A novel vaccine strategy using quick and easy conversion of bacterial pathogens to unnatural amino acid-auxotrophic suicide derivatives.

We propose a novel strategy for quick and easy preparation of suicide live vaccine candidates against bacterial pathogens. This method requires only the transformation of one or more plasmids carrying genes encoding for two types of biological devices, an unnatural amino acid (uAA) incorporation system and toxin-antitoxin systems in which translation of the antitoxins requires the uAA incorporation. Escherichia coli BL21-AI laboratory strains carrying the plasmids were viable in the presence of the uAA, whereas the free toxins killed these strains after the removal of the uAA. The survival time after uAA removal could be controlled by the choice of the uAA incorporation system and toxin-antitoxin systems. Multilayered toxin-antitoxin systems suppressed escape frequency to less than 1 escape per 109 generations in the best case. This conditional suicide system also worked in Salmonella enterica and E. coli clinical isolates. The S. enterica vaccine strains were attenuated with a >105 fold lethal dose. Serum IgG response and protection against the parental pathogenic strain were confirmed. In addition, the live E. coli vaccine strain was significantly more immunogenic and provided greater protection than a formalin-inactivated vaccine. The live E. coli vaccine was not detected after inoculation, presumably because the uAA is not present in the host animals or the natural environment. These results suggest that this strategy provides a novel way to rapidly produce safe and highly immunogenic live bacterial vaccine candidates. IMPORTANCE: Live vaccines are the oldest vaccines with a history of more than 200 years. Due to their strong immunogenicity, live vaccines are still an important category of vaccines today. However, the development of live vaccines has been challenging due to the difficulties in achieving a balance between safety and immunogenicity. In recent decades, the frequent emergence of various new and old pathogens at risk of causing pandemics has highlighted the need for rapid vaccine development processes. We have pioneered the use of uAAs to control gene expression and to conditionally kill host bacteria as a biological containment system. This report proposes a quick and easy conversion of bacterial pathogens into live vaccine candidates using this containment system. The balance between safety and immunogenicity can be modulated by the selection of the genetic devices used. Moreover, the uAA-auxotrophy can prevent the vaccine from infecting other individuals or establishing the environment.

Bovine neutrophils stimulated with Streptococcus uberis induce neutrophil extracellular traps, and cause cytotoxicity and transcriptional upregulation of inflammatory cytokine genes in bovine mammary epithelial cells.

This study aimed to understand the response of neutrophils stimulated by Streptococcus uberis, a major cause of mastitis. It was found that the production of neutrophil extracellular traps (NETs) was induced in milk clots from mastitic milk produced by S. uberis-infected bovine udders. The release of NETs from neutrophils stimulated by S. uberis was investigated. Bovine neutrophils cocultured with S. uberis in vitro released the components of NETs, which contained extracellular DNA and elastase. Bovine mammary epithelial cells (BMECs) incubated in coculture supernatants containing components of NETs, caused cytotoxicity and transcriptional upregulation of inflammatory cytokines, including of interleukin (IL) -1ß, tumor necrosis factor (TNF)-α, IL-6, and IL-8, in BMECs. These findings suggest that bovine neutrophils stimulated by S. uberis induce responses that cause exacerbated inflammation, such as NET formation, cytotoxicity against BMECs, and increased production of inflammatory cytokines. Bovine neutrophil responses stimulated by S. uberis could be involved in the progression of S. uberis-induced mastitis.

Genetics of the rabbit haemorrhagic disease virus strains responsible for rabbit haemorrhagic disease outbreaks in Japan between 2000 and 2020.

Rabbit haemorrhagic disease (RHD) is a highly contagious and fatal disease in rabbits caused by the rabbit haemorrhagic disease virus (RHDV), which includes two genotypes, RHDV-GI.1 and RHDV2-GI.2. RHDVs tend to recombine among different strains, resulting in significant genetic evolution. This study evaluated the genetics of Japanese RHDV strains associated with six outbreaks between 2000 and 2020 using whole-genome sequencing, genomic recombination and phylogenetic analyses. Genomic recombination analysis using near-complete genomic sequences revealed that two Japanese strains detected in 2000 and 2002 were non-recombinant GI.1 (variant RHDVa-GI.1a) strains of different origins, most closely related to strains identified in PR China in 1997 and the USA in 2001, respectively. In contrast, four recent Japanese GI.2 strains detected between 2019 and 2020 were recombinant viruses harbouring structural protein (SP) genes from GI.2 strains and non-SP (NSP) genes from a benign rabbit calicivirus (RCV) strain of genotype RCV-E1-GI.3 (GI.3P-GI.2) or an RHDV G1-GI.1b variant (GI.1bP-GI.2). Phylogenetic analysis based on SP and NSP regions revealed that the GI.1bP-GI.2 recombinant virus detected in Ehime prefecture and the GI.3P-GI.2 recombinant viruses detected in Ibaraki, Tochigi and Chiba prefectures were most closely related to recombinant viruses identified in Australia in 2017 and Germany in 2017, respectively. These results suggested that past RHD outbreaks in Japan did not result from the evolution of domestic RHDVs but rather represented incursions of foreign RHDV strains, implying that Japan is constantly at risk of RHDV incursion from other countries.

Polymer tapered pillar grown directly from an SMF core enables high efficiency optical coupling with a silicon photonics chip.

Silicon photonics technology has attracted considerable attention because of the growing need for high-bit-rate optical interconnections. The low coupling efficiency resulting from the difference in spot size between silicon photonic chips and single-mode fibers remains a challenging issue. This study demonstrated a new, to the best of our knowledge, fabrication method for a tapered-pillar coupling device using a UV-curable resin on a single-mode optical fiber (SMF) facet. The proposed method can fabricate tapered pillars by irradiating only the side of the SMF with UV light; therefore, high-precision alignment against the SMF core end face is automatically achieved. The fabricated tapered pillar with resin cladding has a spot size of 4.46 µm and a maximum coupling efficiency of -0.28 dB with a SiPh chip.

Polymer spot size converter on silicon photonics chip for enabling high coupling to single-mode fiber.

Silicon photonics (SiPh) technology has gained considerable attention as a result of the growing demand for high-bit-rate optical interconnections. Low coupling efficiencies, resulting from the difference in spot size between silicon photonic chips and single-mode fibers (SMFs), remains a challenging issue. To solve this problem, we fabricated a novel, to the best of our knowledge, polymer spot size expander (SSE) device on the end face of a silicon chip. The fabrication of SSEs using self-written waveguide technology and a dipping method using UV-curable resin was highly reproducible. The spot size of the original 3.83 µm of the SiPh chip was expanded to approximately 7.82 µm at a wavelength of 1.55 µm, and the maximum coupling efficiency achieved with the SMF was -0.88 dB. In addition, the -3 dB tolerance of the coupling efficiency along the vertical optical axis was ±4.4 µm.

Bovine leukemia virus-associated B cell lymphoma with severe pleomorphism in a steer.

We examined a 26-month-old steer with neoplastic lesions in the spleen, lymph nodes, heart and kidneys, characterized by pleomorphic lymphoid cells that were immunohistochemically positive for CD20. The presence of bovine leukemia virus (BLV) at >200,000 copies per 100,000 cells by quantitative RT-PCR was considered to be due to random integration of the provirus into the neoplastic cells´ genomes. Inverse PCR identified the presence of one, two, two and three different malignant clones in the heart, spleen, mesenteric node and blood, respectively. Because BLV can rapidly induce lymphoma and a high proviral load facilitates B-cell carcinogenesis, multiclonal tumor development was suspected in the present case.

Histological comparison of malignant epithelioid mesothelioma in young and adult cattle.

The histology and immunohistochemistry of pleomorphic and conventional epithelioid mesotheliomas were examined. The former was detected in two young calves aged 2 and 4 months and was characterized by pleomorphic and atypical cells with decreased expression of cytokeratin 7 (CK7). In contrast, the latter was found in a 31-month-old heifer, consisting of tumor cells uniform in size and shape with CK7 expression in nearly all cells. Production of collagen by tumor cells was demonstrated in both histological types, and was considered to be characteristic of bovine epithelioid mesothelioma. Pleomorphic mesothelioma is far more pleomorphic and mitotically active than conventional mesothelioma, and its normal counterpart may be immature mesothelial cells with high proliferation potential, which exist in fetal life and early calfhood.

An outbreak of rabbit hemorrhagic disease (RHD) caused by Lagovirus europaeus GI.2/rabbit hemorrhagic disease virus 2 (RHDV2) in Ehime, Japan.

A total of ten 1-2-year-old rabbits died within 2 weeks at a facility in Ehime prefecture in May 2019. Necropsy revealed liver discoloration and fragility, hemorrhage of some organs and blood coagulation failure. On histopathologic examination, necrotizing hepatitis was a common finding, together with fibrin thrombi in the small vessels and hemorrhage in some organs. Rabbit hemorrhagic disease (RHD) virus gene was detected in liver samples, and viral particles of approximately 32 nm in diameter were found in the cytoplasm of degenerated hepatocytes by electron microscopy. Phylogenetic analysis based on the partial VP60 gene sequence classified it as Lagovirus europaeus GI.2/RHDV2. This is the first confirmed outbreak of RHD caused by globally emerging GI.2/RHDV2 in Japan.

Repurposing a traditional Japanese method of pest control for wintering pine moths, Komo-trap, for use against summer and winter populations of fall webworms.

BACKGROUND: The fall webworm, Hyphantria cunea Drury (Lepidoptera: Erebidae), is a widespread invasive species. It is native to North America, ranging from southern Canada to northern Mexico. During and after the 1940s, this pest was accidentally introduced in many parts of Europe and Asia. It has now spread to more than 30 countries. The larvae feed on leaves of a wide range of tree species, including ones used as street trees in cities, causing an increase in urban management cost. Although several pest management methods have been employed, pest damage continues especially in newly invaded areas. In this study, we examined the effect and cost-effectiveness of the komo-trap, traditionally used in Japan to reduce the population of larvae of the pine moth Dendrolimus spectabilis Butler (Lepidoptera: Lasiocampidae). This trap, which is safe for people and ecosystems, has not yet been applied to trap the fall webworm. METHODS: In two seasons of 2017, we set komo-traps on street trees in Hakodate City, Japan. We counted the numbers of captured fall webworms compared with controls. We also monitored other species to evaluate any nontarget effects of the trap. RESULTS: One komo, the material cost of which is about 1.10 USD, captured 43.8 fall webworms on average in summer and 27.2 in the fall. The values were significantly larger than those of the controls, which were 0.07 in summer and 0.14 in winter. Bycatch of other species was minimal in summer, whereas in the fall one komo, on average, caught 10.7 woodlice Porcellio sp. or spp. (Isopoda: Porcellionidae). DISCUSSION: The komo-trap is effective in capturing fall webworm. The cost performance of the trap is very favorable, and the nontarget effects can be reduced by using the trap in summer only. The komo-trap would complement other control methods such as tree pruning. Because its cost is low, we recommend that the komo-trap be introduced as a larger-scale trial.

Combined hepatocellular-cholangiocarcinoma in a cow.

We examined a 10-year-old cow in which about half of the liver was displaced by malignant tissue consisting of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC). Cytokeratin (CK) 18 and 7 were expressed in the latter. Metastasis was present in the hepatic, pancreaticoduodenal and mediastinal lymph nodes, where malignant cells had hepatocellular features, but more pleomorphic and atypical than in the primary lesion. Areas composed solely of CC cells or less-differentiated HCC cells were observed. In contrast, well-differentiated HCC cells were almost always admixed with the other two types, and may have had the ability to transform into CC cells and to dedifferentiate into less-differentiated cells. This report suggests that CK18 is an excellent marker for biliary differentiation in cattle.

Erythroblastic sarcoma in the thoracic cavity of a cow.

Erythroblastic sarcoma in a 10-year-old Japanese Black cow with anemia is described. Tumor masses or nodules were located mainly in the thoracic cavity, and some lymph nodes were slightly enlarged. Although neoplastic involvement of the bone marrow was detected, the cow was not leukemic. The diagnosis was made based on the localized distribution of neoplastic lesions, no increase of intravascular nucleated cell number, deeply eosinophilic cytoplasm in some tumor cells, and frequent immunoreactivity of the tumor cells for hemoglobin. The tumor cells were characterized by marked pleomorphism and atypia; such morphological deviation from their normal counterparts may be connected with functional deviation resulting in the sarcomatous growth of these erythroid cells.

Male-specific mortality biases secondary sex ratio in Eurasian tree sparrows Passer montanus.

Sex allocation theory predicts that parents bias the offspring sex ratio strategically. In avian species, the offspring sex ratio can be biased at multiple growth stages, although the mechanisms are not well known. It is crucial to reveal a cause and timing of biased offspring sex ratio. We investigated (i) offspring sex ratio at multiple growth stages, from laying to fledging; and (ii) the stage at which offspring sex ratio became biased; and (iii) the cause of biased offspring sex ratio in Eurasian tree sparrows Passer montanus. Sex determination of 218 offspring, including hatchlings and unhatched eggs from 41 clutches, suggested that the offspring sex ratio was not biased at the egg-laying stage but was significantly female-biased after the laying stage due to higher mortality of male embryos. Half of the unhatched eggs showed no sign of embryo development (37/74, 50.00%), and most undeveloped eggs were male (36/37, 97.30%). Additional experiments using an incubator suggested that the cause of embryo developmental failure was a lack of developmental ability within the egg, rather than a failure of incubation. This study highlights the importance of clarifying offspring sex ratio at multiple stages and suggests that offspring sex ratio is adjusted after fertilization.

Radioactive contamination of nest materials of the Eurasian Tree Sparrow Passer montanus due to the Fukushima nuclear accident: The significance in the first year.

The 2011 Fukushima nuclear accident contaminated large areas of eastern and northeastern Japan, releasing vast amounts of radiation. Here we investigated radioactive contamination of the nest materials of the Eurasian Tree Sparrow Passer montanus from the breeding season of 2011 directly after the accident to the next breeding season of 2012 at two sites. In Tokyo (222 km southwest of the plant), ambient dose rates in the nestboxes were lower than those in Ibaraki (175 km southwest of the plant), where the levels of 2011 were higher than those of 2012. Further, the amount of radioactive Cs in each nest increased with the increase in nest weight, with a higher increment at Ibaraki than at Tokyo. These data suggested higher nest contamination levels in the breeding season directly after a nuclear accident than in later seasons, and an increment of nest contamination levels via nest materials of birds.

Application of a SYBR®Green one step real-time RT-PCR assay to detect type 1 porcine reproductive and respiratory syndrome virus.

The emergence in Japan of field isolates of type 1 porcine reproductive and respiratory syndrome virus (PRRSV) suggests problems with control. We therefore developed a one-step real-time reverse transcription polymerase chain reaction (qRT-PCR) with improved sensitivity that detects as little as 1 × 10(-2) TCID50/ml of viral RNA. We tested serum samples collected in January and September 2008, October 2009 and January 2011 from a farm with an outbreak and found infected pigs between January and September 2008, but not in January 2011. Further, between 2008 and 2011, we did not detect infection in pigs at 8 nearby farms or in 2,052 serum samples collected from pigs from 74 farms in 12 prefectures. This assay should help prevent future outbreaks.

Toxicological evaluation and bioaccumulation potential of lolitrem B, endophyte mycotoxin in Japanese black steers.

Lolitrem B, a causative toxin for ryegrass staggers, is produced by Neotyphodium lolii infecting perennial ryegrass (Lolium perenne). Japanese black cattle have been suspected to be more sensitive to lolitrem B than to other strains, and there has been a concern about the public health hazard of eating beef contaminated with lolitrem B. We carried out a feeding experiment to examine the sensitivity of Japanese black cattle to lolitrem B and the residual level of lolitrem B in several animal tissues. Japanese black steers were fed a 0, 500, 750, 1000, 1500 or 2000 µg kg(-1) diet of lolitrem B provided by endophyte-infected perennial ryegrass straw for 12 weeks. All six animals in the 2000 µg kg(-1) diet group exhibited ryegrass staggers symptoms. Furthermore, two out of three animals in the 1500 µg kg(-1) diet group, three out of six animals in the 1000 µg kg(-1) diet group and one out of three animals in the 750 µg kg(-1) diet group presented clinical signs of ryegrass staggers. These results suggest that a daily intake of 18 µg kg(-1) body weight of lolitrem B can produce ryegrass staggers in Japanese black steers. Perirenal fat tissues of the steers from those groups having one or more animals exhibiting ryegrass staggers symptoms contained approximately 150 ng g(-1) of lolitrem B, while only small amounts of lolitrem B were detected in muscle, liver and kidney. Because the residual amount of lolitrem B in tissues of Japanese black cattle is small, the exposure to lolitrem B in consumers of the beef is likely to be low.

Plasmid-mediated florfenicol resistance in Mannheimia haemolytica isolated from cattle.

The aim of this study was to analyse a florfenicol-resistant Mannheimia haemolytica isolated from a calf to determine the genetic basis of its florfenicol-resistance. The antimicrobial susceptibility and plasmid content of the isolate were determined. A florfenicol resistant plasmid carrying the floR gene was identified by PCR and transformed into Escherichia coli JM109 and HB101 strains. The plasmid was then mapped and sequenced completely. The isolate was resistant to chloramphenicol, florfenicol, oxytetracycline, kanamycin, dihydrostreptomycin, nalidixic acid, ampicillin, and amoxicillin; it carried a floR plasmid of 7.7kb, designated pMH1405. The mobilisation and replication genes of pMH1405 showed extensive similarity to the 5.1-kb pDN1 plasmid from Dichelobacter nodosus and the 10.8-kb pCCK381 plasmid from Pasteurella multocida. An adjacent 2.4-kb segment was highly homologous to the TnfloR region of the E. coli BN10660 plasmid. A plasmid-mediated floR gene was responsible for florfenicol resistance in the bovine respiratory tract pathogen M. haemolytica. The pMH1405 plasmid is the smallest floR-carrying plasmid reported to date. To the best of our knowledge, this is the first report of a florfenicol-resistant gene in M. haemolytica.

Genetic analysis of ORF5 in porcine reproductive and respiratory syndrome virus in Japan.

In recent years, no reports regarding genetic information on porcine reproductive and respiratory syndrome virus (PRRSV) with a focus on Japan have been published. To clarify the effect of time on PRRSV genomic evolution, we sequenced the open reading frame 5 (600 or 603 bases) obtained from Japanese PRRSV isolates for three periods (1992-1993, 2000-2001, and 2007-2008) and compared their phylogenetic relationships. Assessment of mean pairwise homology of nucleotide sequences of PRRSV isolates indicated a trend towards increasing heterogeneity over time. In addition, we newly detected a virus classified in cluster IV, indicative of the increasing genetic variation of PRRSV in Japan.

The effects of acute exposure to deoxynivalenol on some inflammatory parameters in miniature pigs.

Seven miniature pigs were injected intravenously with deoxynivalenol (DON) at 1 mg/kg body weight; afterward, the number of leukocytes in peripheral blood, the luminol-dependent chemiluminescence of neutrophils, the serum or plasma concentration of cytokines and acute-phase proteins were evaluated to determine the effects of acute exposure to DON on inflammatory responses. White blood cell counts were transiently increased at 3, 6, and 12 hr post-injection (PI) due to the increased number of neutrophils. The luminol-dependent chemiluminescence value of neutrophils was significantly elevated at 24 hr PI, indicating the activation of the bactericidal function of neutrophils. Significant increases of interleukin (IL)-8 and tumor necrosis factor-α at 3 hr PI and IL-6 at 6 hr PI were detected in the serum. The concentration of haptoglobin and serum amyloid A was significantly increased at 24 hr PI. These results suggest that acute exposure to DON induced a temporary recruitment of neutrophils in the peripheral blood by IL-8 and subsequent activation of the bactericidal function, and a transient increase of proinflammatory cytokines and acute-phase proteins, indicating the immunomodulatory effects of DON in pigs.

Induction of apoptotic lesions in liver and lymphoid tissues and modulation of cytokine mRNA expression by acute exposure to deoxynivalenol in piglets.

Six 1-month-old piglets were intravenously injected with deoxynivalenol (DON) at the concentration of 1 mg/kg body weight, with three pigs each necropsied at 6 and 24 h post-injection (PI) for investigation of hepatotoxicity and immunotoxicity with special attention to apoptotic changes and cytokine mRNA expression. Histopathological examination of the DON-injected pigs revealed systemic apoptosis of lymphocytes in lymphoid tissues and hepatocytes. Apoptosis of lymphocytes and hepatocytes was confirmed by the TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method and immunohistochemical staining against singlestranded DNA and cleaved caspase-3. The number of TUNELpositive cells in the thymus and Peyer's patches of the ileum was increased at 24 h PI compared to 6 h PI, but the peak was at 6 h PI in the liver. The mRNA expression of interleukin (IL)-1beta, IL-6, IL-18, and tumor necrosis factor (TNF)-alpha in the spleen, thymus and mesenteric lymph nodes were determined by semi-quantitative RT-PCR, and elevated expression of IL-1beta mRNA at 6 h PI and a decrease of IL-18 mRNA at 24 h PI were observed in the spleen. IL-1beta and IL-6 mRNA expressions increased significantly at 6 h PI in the thymus, but TNF-alpha decreased at 6 h PI in the mesenteric lymph nodes. These results show the apoptosis of hepatocytes suggesting the hepatotoxic potential of DON, in addition to an immunotoxic effect on the modulation of proinflammatory cytokine genes in lymphoid organs with extensive apoptosis of lymphocytes induced by acute exposure to DON in pigs.

Detection of Salmonella in Swine fecal samples by flow-through immunocapture.

This study reports the preliminary evaluation of flow-through immunocapture (FTI) followed by real-time PCR (FTI-PCR) for the detection of different serotypes of Salmonella in pig fecal specimens. The FTI-PCR method was compared with the direct plating of FTI beads on xylose lysine desoxycholate agar, real-time PCR, and the conventional culturing methods, Rappaport-Vassiliadis and modified semisolid Rappaport-Vassiliadis. Artificially contaminated swine fecal specimens were used to evaluate and compare these methods. The results of our comparisons indicate that the FTI-PCR, FTI-plating, and modified semisolid Rappaport-Vassiliadis culture methods were accurate, specific, and sensitive. Moreover, FTI-PCR was the fastest method, providing results in fewer than 20 h, as opposed to the 48 to 96 h required for the other methods. Our results indicate that FTI-PCR could be a useful tool for detecting Salmonella in swine fecal specimens at a lower limit of 1.0 CFU/g.