Isolation and propagation of bovine blood-derived macrophages using a mixed culture with bovine endothelial B46 cells.

Macrophages are innate immune cells with multiple functions such as phagocytosis, cytokine production, and antigen presentation. Since macrophages play critical roles in some bacterial infectious diseases in cattle, including tuberculosis, paratuberculosis, and brucellosis, the in vitro culturing of bovine macrophages is useful for evaluating host-pathogen interactions at the cellular and molecular levels. We have previously reported the establishment of two immortalized bovine liver sinusoidal cell lines, endothelial B46 cells and myofibroblast-like A26 cells (Cell Biology International, 40, 1372-1379, 2016). In this study, we investigated the use of these cell lines as feeder cells that support the proliferation of bovine blood-derived macrophages (BBMs). Notably, the B46 cell line efficiently acts as feeder cells for the propagation of BBMs. Compared with primary cultured vascular endothelial cells, the infinite proliferation ability of B46 cells is more beneficial for preparing confluent feeder layers. In conclusion, this study provides a simple and efficient protocol for the isolation and propagation of BBMs using a primary mixed culture of bovine whole blood with B46 feeder cells. Isolated BBMs are expected to be useful for developing in vitro models for studying the interactions between bovine pathogens and host immune cells.

Variants in KIF2A cause broad clinical presentation; the computational structural analysis of a novel variant in a patient with a cortical dysplasia, complex, with other brain malformations 3.

Cortical dysplasia, complex, with other brain malformations 3 (CDCBM3) is a rare autosomal dominant syndrome caused by Kinesin family Member 2A (KIF2A) gene mutation. Patients with CDCBM3 exhibit posterior dominant agyria/pachygyria with severe motor dysfunction. Here, we report an 8-year-old boy with CDCBM3 showing a typical, but relatively mild, clinical presentation of CDCBM3 features. Whole-exome sequencing identified a heterozygous mutation of NM_001098511.2:c.1298C>A [p.(Ser433Tyr)]. To our knowledge, the mutation has never been reported previously. The variant was located distal to the nucleotide binding domain (NBD), in which previously-reported variants in CDCBM3 patients have been located. The computational structural analysis showed the p.433 forms the pocket with NBD. Variants in KIF2A have been reported in the NBD for CDCBM3, in the kinesin motor 3 domain, but not in the NBD in epilepsy, and outside of the kinesin motor domain in autism spectrum syndrome, respectively. Our patient has a variant, that is not in the NBD but at the pocket with the NBD, resulting in a clinical features of CDCBM3 with mild symptoms. The clinical findings of patients with KIF2A variants appear restricted to the central nervous system and facial anomalies. We can call this spectrum "KIF2A syndrome" with variable severity.

A frameshift mutation in the rRNA large subunit methyltransferase gene rlmA II determines the susceptibility of a honey bee pathogen Melissococcus plutonius to mirosamicin.

American foulbrood (AFB) and European foulbrood (EFB) caused by Paenibacillus larvae and Melissococcus plutonius, respectively, are major bacterial infections of honey bees. Although macrolides (mirosamicin [MRM] and tylosin) have been used to prevent AFB in Japan, macrolide-resistant P. larvae have yet to be found. In this study, we revealed that both MRM-resistant and -susceptible strains exist in Japanese M. plutonius and that a methyltransferase gene (rlmA II ) was disrupted exclusively in MRM-susceptible strains due to a single-nucleotide insertion. The M. plutonius RlmAII modified G748 of 23S rRNA, and the deletion of rlmA II resulted in increased susceptibility to MRM and the loss of modification at G748, suggesting that methylation at G748 by RlmAII confers MRM resistance in M. plutonius. The single-nucleotide mutation in MRM-susceptible strains was easily repaired by spontaneous deletion of the inserted nucleotide; however, intact rlmA II was only found in Japanese M. plutonius and not in a Paraguayan strain tested or any of the whole-genome-sequenced European strains. MRM has been used in apiculture only in Japan. Although M. plutonius is not the target of this drug, the use of MRM as a prophylactic drug for AFB may have influenced the antibiotic susceptibility of the causative agent of EFB.

Vaginitis with purulent vaginal discharge caused by artificial insemination using frozen Histophilus somni-contaminated semen.

In April 2020, two cows in Japan, developed reproductive disorders accompanied by vaginitis with purulent discharge within 3 days of artificial insemination (AI) with the same lot of frozen semen. Histophilus somni was isolated from the vaginal swabs of both cows as well as from the same lot of frozen semen used for the AI. This incident marks the first reported case of H. somni infection in cattle through AI. The major outer membrane protein gene sequences and pulsed-field gel electrophoresis profiles of the isolates were identical. Moreover, we investigated the antimicrobial activity of 12 frozen semen straws against an H. somni isolate using a disk diffusion test. These straws were sourced from five AI centers and included the same lot of semen used for the AI. Although the composition of semen diluents from individual AI centers is not publicly available, both the same lot of frozen semen used in the AI and other lots produced by the same manufacturer showed lower antimicrobial activity than semen from other manufacturers. These results strongly suggest that the two vaginitis were caused by AI using H. somni-contaminated frozen semen because of insufficient antimicrobial activity to inhibit bacterial growth. The minimum inhibitory concentrations of the six antimicrobials recommended for addition to frozen semen in isolates were below the recommended concentrations, suggesting that proper addition could have prevented this incident. This highlights the importance of conducting periodical checks on the antibacterial activity of frozen semen to prevent the transmission of pathogens via AI.

Hematite (α-Fe2O3) with Oxygen Defects: The Effect of Heating Rate for Photocatalytic Performance.

Hematite (α-Fe2O3) emerges as an enticing material for visible-light-driven photocatalysis owing to its remarkable stability, low toxicity, and abundance. However, its inherent shortcomings, such as a short hole diffusion length and high recombination rate, hinder its practical application. Recently, oxygen vacancies (Vo) within hematite have been demonstrated to modulate its photocatalytic attributes. The effects of Vo can be broadly categorized into two opposing aspects: (1) acting as electron donors, enhancing carrier conductivity, and improving photocatalytic performance and (2) acting as surface carrier traps, accelerating excited carrier recombination, and deteriorating performance. Critically, the generation rate, distribution, role, and behavior of Vo significantly differ for synthesis methods due to differences in formation mechanisms and oxygen diffusion. This complexity hampers simplified discussions of Vo, necessitating careful investigation and nuanced discussion tailored to the specific method and conditions employed. Among various approaches, hydrothermal synthesis offers a simple and cost-effective route. Here, we demonstrate a hydrothermal synthesis method for Vo introduction to hematite using a carbon source, where variations in the heating rate have not been previously explored in terms of their influence on Vo generation. The analyses revealed that the concentration of Vo was maximized at a heating rate of 16 °C/min, indicative of a high density of surface defects. With regard to photocatalytic performance, elevated heating rates (16 °C/min) fostered the formation of Vo primarily on the hematite surface. The photocatalytic activity was 7.1 times greater than that of the sample prepared at a low heating rate (2 °C/min). These findings highlight the crucial role of surface defects, as opposed to bulk defects, in promoting hematite photocatalysis. Furthermore, the facile control over Vo concentration achievable via manipulating the heating rate underscores the promising potential of this approach for optimizing hematite photocatalysts.

Effects of dehydration on immune functions after a judo practice session.

We investigated the effects of dehydration after a judo practice session on player muscle and immune functions. Subjects included 25 female university judoists. Investigations were performed before and after 2.5 h of regular judo practice. Body composition, serum enzymes (myogenic enzymes, immunoglobulins and complements), neutrophils counts, reactive oxygen species (ROS) production capability, and phagocytic activity (PA) were measured. Subjects were divided into two groups according to level of dehydration after practice (mild dehydration and severe dehydration groups) and results were compared. Creatine kinase was found to increase significantly after practice. In addition, neutrophil count also increased significantly after practice in both groups. The changing ratios of IgA, IgG and C3 observed in the mild dehydration group were significantly higher than those in the severe dehydration group. In the severe dehydration group, post-practice PA/neutrophil had decreased significantly. Significant positive correlations were found between severity of dehydration and changing ratios of IgA, IgG, IgM, C3, C4 and ROS production capabilities, whereas no significant association was seen with PA and/or serum SOD activity. These results suggest that dehydration resulted in immunosuppression, including decreased neutrophil function.

Changes in immune functions during a peaking period in male university soccer players.

The concept of peaking ensures that athletes have trained to attain their absolute peak performance levels prior to a competition. This study investigates the effects of peaking on the functions of neutrophils and lymphocytes in university soccer players during a five-day soccer training camp followed by two weeks of tapering. The study subjects were 22 soccer players who were members of a university soccer club. We carried out our investigation during a five-day training camp and two weeks after the training camp (i.e. the tapering period). We measured body composition, immune-related parameters (leukocyte count, neutrophil count, lymphocyte count, immunoglobulins and complements), myogenic enzymes, superoxide dismutase activity (SOD) and neutrophil functions [reactive oxygen species (ROS) production, phagocytic activity, serum opsonic activity and lymphocyte subtypes]. Leukocyte and neutrophil counts tend to increase after the training camp compared with values before the training camp, and recovered during the conditioning period, although the final values were still lower than those before the training camp. The amount of ROS production per neutrophil and level of SOD decreased significantly during the conditioning period compared with before the training camp (p < 0.05, p < 0.01, respectively). Levels of Th1 cells decreased significantly during the conditioning period compared with the training camp (p < 0.05). In conclusion, the period of two weeks for the tapering period was considered insufficient to allow the athletes' immune function to recover completely after an intensive training camp.

Draft genome sequence of Pasteurella multocida strain BD1769 with untypable capsular serotype isolated from a layer chicken.

We report the draft genome sequence of Pasteurella multocida strain BD1769 (GenBank accession numbers JARFTQ010000001-JARFTQ010000021) isolated in 2021 from a layer chicken in Japan. No gene locus for capsular biosynthesis was annotated in the genome of this strain.

Development of a Streptococcus pluranimalium-specific PCR assay.

Streptococcus pluranimalium, an emerging zoonotic pathogen associated with infections in various animal species and humans, cannot be reliably identified by phenotypic characterization using the commercial kits routinely used in laboratories. We herein developed the first S. pluranimalium-specific PCR assay useful for the easy and reliable identification of this species.

Antimicrobial resistance and associated genetic background of Histophilus somni isolated from clinically affected and healthy cattle.

Histophilus somni, a member of the Pasteurellaceae family, causes various diseases, including thrombotic meningoencephalitis and respiratory diseases. Here, 166 isolates recovered from Japanese cattle with various diseases between the late 1970s and the 2010s were subjected to susceptibility testing against 14 antimicrobials (ampicillin, amoxicillin, cefazolin, ceftiofur, kanamycin, streptomycin, nalidixic acid, enrofloxacin, danofloxacin, florfenicol, erythromycin, tylosin, oxytetracycline, and fosfomycin). The proportions of antimicrobial-resistant/intermediate isolates were low in the total isolates, with resistance rates ranging from 0% for ceftiofur and florfenicol to 13.2% for ampicillin. However, relatively high minimum inhibitory concentrations (MICs) and resistance/intermediate rates were observed in the isolates from cattle with respiratory diseases; i.e., 21/53 isolates (39.6%) showed resistance or intermediate to one or more antimicrobials for treatment of respiratory diseases, and the resistance/intermediate rates to oxytetracycline, kanamycin, ampicillin, amoxicillin, nalidixic acid, and danofloxacin were 28.3, 24.5, 24.5, 13.2, 1.9, and 1.9%, respectively. Isolates with high MICs tended to possess antimicrobial resistance genes, which may confer antimicrobial resistance phenotypes. In particular, all isolates with MICs of ampicillin/amoxicillin, kanamycin, and oxytetracycline ≥2 µg/mL, ≥512 µg/mL, and ≥4 µg/mL possessed bla ROB - 1, aphA-1, and tetH/tetR, respectively, whereas isolates whose MICs were lower than the above-mentioned values did not possess these resistance genes. These results suggest that the resistance genes detected in this study are primarily responsible for the reduced susceptibility of H. somni strains to these antimicrobials. As integrative and conjugative element (ICEs)-associated genes were detected only in genetically related isolates possessing antimicrobial resistance genes, ICEs may play an important role in the spread of resistance genes in some genetic groups of H. somni strains.

Complete Genome Sequence of Mycobacterium avium subsp. paratuberculosis Strain 42-13-1, Isolated in Japan.

Here, we report the complete genome sequence of Mycobacterium avium subsp. paratuberculosis strain 42-13-1, isolated from cattle presenting with chronic diarrhea caused by Johne's disease in Japan, which was assembled via long- and short-read hybrid assembly.

Immunohistochemical and molecular analysis of Pasteurella multocida in a rabbit with suppurative pleuropneumonia.

A 1-month-old rabbit, imported as a pet by a distributor, died suddenly in the quarantine period in Japan due to suppurative pleuropneumonia. A bacterial isolate from its right lung was identified as Pasteurella multocida serotype A: 11. The isolate was classified as ST204 using the RIRDC scheme of multilocus sequence typing, suggesting that the isolate was genetically related to European isolates of the same sequence type listed in the PubMLST database and not to four other isolates that originated from past imported rabbits. In the immunohistochemical assay, an antiserum recognizing the somatic serotype 11 antigen generated from chicken could specifically detect P. multocida, indicating that the antiserum for somatic serotyping was useful for immunohistochemical diagnosis of rabbit pasteurellosis.

Molecular characterization of Brucella ceti from a bottlenose dolphin (Tursiops truncatus) with osteomyelitis in the western Pacific.

Although the presence of Brucella spp. in the western Pacific has been suggested by epidemiological studies on cetaceans, it has not been confirmed by bacterial isolation. Here, for the first time, we report that a marine Brucella strain was isolated in the western Pacific from a bottlenose dolphin with osteomyelitis. The isolate from the lesion was confirmed to be B. ceti of sequence type 27 by multilocus sequence typing and Bruce-ladder PCR. Infrequent-restriction-site PCR and omp2 gene sequencing revealed that molecular characteristics of this isolate were similar to those of Brucella DNA previously detected from minke whales in the western North Pacific. These results suggest that genetically related Brucella strains circulate in cetacean species in this region.

Draft Genome Sequence of Brucella ceti Isolated in the Western Pacific Ocean.

In 2018, Brucella ceti was isolated from a bottlenose dolphin from the western Pacific Ocean. Here, we report a draft genome sequence of the isolate BD1442 of sequence type 27, which is the only sequence type known to have been isolated from human clinical cases.

Avian influenza and Newcastle disease viruses from northern pintail in Japan: isolation, characterization and inter-annual comparisons during 2006-2008.

Since wild ducks constitute a vital element in the epizootiology of avian influenza viruses (AIVs) as well as avian paramyxoviruses (APMVs) and play a key role in the ecology and inter-species transmission of these viruses, it is crucial to elucidate the diversity and prevalence of these viruses within these bird populations. This report shows the presence, antigenic diversity, and inter-annual prevalence variations of AIVs in apparently healthy northern pintail (Anas acta) wintering in Japan. We also provide evidence that this host carries APMV-1: Newcastle disease virus (NDV) and other haemagglutinating viruses. Composite samples (n=2381) of fresh fecal materials were collected from northern pintail during November 2007-March 2008 at different locations of Tohoku district, main Island, Japan. We isolated 47 haemagglutinating viruses, out of which 25 were identified as AIVs, representing 9 combinations of 5 different haemagglutinin (HA) and 6 neuraminidase (NA) subtypes. Both H5 and H7 subtypes were identified and found to be low pathogenic. A further 11 viruses were grouped into APMV-1 (NDV). The rest of the viruses (n=11) remained to be identified. Some of the HA subtypes and NA subtypes detected during the first season reoccurred in the second season, as well as some of their combinations; yet, several new subtypes and combinations appeared during the second season. These findings indicate that different subtypes of AIVs, NDV and other haemagglutinating viruses circulate subclinically in the pintail populations sampled. Pintails should be regarded, potentially, as important spreaders of AIVs and NDVs, particularly due to their extensively ramified flyways, which include various inter-continental routes.

Novel reovirus isolation from an Ostrich (Struthio camelus) in Japan.

An orthoreovirus was isolated from an Ostrich (Struthio camelus) and rapidly identified as orthoreovirus by the rapid determination of viral RNA sequences (RDV) system and electron microscopy. Phylogenetic analysis of the sigma A protein indicated that the isolate belonged to avian species and was closely related to chicken orthoreovirus strain 138. The results of the present study indicated that an ostrich orthoreovirus is slight different from other chicken orthoreoviruses and provided evidence of diversity among avian orthoreoviruses. To our knowledge, this is the first genetic report of an orthoreovirus isolated from an ostrich.

Ceramic powder made from chicken feces: anti-viral effects against avian influenza viruses.

Ceramic powder prepared by sintering of chicken feces, when mixed with avian influenza viruses or an avian adenovirus, inactivated these organisms to below detection levels. When the ceramic powder was mixed with double-distilled water, the pH of the water rose to 10 but the aqueous phase did not show any antivirus activity. After 10 washings with water or five washings with 1M Tris-HCl (pH 8.0), the ceramic powder still retained antivirus activity. Antivirus activity was not affected by the presence of organic material (33% fetal calf serum). When chicks were fed food containing 5% ceramic powder, there was no difference in body weight between normal feeding and the ceramic-mixture feeding. The mode of action of the ceramic powder remains unknown, but it possibly works by adsorbing the virus. These results show that the ceramic powder has antiviral activities and is a potentially useful tool against avian influenza on poultry farms.

High rate misidentification of biochemically determined Streptococcus isolates from swine clinical specimens.

In this study, 22 bacterial isolates from swine necropsy specimens, which were biochemically identified as Streptococcus suis and other Streptococcus species, were re-examined using species-specific PCR for authentic S. suis and 16S rRNA gene sequencing for the verification of the former judge. Identification of S. suis on the basis of biochemical characteristics showed high false-positive (70.6%) and false-negative (60%) rates. The authentic S. suis showed various capsular polysaccharide synthesis gene types, including type 2 that often isolated from human cases. Five of 22 isolates did not even belong to the genus Streptococcus. These results suggested that the misidentification of the causative pathogen in routine veterinary diagnosis could be a substantial obstacle for the control of emerging infectious diseases.

A novel missense PTEN mutation identified in a patient with macrocephaly and developmental delay.

Phosphatase and tensin homolog (PTEN) plays an important role in tumor suppression. A germline mutation in the PTEN gene induces not only PTEN hamartoma tumor syndrome, including Cowden syndrome, but also macrocephaly/autism syndrome. Here, we describe a boy with macrocephaly/autism syndrome harboring a novel missense heterozygous PTEN mutation, c.959T>C (p.Leu320Ser). Interestingly, a previously reported nonsense mutation resulting in p.Leu320X was found in Cowden syndrome patients. Our case may be suggestive of a genotype-phenotype correlation.

[Monostotic fibrous dysplasia of the cranial vault: a case report].

Fibrous dysplasia, when it occurs in the craniofacial region, mostly involves the skull base and is rarely localized in the cranial vault. Although there have been several reports on magnetic resonance imaging (MRI) findings of fibrous dysplasia involving the skull base, cases occurring in the cranial vault have seldom been reported. We describe here a rare case of monostotic fibrous dysplasia that occurred in the parietal bone and discuss the characteristics of the MRI findings. A 47-year-old female was admitted to our hospital with a complaint of vertigo. A computed tomography (CT) scan did not reveal any intraparenchymal lesions in either the infra- or supratentorium, and her vertigo improved immediately without any treatments. However, a solitary osteolytic lesion was found incidentally in the left parietal bone. MRI showed that the lesion demonstrated hypointensity on T1-weighted images and hyperintensity on T2-weighted images, and was enhanced heterogeneously following injection of Gadolinium-DTPA. Removal of the parietal bone containing the lesion was performed according to the patient's wishes. The histopathological findings of the removed tissues corresponded to fibrous dysplasia. Although it is well known that craniofacial fibrous dysplasia demonstrates iso- or hypointensity on T1- as well as T2-weighted images, in the present case, the lesion showed apparent hyperintensity on T2-weighted images. These findings suggest that fibrous dysplasia can display various MR intensities depending on its origin.