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Porcine epidemic diarrhoea virus induces cell-cycle arrest through the DNA damage-signalling pathway
2020
Luo, Yi-Ran | Zhou, Shu-Ting | Yang, Liang | Liu, Yuan-Ping | Jiang, Sheng-Yao | Dawuli, Yeliboli | Hou, Yi-Xuan | Zhou, Tian-Xing | Yang, Zhi-Biao
Porcine epidemic diarrhoea virus (PEDV) infection causes watery diarrhoea, vomiting, anorexia, and weight loss, especially among neonatal piglets, inflicting on them morbidity and mortality potentially reaching 90%–100%. Despite it being known that certain mammalian cell phases are arrested by PEDV, the mechanisms have not been elucidated, and PEDV pathogenesis is poorly understood. This study determined the effect of an epidemic PEDV strain on cell cycle progression. We observed the effect of the PEDV SHpd/2012 strain on an infected Vero cell cycle through flow cytometry and Western blot, investigating the interrelationships of cell-cycle arrest, the DNA damage–signalling pathway caused by PEDV and the phosphorylation levels of the key molecules Chk.2 and H2A.X involved upstream and downstream in this pathway. PEDV induced Vero cell-cycle arrest at the G1/G0 phase. The phosphorylation levels of Chk.2 and H2A.X increased with the prolongation of PEDV infection, and no significant cell-cycle arrest was observed after treatment with ATM or Chk.2 inhibitors. The proliferation of PEDV was also inhibited by treatment with ATM or Chk.2 inhibitors. PEDV-induced cell-cycle arrest is associated with activation of DNA damage–signalling pathways. Our findings elucidate the molecular basis of PEDV replication and provide evidence to support further evaluation of PEDV pathogenesis.
Afficher plus [+] Moins [-]Development and application of a TaqMan-MGB real-time RT-PCR assay for the detection of porcine epidemic diarrhoea virus strains in China
2016
Hou, Yi-Xuan | Xie, Chun | Wang, Kang | Zhao, Yu-Ting | Xie, Yang-Yang | Shi, Hong-Yan | Chen, Jian-Fei | Feng, Li | Tong, Guang-Zhi | Hua, Xiu-Guo | Yuan, Cong-Li | Zhou, Yan-Jun | Yang, Zhi-Biao
Introduction: A real-time RT-PCR method for identification and quantification of porcine epidemic diarrhoea virus (PEDV) strains in China was developed.Material and Methods: Based on the conserved sequence of the PEDV nucleocapsid (N) gene, a primer pair and probe were designed to establish a TaqMan-MGB real-time RT-PCR assay for quantitative detection of the virus. The sequence was cloned into the pMD18-T vector and a series of diluted recombinant plasmids were used to generate a standard curve with an R2 value of 0.999.Results: The developed quantitative PCR assay detected viral titres as low as 0.1 TCID₅₀ with high specificity and no cross-reaction with other porcine viruses (PoRV, TGEV, PRRSV, or CSFV). The intra-batch and inter-batch coefficients of variation were both less than 1%, which indicated good reproducibility. Thirty clinical diarrhoea samples obtained from pigs in Shanghai and Fujian were analysed using this quantitative PCR assay. Out of these samples, 93.3% were found to be PEDV positive.Conclusion: This approach is suitable for clinical sample identification and pathogenesis studies.
Afficher plus [+] Moins [-]Epidemiological and histopathological analysis of 40 apocrine sweat gland carcinomas in dogs: a retrospective study
2016
Kycko, Anna | Jasik, Agnieszka | Bocian, Łukasz | Otrocka-Domagała, Iwona | Mikiewicz, Mateusz | Smiech, Anna | Łopuszyński, Wojciech | Dolka, Izabella | Nowak, Marcin | Madej, Janusz A.
Introduction: Apocrine sweat gland carcinomas (ASGCs) are malignant neoplasms of dogs and other animals, rarely reported worldwide. The aim of this study was to summarise the occurrence of this cancer in a population of dogs in Poland between 2009 and 2014 with regards to histological features and body location of the tumours, as well as age, sex and breed of the cancer-affected dogs.Material and Methods: The study involved 40 canine ASGC cases diagnosed in five national veterinary pathology laboratories. The material was processed according to routine histological methods.Results: Histological types of the tumours involved simple and complex apocrine carcinoma of cystic/papillary (62.5%), solid (15%), and tubular type (12.5%), as well as apocrine ductal carcinoma (10%). The epidemiological analysis revealed peak incidence of the cancer in dogs between 8 and 14 years of age, with the most commonly affected sites being forelimbs and thorax. The highest number of the cancer cases was diagnosed in mixed breed dogs and German Shepherds; no sex predilection was noted.Conclusion: To the authors’ knowledge, this is the first report recounting the study on canine malignant apocrine sweat gland tumours in Poland providing detailed phenotypical and histological data, which are otherwise rarely described in veterinary literature. This type of cancer appears to be diagnosed more frequently in dogs than in humans. Being an easily accessible material for research, canine ASGCs might serve as a relevant animal model for studies related to pathogenesis of sweat gland tumours.
Afficher plus [+] Moins [-]Identification of novel pathways in pathogenesis of ketosis in dairy cows via iTRAQ/MS
2016
Shu, Shi | Xu, Chuchu | Xia, Cheng | Xiao, Xinhuan | Wang, Gang | Fan, Ziling | Cao, Yu | Wang, Yanhui | Zhang, Hongyou
Introduction: To identify novel pathways involved in the pathogenesis of ketosis, an isobaric tag for relative and absolute quantitation/mass spectrometry was used to define differences in protein expression profiles between healthy dairy cows and those with clinical or subclinical ketosis.Material and Methods: To define the novel pathways of ketosis in cattle, the differences in protein expression were analysed by bioinformatics. Go Ontology and Pathway analysis were carried out for enrich the role and pathway of the different expression proteins between healthy dairy cows and those with clinical or subclinical ketosis.Results: Differences were identified in 19 proteins, 16 of which were relatively up-regulated while the remaining 3 were relatively down-regulated. Sorbitol dehydrogenase (SORD) and glyceraldehyde-3-phosphate dehydrogenase (G3PD) were up-regulated in cattle with ketosis. SORD and G3PD promoted glycolysis. These mechanisms lead to pyruvic acid production increase and ketone body accumulation.Conclusion: The novel pathways of glycolysis provided new evidence for the research of ketosis.
Afficher plus [+] Moins [-]Selected aspects related to epidemiology, pathogenesis, immunity, and control of African swine fever
2016
Woźniakowski, Grzegorz | Frączyk, Magdalena | Niemczuk, Krzysztof | Pejsak, Zygmunt
African swine fever (ASF) is currently one of the most severe viral infections of domestic pigs, wild boars, and other hosts belonging to Suidae family. ASF is also considered as the most complex and devastating infectious and haemorrhagic disease of swine due to its severe socio-economic impact and transboundary character. ASF it is a notifiable disease and due to the lack of specific treatment and vaccine, the disease can be only limited by the administrative measures comprising wild boar hunting and stamping out of affected pigs. ASF occurred for the first time in Kenya in 1921 while in Europe (Portugal) the virus was detected at the end of the 1950s. In spite of successful eradication of this threat in a number of affected regions, the virus remains endemic in both feral and domestic pigs in Africa and Sardinia. The ‘new era’ of ASF started in 2007 after its re-introduction to Georgia. Following its intensive expansion, the virus spread to other Caucasian countries, including the territory of the Russian Federation. In 2014 the virus reached Ukraine, Belarus, and, consequently, European Union countries: Lithuania, Latvia, Estonia, and Poland. The occurrence of ASF in wild boars and pigs had a severe impact on both epidemiology and economy because of the national and international transport and trade consequences. Up to date, starting from the February 2014, eighty ASF cases in wild boar and three outbreaks in domestic pigs have been diagnosed. Taking into account the diverse rate of spread in Poland, this review aims to present and discuss the current state of knowledge on ASF including its epidemiology, pathology, transmission, and perspectives of control.
Afficher plus [+] Moins [-]Histopathological, immunohistochemical, and parasitological studies on pathogenesis of Coenurus cerebralis in sheep
2018
Rahsan, Yilmaz | Nihat, Yumusak | Bestami, Yilmaz | Adnan, Ayan | Nuran, Aysul
This study consisted in histopathological and immunohistochemical examinations of the central nervous system of 15 sheep suspected of infection with Coenurus cerebralis. The sheep displayed compulsive circling and were submitted for necropsy in 2012–2016. Species identification was made on the basis of the PCR analysis and parasitological examination of the cysts. Coenurus cerebralis cysts were detected only in the cerebral tissue of 13 sheep and in the cerebral and cerebellar tissues of 2 animals. Out of the 33 parasite cysts, most (21.21%) were located in the right and left frontal lobes of the cerebrum. The largest cyst measured 6 × 5 cm and the smallest cyst was 2 × 2 cm in size. The highest and lowest numbers of scolices were 55 and 21, and the number of rostellar hooks ranged between 22 and 30. Histopathological examination revealed the presence of typical parasitic granulomatous inflammatory foci. Immunohistochemical staining showed that most common in the periphery of the parasite cysts were, in descending order by cell number, GFAP, CD163, CD3, and CD79α-positive cells. The study confirms the role of cellular defence mechanisms in the pathogenesis of Coenurus cerebralis infection in sheep.
Afficher plus [+] Moins [-]Chlamydia psittaci reference genes for normalisation of expression data differ depending on the culture conditions and selected time points during the chlamydial replication cycle
2016
Van Lent, Sarah | Vanrompay, Daisy
Introduction: Chlamydia psittaci is a gram-negative obligate intracellular pathogen of birds. Poultry infections lead to economic losses and can be transmitted to humans. No vaccine is available and the bacterium-host cell interaction is not completely understood. Replicating bacteria cause pneumonia, but C. psittaci can also be non-replicating and persistent inside the cytoplasm of avian cells. RT-qPCR provides insight into the molecular pathogenesis of both active replicating and persistent Chlamydia psittaci in birds, but requires identification of stably expressed reference genes to avoid biases. Material and Methods: We investigated the expression stability of 10 C. psittaci candidate reference genes for gene expression analysis during normal growth and penicillin-induced persistence. C. psittaci Cal10 was cultured in HeLa229 and RNA was extracted. The expression level of each candidate was examined by RT-qPCR and Cq values were analysed using geNorm. Results: The genes tyrS, gidA, radA, and 16S rRNA ranked among the most stably expressed. The final selected reference genes differed according to the bacterial growth status (normal growth versus persistent status), and the time points selected during the duration of the normal chlamydial developmental cycle. Conclusion: The study data show the importance of systematic validation of reference genes to confirm their stability within the strains and under the conditions selected.
Afficher plus [+] Moins [-]The genetic variability of small-ruminant lentiviruses and its impact on tropism, the development of diagnostic tests and vaccines and the effectiveness of control programmes
2023
Olech Monika
Maedi-visna virus and caprine arthritis encephalitis virus are two closely related lentiviruses which cause multisystemic, progressive and persistent infection in goats and sheep. Because these viruses frequently cross the species barrier, they are considered to be one genetic group called small-ruminant lentiviruses (SRLV). They have in vivo tropism mainly for monocytes and macrophages and organ tropism with unknown mechanisms. Typical clinical signs are pneumonia in sheep, arthritis in goats, and mastitis in both species. Infection with SRLV cannot currently be treated or prevented, and control programmes are the only approaches to avoiding its spread. These programmes rely mainly on annual serological testing and elimination of positive animals. However, the high genetic and antigenic variability of SRLV complicate their early and definitive diagnosis. The objective of this review is to summarise the current knowledge of SRLV genetic variation and its implications for tropism, the development of diagnostic tests and vaccines and the effectiveness of control and eradication programmes.
Afficher plus [+] Moins [-]Establishment of a canine lens epithelial cell line
2021
Matsuda, Akira | Shimizu, Yuki | Kanda, Teppei | Ohnishi, Akihiro | Maeta, Noritaka | Miyabe, Masahiro | Saeki, Kaori | Itoh, Yoshiki
Some immortalized lens epithelial cell lines have been established and are useful for molecular analysis. The establishment of additional cell lines must, however, enable a variety of in-vitro examinations. The objective of this study was to establish a new canine lens epithelial cell line by isolating CLC-1 cells from the lens tissue of a dog with cataracts. In CLC-1 cells, transforming growth factor beta (TGF-β) treatment significantly decreased gene expression of an epithelial marker and elevated that of mesenchymal markers; these characteristics are similar to those of a human lens epithelial cell line. Interestingly, CLC-1 cells exhibited lower expression of an epithelial marker and higher expression of mesenchymal markers than an anterior lens capsule. These results suggest that CLC-1 cells were derived from a cell population that was committed to epithelial-mesenchymal transition in cataract lens tissue. In conclusion, CLC-1 cells could be useful for analyzing molecular pathogenesis in canine cataracts.
Afficher plus [+] Moins [-]Assessment of luteinizing hormone receptor expression in structural support tissues of canine hip and femorotibial joints
2020
Kiefel, Claire A. | Kutzler, Michelle A.
OBJECTIVE To determine whether luteinizing hormone receptors (LHRs) are expressed in canine femoral head subchondral bone (FHSB), hip joint round ligament (RL), cranial cruciate ligament (CCL), and femorotibial joint synovium (FJS) specimens. SAMPLE 1 specimen each of the FHSB, RL, CCL, and FJS obtained from the left hind limbs of 19 fresh canine cadavers. PROCEDURES 1 section of each FHSB, RL, CCL, and FJS specimen was processed with rabbit polyclonal IgG anti-human LHR antibody, and 1 section was treated with negative control reagents. Percentage immunoexpression of LHRs in FHSB and FJS sections was analyzed by assessment of 100 bone marrow cells or synoviocytes in 3 adjacent hpf (400×). In each RL and CCL section, immunoexpression of LHRs in fibrocytes was semiquantitatively analyzed on the basis of the mean of the product of percentage staining score (from 0 [no staining] to 3 [> 50% of cells stained]) and staining intensity score (from 0 [no staining] to 2 [moderate to strong staining]) for 3 adjacent hpf. RESULTS All tissues examined had variable LHR expression. Expression of LHRs in FHSB, CCL, or FJS specimens did not differ between sexes or between sexually intact and gonadectomized dogs. However, RL specimens from female dogs had significantly greater LHR expression scores, compared with findings for male dogs. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that LHRs are expressed in structural support tissues of canine hip and femorotibial joints. Further research is required to determine the LHRs' function, mechanism of action, and potential contribution to the pathogenesis of hip dysplasia or CCL rupture in dogs.
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