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Correlation Between Infectivity and qRT-PCR Values for Murine Norovirus Recovered from Frozen Berries.

Human norovirus (HuNoV) is the leading cause of acute gastroenteritis globally, with frozen berries frequently implicated in foodborne outbreaks. Current surveillance relies on quantitative reverse transcription PCR (qRT-PCR), which cannot differentiate between infectious and non-infectious viral particles, complicating risk assessment. This study is aimed to establish the minimum viral load on frozen berries detectable by qRT-PCR that corresponds to infectious virus, using murine norovirus (MNV) as a surrogate for HuNoV. Frozen raspberries were artificially inoculated with serial dilutions of MNV (7.1-1.0 log PFU/25 g) and processed using the ISO 15216:2017 method. Infectious virus was quantified by plaque assay, and viral RNA was detected by qRT-PCR. The limit of detection (LOD) for cell culture was 3.1 log PFU/25 g, whereas qRT-PCR extended sensitivity to 1.0 log PFU/25 g (Ct value at 36.7 ± 0.6), representing a 2-log difference. Recovery rates for infectious virus exceeded the ISO 15,216 minimum threshold (1%), and PCR inhibition was negligible. We next examined the extraction efficiency for both infectious MNV and its genetic material from frozen strawberries at inoculation levels higher than the LOD, and observed that the viral recovery from frozen strawberries is very similar to viral recovery from frozen raspberries with no significant differences between them. The disparity between LODs indicates that a substantial proportion of MNV genomes detected by qRT-PCR do not represent infectious particles, aligning with previous findings that one PFU may correspond to multiple genome copies. Given that many surveillance studies report high Ct values (> 35), our data suggest that such detections may not indicate viable virus, underscoring the importance of contextualizing qRT-PCR results with epidemiological evidence. These findings highlight the need for cautious interpretation of surveillance data, particularly for public health decision-making.

Norovirus

Aflatoxins and their biosynthetic precursors in lotus seeds: simultaneous UPLC-MS/MS determination, contamination profiling, and matrix-specific accumulation during Aspergillus flavus infection.

Aflatoxin (AF) contamination poses a severe global threat to food and medicinal material safety, yet existing research focuses on terminal AF metabolites while neglecting residual biosynthetic precursors, leading to potential underestimation of contamination risks. In this study, a UPLC-MS/MS method was established for the simultaneous quantification of six AFs and their five precursors in lotus seeds, with optimization of mass spectrum parameters, chromatographic separation conditions, and sample pretreatment. Method validation confirmed linearity (R2&#xa0;>&#xa0;0.99), LODs (0.03-0.36&#xa0;&#x3bc;g/kg), and recoveries (76.53%-120.0%, RSD&#xa0;<&#xa0;15%). Analysis of 41 natural lotus seed samples revealed a 63.4% AF contamination rate, dominated by B-group AFs, while O-methylsterigmatocystin (OMST) and versicolorin hemiacetal (VOH) were identified as the primary co-residual precursors with co-occurrence rates &#x2265; 50%. Notably, AFM1 was predominantly detected in natural samples with AFB1 concentrations exceeding 100&#xa0;&#x3bc;g/kg. Artificial inoculation experiments further demonstrated that sterilization and sealing conditions modulated AF biosynthesis in lotus seeds, with non-sterilized and non-sealed groups showing delayed fungal metabolism and lower toxin accumulation. A significant linear correlation was observed between AFM1 and AFB1 levels (r&#xa0;=&#xa0;0.94) in infected samples, demonstrating their accumulation levels are coupled with fungal overall metabolic flux. Given the high co-occurrence rate of OMST/VOH with AFB1 in natural samples, their individual and combined toxicities require in-depth investigation. This work deciphers matrix-specific AF dynamics in lotus seeds, supporting regulatory standard refinement (e.g., precursor inclusion) and targeted control (e.g., time-sensitive drying after harvest). Further studies will focus on exploring the molecular mechanisms of substrate-dependent AF synthesis.

Aflatoxins

Host Range and Chemical Control of Cercospora citrullina, the Causal Agent of Watermelon Spot Disease.

This study systematically evaluated cultivation requirements, host range, and chemical control options for Cercospora citrullina causing watermelon spot disease. Among 11 chemically defined media tested, corn meal agar medium supported optimal mycelial growth of C. citrullina, with an average radial growth rate of 56.72 &#xb1; 1.45 mm under controlled conditions (25&#xb0;C, darkness). Host range determination via artificial inoculation of 19 plant species confirmed that the host range of the strain UNL090101 is limited to the Cucurbitaceae species tested, with watermelon (Citrullus lanatus) exhibiting the highest susceptibility, followed by melon (Cucumis melo) and cucumber (Cucumis sativus). Fungicide screening of 17 commercial formulations identified 40% iminoctadine tris (albesilate) WP (EC50 = 2.82 &#x3bc;g&#xb7;liter-1) and 64% mancozeb + 8% cymoxanil (WS) (EC50 = 48.75 &#x3bc;g&#xb7;liter-1) as the most effective treatments, achieving control efficacies of 74.47 and 58.62%, respectively. These findings provide actionable guidelines for optimizing crop rotation, intercropping strategies, and fungicide selection in watermelon production systems.

Cercospora citrullina

A simplified procedure for the examination of drinking water for bacteria of public health significance: the differential hydrobacteriogramme.

A new method is described which can be used for the examination of piped drinking water. It is also suitable for monitoring water which was initially of potable quality, and is intended for reuse in the food industry. The method is based on CLARK's "P-A test" and, because this allows many bacterial types to be detected, i.e. Enterobacteriaceae, E. coli, P. aeruginosa, Aeromonadaceae and LANCEFIELD group D streptococci it is called differential hydrobacteriogramme. A preliminary resuscitation treatment to revive sublethally injured cells is essential in this procedure. In earlier work this was attained by adding an equal volume of double strength nutrient broth and later double strength MACCONKEY purple broth, making the method somewhat bulky. In the new procedure, after the resuscitation step, a concentrated bile salts/indicator solution is added, allowing subsequent selective enrichment of the taxa sought. Positive enrichment cultures are examined for these organisms by the procedures summarized in Fig. 1. The new method, when tested on approx. 150 artificially inoculated and 92 natural samples, showed the same productivity and selectivity as the one introduced earlier. The new method is recommended for routine monitoring purposes, because it is less bulky.

Aeromonas

Genome-wide association study combined with multi-assay phenotyping identifies a novel anthracnose resistance locus in apple.

BACKGROUND: Apple anthracnose, a disease complex that includes Glomerella leaf spot (GLS) and bitter rot caused by Colletotrichum species, is a major disease affecting apple production worldwide. In this study, we combined multi-year field evaluations with controlled inoculation assays to identify genomic regions associated with anthracnose resistance in apple. RESULTS: A total of 440 apple genotypes, including 411&#xa0;F&#x2081; progenies derived from six parental crosses and 29 cultivars, were evaluated under natural orchard conditions and through artificial fruit and leaf inoculation assays using wound and non-wound methods. Disease severity varied substantially between years, particularly under contrasting environmental conditions, indicating strong genotype-by-environment interactions. Genome-wide association analysis (GWAS) using field-derived disease severity scores from 2019 identified a significant quantitative trait locus (QTL) on chromosome 15 (~&#x2009;31.8&#xa0;Mb) associated with reduced anthracnose severity. This locus was distinct from the previously reported Rgls/MdTNL1 region on chromosome 15 (~&#x2009;2-5&#xa0;Mb), suggesting the presence of a novel resistance-associated locus. In contrast, no genome-wide significant associations were detected from artificial inoculation datasets. CONCLUSIONS: These findings demonstrate the importance of field-based, multi-environment phenotyping for detecting field-relevant resistance loci and improving understanding of the genetic architecture underlying anthracnose resistance in apple.

Malus

Effect of chlorhexidine on plaque development in an artificial mouth.

The effect of chlorhexidine on the development of plaque, resulting from the inoculation with saliva of a tooth mounted in an artificial mouth, has been studied. The agent delayed the formation of plaque and also inhibited changes in pH and Eh, whether sucrose was present or not. Applied as a rinse at intervals of 12 h it prevented formation of plaque and pH and Eh remained constant. Moreover, chlorhexidine inhibited pH and Eh changes in established plaque. A single application of a gel containing chlorhexidine also greatly inhibited plaque development.

Administration, Topical

[Technical note on the isolation of arboviruses by inoculation in young mice: preparation of the mosquito brei].

Studying the effects of centrifugation and deep freezing on the quantity of yellow fever virus in a grinded pool of mosquitoes, the authors followed the mortality rate of inoculated baby mice with twenty five artificially infected mosquitoes treated in four different ways. The statistical analysis of the results show that centrifugation and deep freezing have both an effect on the titer of virus and that the addition of the two treatments have an effect superior to the addition of the separate effects of each of them. The authors propose a new technic for the preparation of pools of mosquitoes, without centrifugation or deep freezing.

Animals

[Media with yeast RNA for isolating Shigella flexneri from feces].

The authors demonstrated a considerable, in comparison with the control, increase of the biomass of Sh. flexneri causative agent, when growing in meat-peptone broth with 1-4 mg of NaRNA, and of the number of colonies in the feces of the patients and of healthy persons artificially contaminated with Sh. flexneri when inoculated in Ploskirev's agar or Endo medium with 4 mg/ml of NaRNA. Using media with NaRNA for laboratory diagnosis of dysentery is recommended.

Culture Media

Data-driven approaches in green microbiology: strategies for plant growth-promoting bacteria.

Plant growth-promoting bacteria (PGPB) are gaining attention as scalable biological solutions to enhance crop productivity and resilience. However, accurately identifying and characterizing PGPB remains challenging, particularly under variable environmental conditions where microbial functions are context-dependent and shaped by complex plant-microbe interactions. Advances in high-throughput sequencing have shifted the field from culture-dependent approaches to genome-informed strategies, enabling large-scale taxonomic and functional profiling. Although trait-based databases support the prediction of plant-beneficial genes, they capture only a fraction of the underlying biological complexity and often require labor-intensive analyses. Machine learning (ML) and deep learning (DL) have emerged as powerful tools to integrate genomic, physiological, and ecological data, enabling the prioritization of candidate strains with plant growth-promoting potential. To evaluate advances in the field, we conducted a systematic review of studies integrating ML and DL with PGPB characterization, assessing algorithm selection, performance, and target plant systems. Across 248 observations, only 6.0% of studies directly addressed PGPB screening, whereas the majority (77.4%) focused on plant disease detection, revealing a substantial gap in the application of AI to beneficial microorganisms for plant growth. Convolutional neural networks (CNNs) were the most frequently applied algorithms, largely driven by image-based phenotyping tasks. Overall, the field is constrained by limited datasets, high computational demands, and challenges in modeling multispecies and host-associated interactions. We highlight the need for integrative and interpretable ML and DL frameworks that bridge genomic data and functional validation. Such approaches represent a promising path toward scalable, data-driven discovery and deployment of bioinoculants in sustainable agriculture.

Agriculture

Diarrhoea in piglets inoculated with rotavirus.

A rotavirus isolated from a field outbreak of diarrhoea in artificially reared piglets was purified, filtered and administered orally to gnotobiotic and conventional piglets. Four successive passages of the virus in gnotobiotic piglets produced severe diarrhoea within 20 to 24 hours of administration. The diarrhoea lasted several days causing dehydration, emaciation, loss of body weight and some deaths. Virus was demonstrated in the faeces of the infected piglets by electron microscopy. Conventionally reared piglets developed little or no diarrhoea when given virus, whereas artificially reared piglets developed moderate to severe diarrhoea which lasted from 3 to 8 days with some deaths. No clinical disease was obvious in surviving piglets following challenge with the virus 10 or 17 days after initial infections.

Animal Nutritional Physiological Phenomena

Infectious bovine keratoconjunctivitis I. Experimental production.

One or both eyes of 20 calves were inoculated one or more time with variou(s combinations of microorganism (live oor killed Moraxella bovis, infectious bovine rhinotracheitis virus, bovine adenovirus, bovine parainfluenza-3 virus and Mycoplasma bovoculi) by conjunctival instillation or direct inoculation of the conjunctivea or cornea. The eyes of all the calves received natural or artificial ultraviolet irradiation. Neither the adenovirus nor parainfluenza-3 virus became established in the eye or produced keratoconjunctivitis. Both M. bovis and infectious bovine rhinotracheitis virus became established in the bovine eye and produced disease. Subconjunctival or intracorneal inoculation of M. bovis caused a severe disease, simulating natural infectious bovine keratoconjunctivitis. Only the intracorneal inoculation of mycoplasma produced severe keratoconjunctivits. Eyes that on initial exposure to M. bovis became severly inflamed were more resistant to a second or third exposure to M. bovis, presumably by enhanced local defence mechanisms.

Adenoviridae Infections

The spread of a pathogenic and an apathogenic strain of Newcastle disease virus in the chick embryo as depending on the protease sensitivity of the virus glycoproteins.

The pathogenic strain Italien and the apathogenic strain Ulster of Newcastle disease virus have been compared with respect to organ tropism and spread of infection in 11-day-old chick embryos. After infection of the endodermal layer of the chorioallantoic membrane by intra-allantoic inoculation with strain Italien, high virus titres are found in all extra-embryonic membranes and fluids and in the embryo itself. Infection results in early death of the embryo. In contrast, after infection with strain Ulster by the same route of inoculation, high virus titres are found only in the allantoic sac and embryos are not killed. Inoculations with strain Italien on to the ectodermal layer through an artificial air sac results in rapid spread of infection in the chorioallantoic membrane and the embryo dies before the virus invades other tissues including the embryo. Under the same conditions of infection, strain Ulster neither spreads within chorioallantoic membrane nor does it kill the embryo. Virus spread in each germinal layer of the chorioallantoic membrane was analysed by immune fluorescence. These studies showed that endoderm as well as mesoderm and ectoderm allowed the spread of strain Italien, whereas only the endoderm is permissive for strain Ulster. These differences in host range are based upon differential activation of the virus glycoproteins by proteolytic cleavage. The glycoproteins of strain Italien are cleaved in each germinal layer, whereas those of strain Ulster are cleaved only in endoderm. These studies demonstrate that, in the system analysed here, spread of infection and organ tropism are important factors for pathogenicity and both of these factors are determined by the susceptibility of the virus glycoproteins to proteolytic cleavage.

Allantois

The effect of natural and artificial breeding using bulls infected with, or semen contaminated with, infectious bovine rhinotracheitis virus.

Ten cows and heifers (Group B) were inoculated into the uterus at oestrus with semen followed by IBR virus for the first insemination and semen alone if a second insemination was necessary. All animals developed infectious pustular vulvo-vaginitis (IPV), and 2 cows conceived to the first and 2 to the second insemination (pregnancy rate of 40 percent requiring 4.5 services per conception). This group was compared with 10 control animals (Group A) which were treated similarly but received tissue culture fluid instead of virus at the first insemination. Group A had a pregnancy rate of 90 percent requiring 1.7 services per conception. Natural mating of 4 bulls with preputial infections due to infectious bovine rhinotracheitis (IBR) virus with 9 susceptible cows and heifers (group D), resulted in the production of lesions of IPV. The IPV infection did not affect their fertility (pregnancy rate of 89 percent requiring 1.4 services per conception) when it was compared to a similar group of females (group C) mated to the same bulls prior to infection with IBR virus (pregnancy rate of 100 percent requiring 1.2 services per conception). The 6 animals in Group B that were not pregnant and returned to oestrus 3 times were found on slaughter to have endometritis, salpingitis and vaginitis. A high incidence, 5 out of 18 (28 percent), of shortened oestrous cycles (less than 18 days) was a feature of the breeding pattern of this group. The undesirable consequences of distributing semen contaminated with IBR virus from artificial insemination centres are apparent.

Animals

Detection of arbovirus antibodies in avian sera by the complement fixation-inhibition test.

The complement fixation-inhibition (CFI) test was described for the detection of antibodies to arboviruses in bird sera. The CFI antibody present in bird sera inhibited the standard complement-fixation reaction of a reference complement-fixing antigen-antibody pair. Using reference antigens (St. Louis encephalitis, eastern equine encephalomyelitis, western equine encephalomyelitis, and yellow fever) prepared from infected mouse brains and reference antisera prepared in rabbits or horses, reproducible CFI antibody titers were obtained in artificially immunized chickens. Time-course studies on the CFI immune response in birds inoculated with live St. Louis encephalitis virus indicated that the CFI antibody was distinct from the antibody detected by the hemagglutination-inhibition test.

Animals

Macrophage activation and antitumor activity of a Brucella abortus ether extract, Bru-Pel.

Bru-Pel and Brucella abortus lipopolysaccharide (LPS) were tested for both macrophage activation and antitumor activity in an artificial metastasis model. Resting macrophages were rendered nonspecifically tumoricidal for MBL-2 lymphoblastic leukemia target cells by exposure to Bru-Pel at greater than or equal to 1 ng/ml of culture medium. B. abortus LPS failed to activate macrophages in vitro at all concentrations tested. Ip treatment of homozygous nude mice with Bru-Pel induced cytotoxic macrophages, indicating that Bru-Pel activated macrophages through a thymic-independent process. An artificial metastasis model was developed where single-cell suspensions of Madison 109 lung carcinoma were inoculated iv into syngeneic BALB/c mice. Bru-Pel, but not B. abortus LPS, strikingly inhibited tumor-colony formation in the lungs. Although Bru-Pel contains endotoxin, the data demonstrate that endotoxin is apparently not the active component by which Bru-Pel activates macrophages and enhances host resistance to cancer.

Animals

Host treatments affecting artificial pulmonary metastases: interpretation of loss of radioactively labelled cells from lungs.

The effect has been examined of various host treatments (C. parvum injection, immunization, thoracic irradiation, cyclophosphamide injection, and anticoagulation) on both lung colony formation and clearance of radioactive cells from the lungs after i.v. injection of tumour cells. Two tumour-host models have been used: the non-immunogenic KHT tumour in C3H/Km mice, and the immunogenic EMT6 tumour in BALB/c/Ka mice. Even for the at most weakly immunogenic KHT tumour, the number of artificial pulmonary metastases could be modified by a factor of up to 10(4) by different host treatments before i.v. inoculation of tumour cells. For all pretreatments except immunization, the shape of the curve of loss of radioactivity from the lungs vs time was biphasic, with an initial steep portion representing intravascular death of the tumor cells, followed 1--2 days after tumour-cell injection by a shallow exponential curve. It was concluded that the shallow slope represented spontaneous death of tumour cells in the perivascular tissues. Essentially all the injected tumour cells lodged initially in the lungs, and this was unaffected by the different host treatments. Furthermore, except for specific immunization, cell death in the perivascular tissues was also unaffected by host treatment. However, the survival of the tumour cells during the 24 h after injection (before they became extravascular) was extremely dependent on the particular host pretreatment. It would appear from these studies that host treatments such as C. parvum injection or anticoagulation can markedly affect the number of blood-borne pulmonary metastases, but they will only be effective if given before the tumor cells arrive in the lung vasculature.

Animals

Infection of artificial air pouches in the connective tissue of mice with Neisseria gonorrhoeae.

Artificial air pouches in the connective tissue of mice were evaluated as a means of studying Neisseria gonorrhoeae infections. Animals inoculated with type-1 N. gonorrhoeae cells developed an infection characterised by infiltration of polymorphonuclear leucocytes. Viable cocci could be recovered from the air pouches for up to 10 days after infection and intracellular cocci were evident in electronmicrographs within connective-tissue fibroblasts for at least 35 days, indicating that a persistent infection had been established. The mouse air pouch should be of value in the study of gonococcal and other infections.

Animals