Pathological analysis of batch safety testing of veterinary vaccines using small laboratory animals.

Batch safety tests (BSTs) of veterinary vaccines are conducted using small laboratory animals to assure the safety of vaccines according to several criteria, including clinical signs and change in body weight. Although the latter is used as an evaluation index in BSTs, there have been no reports on the internal changes that affect body weight during the test period. Therefore, we analyzed BST via pathological examination of the tested animals. Here, BSTs were performed for 176 batches using mice and 126 batches using of guinea pigs. Most of the gross findings could be classified into four lesion types (nodules, adhesions, ascites, no apparent signs), with only one vaccine inducing lesions that could not be classified into any of these four types. Histopathological examination revealed that the reactions caused by BST were pyogenic and/or granulomatous inflammation. Nodular or adhesive lesions comprised more severe pyogenic granulomatous inflammation than ascites or cases with no apparent gross lesions. These nodular or adhesive lesions were more frequently induced by vaccines that contained an adjuvant than by vaccines that did not contain an adjuvant. The cases with "exceptional" gross findings histologically presented severe necrosis of the hematopoietic system. Additional testing showed that these "exceptional" lesions were induced when a specific type of light liquid paraffin was injected along with other vaccine additives. Our results show that body weight loss and/or lesions during BST were induced by proinflammatory properties of the tested vaccines and that BST is a sensitive method for detecting unexpected effects of vaccine components.

The formation process of button ulcers in pigs experimentally infected with a subgenotype 2.1 isolate of classical swine fever virus.

We evaluated the role of classical swine fever virus (CSFV) in the formation of button ulcers in the mucosa of the gastrointestinal tract. Histopathological and immunohistochemical analyses of pigs experimentally infected with a subgenotype 2.1 isolate of CSFV, which was isolated in Japan in 2019, revealed follicular necrosis in the submucosal mucosa-associated lymphoid tissue and herniation of crypts as factors that contribute to the development of button ulcers during CSFV infection. These findings indicate that CSFV induces follicular necrosis and is one of the causative agents of button ulcers in pigs.

Isolation of highly pathogenic H5N6 avian influenza virus in Southern Vietnam with genetic similarity to those infecting humans in China.

Since 2013, H5N6 highly pathogenic avian influenza viruses (HPAIVs) have been responsible for outbreaks in poultry and wild birds around Asia. H5N6 HPAIV is also a public concern due to sporadic human infections being reported in China. In the current study, we isolated an H5N6 HPAIV strain (A/Muscovy duck/Long An/AI470/2018; AI470) from an outbreak at a Muscovy duck farm in Long An Province in Southern Vietnam in July 2018 and genetically characterized it. Basic Local Alignment Search Tool (BLAST) analysis revealed that the eight genomic segments of AI470 were most closely related (99.6%-99.9%) to A/common gull/Saratov/1676/2018 (H5N6), which was isolated in October 2018 in Russia. Furthermore, AI470 also shared 99.4%-99.9% homology with A/Guangxi/32797/2018, an H5N6 HPAIV strain that infected humans in China in 2018. Phylogenetic analyses of the entire genome showed that AI470 was directly derived from H5N6 HPAIVs that were in South China from 2015 to 2018 and clustered with four H5N6 HPAIV strains of human origin in South China from 2017 to 2018. This indicated that AI470 was introduced into Vietnam from China. In addition, molecular characteristics related to mammalian adaptation among the recent human H5N6 HPAIV viruses, except PB2 E627K, were shared by AI470. These findings are cause for concern since H5N6 HPAIV strains that possess a risk of human infection have crossed the Chinese border.

Incidental induction of secondary bowel disorders in guinea pigs during a batch safety test of veterinary vaccines.

Veterinary vaccines are subjected to a safety testing using laboratory animals via intraperitoneal injection per batch. From April 2010 to March 2011, 7 guinea pigs in 4 batch tests exhibited unrecoverable weight loss and/or were found dead. Six guinea pigs had developed intussusception, whereas another one had developed an intestinal obstruction consequent to adhesion. A histopathology revealed that these lesions were associated with inflammatory foci. Other animals than the 7 guinea pig also developed similar inflammatory foci but did not develop bowel disorders. In the retesting of these batches, animals did not exhibited clinical signs, though inflammatory foci were detected. The clinical signs, detected in the primary test, might be due to bowel disorders secondary to an inflammatory response, rather than toxicity.

Comparative analysis of in vitro neurotoxicity of methylmercury, mercury, cadmium, and hydrogen peroxide on SH-SY5Y cells.

Mercury (Hg) and cadmium (Cd) are the major toxic heavy metals and are known to induce neurotoxicity. Although many studies have shown that several heavy metals have neurotoxic effects, the cellular and molecular mechanisms thereof are still not clear. Oxidative stress is reported to be a common and important mechanism in cytotoxicity induced by heavy metals. However, the assays for identifying toxic mechanisms were not performed under the same experimental conditions, making it difficult to compare toxic properties of the heavy metals. In this study, we investigated the mechanisms underlying neurotoxicity induced by heavy metals and H2O2, focusing on cell death, cell proliferation, and oxidative stress under the same experimental condition. Our results showed that MeHg caused lactate dehydrogenase (LDH) release, caspase activation and cell-cycle alteration, and ROS generation in accordance with decreased cell viability. HgCl2 caused LDH release and cell-cycle alteration, but not caspase activation. CdCl2 had a remarkable effect on the cell cycle profiles without induction of LDH release, caspase activation, or ROS generation. Pretreatment with N-acetyl-l-cysteine (NAC) prevented the decrease in cell viability induced by MeHg and HgCl2, but not CdCl2. Our results demonstrate a clear difference in neurotoxic mechanisms induced by MeHg, HgCl2, CdCl2 or H2O2 in SH-SY5Y cells. Elucidating the characteristics and mechanisms of each heavy metal under the same experimental conditions will be helpful to understand the effect of heavy metals on health and to develop a more effective therapy for heavy metal poisoning.

MARCKS is involved in methylmercury-induced decrease in cell viability and nitric oxide production in EA.hy926 cells.

Methylmercury (MeHg) is a persistent environmental contaminant that has been reported worldwide. MeHg exposure has been reported to lead to increased risk of cardiovascular diseases; however, the mechanisms underlying the toxic effects of MeHg on the cardiovascular system have not been well elucidated. We have previously reported that mice exposed to MeHg had increased blood pressure along with impaired endothelium-dependent vasodilation. In this study, we investigated the toxic effects of MeHg on a human endothelial cell line, EA.hy926. In addition, we have tried to elucidate the role of myristoylated alanine-rich C kinase substrate (MARCKS) in the MeHg toxicity mechanism in EA.hy926 cells. Cells exposed to MeHg (0.1-10 µM) for 24 hr showed decreased cell viability in a dose-dependent manner. Treatment with submaximal concentrations of MeHg decreased cell migration in the wound healing assay, tube formation on Matrigel and spontaneous nitric oxide (NO) production of EA.hy926 cells. MeHg exposure also elicited a decrease in MARCKS expression and an increase in MARCKS phosphorylation. MARCKS knockdown or MARCKS overexpression in EA.hy926 cells altered not only cell functions, such as migration, tube formation and NO production, but also MeHg-induced decrease in cell viability and NO production. These results suggest the broad role played by MARCKS in endothelial cell functions and the involvement of MARCKS in MeHg-induced toxicity.