isolates comprising the bla_OXA-23 carbapenemase gene and seventeen isolates belonging to the Acb complex were also isolated. This is the first investigation for MDR pathogens in DDFs, a close human–animal interaction environment. Though the role of DDFs in the transmission and spreading of MDR bacteria is not known, the findings should contribute to the public awareness and underscore the necessity of adequate cleaning protocols. Full article