Development and validation of a chemiluminescent western blot assay for glanders (Burkholderia mallei) serodetection.

Glanders, caused by Burkholderia mallei, is a zoonotic disease of equids. Serologic testing for glanders is required by disease-free countries before international movement of equids. The World Organisation for Animal Health Terrestrial Manual recommends the complement fixation test (CFT) for clearance of individual animals for movement, but the CFT is prone to false-positive results. A colorimetric western blot (WB) assay was developed and validated to resolve false-positive CFT results; however, that assay is relatively time-consuming, and the interpretation is subjective. We present here a procedurally similar chemiluminescent WB assay that performs comparably to the validated colorimetric WB assay and offers noticeable benefits of decreased time-to-result and greater ease of interpretation.

Recombinantly expressed virus-like particles (VLPs) of canine circovirus for development of an indirect ELISA.

Canine circovirus (CanineCV) is an emerging pathogen in domestic dogs, detected in multiple countries in association with varying clinical and pathological presentations including diarrhoea, vasculitis, granulomatous inflammation, and respiratory signs. Understanding the pathology of CanineCV is confounded by the fact that it has been detected in asymptomatic dogs as well as in diseased dogs concurrently infected with known pathogens. Recombinantly expressed self-assembling Virus-like particles (VLPs) lack viral genomic material but imitate the capsid surface conformations of wild type virion, allowing arrays of biological applications including subunit vaccine development and immunodiagnostics. In this study, full length CanineCV capsid gene was expressed in Escherichia coli followed by two-step purification process to yield soluble capsid protein in high concentration. Transmission electron microscopy (TEM) confirmed the capsid antigen self-assembled into 17-20 nm VLPs in glutathione S-transferase (GST) buffer, later utilised to develop an indirect enzyme-linked immunosorbent assay (iELISA). The respective sensitivity and specificity of the proposed iELISA were 94.10% and 88.40% compared with those obtained from Western blot. The mean OD450 value for western blot positive samples was 1.22 (range 0.12-3.39) and negative samples was 0.21 (range 0.07-0.41). An optimal OD450 cut-off of 0.35 was determined by ROC curve analysis. Median inter-assay and intra-assay validation revealed that the iELISA test results were reproducible with coefficients of variation 7.70 (range 5.6-11.9) and 4.21 (range 1.2-7.4). Our results demonstrated that VLP-based iELISA is a highly sensitive method for serological diagnosis of CanineCV infections in dogs, suitable for large-scale epidemiological studies.

Secretory Excretory and Somatic Immunogenic Antigens Profiles of Adult Fasciola spp.

Fascioliasis is a common human-animal disease that is reported in most parts of the world. Fascioliasis is also prevalent in different provinces of Iran. Since it has done no study on the excretory/secretory and somatic immunogenic antigens profiles of adult Fasciola in Iran, the present study was performed on the Fasciola spp. collected from Mazandaran province. For this purpose, the Fasciola worm was isolated from the liver of infected sheep, then its excretory/secretory and somatic antigens were prepared from adult worms. The protein of the samples was measured by the Lowry method. Then, somatic and secretory excretions were examined by SDS-PAGE and the protein profile of the two substances was determined. To evaluate the immunogenicity, the somatic and secretory excretions antigens of Fasciola spp. were injected into white rabbits and after boosting, the blood serum of the rabbits was collected and then Western blotting was performed on them and the results were evaluated. According to the results of Western blotting, 11 somatic antigen bands with a molecular weight of 149, 122, 99, 85, 75, 65, 50, 46, 40, 37, 30 kDa and 12 protein bands of excretory/secretory antigens with molecular weights of 100, 82, 75, 70, 58, 55, 47, 40, 38, 37, 30,25 kDa were observed in adult Fasciola spp. that immunogenic, which appear to have a protective effect or can be used to prepare a diagnostic kit.

The histologic comparison of submandibular gland between yak and yellow cattle.

The purpose of this study was to compare between the histochemical characteristics and the expression of epidermal growth factor (EGF) and it's receptor (EGFR) in the submandibular gland (SMG) of adult yaks and yellow cattle. The SMG tissues of yaks and yellow cattles were collected for histochemical, immunohistochemical (IHC), immunofluorescence (F-IHC)，real-time quantitative polymerasechain reaction (RT-qPCR) and Western blotting methods. The results showed that the striated ducts of SMG were highly developed and connected to the intercalated ducts, which were shorter and directly connected to the acini. Compared with yellow cattle, yak SMG contains more mucous acini. Immunofluorescence showed significant expression of EGF and its receptor in both striated and intercalated ducts of these two species of cattle. Statistical analysis divulged that the distribution density of EGF and EGFR in the SMG of the yak was both significantly higher than that in yellow cattle (p < 0.05). Furthermore, the mRNA expression of EGF and EGFR in yak SMG was also higher than that in yellow cattle. The above results indicated that the intercalated ducts and striated ducts are the main expression sites of EGF and EGFR, the acidic mucin and EGF secreted from SMG of yak were more than that from yellow cattle. The results of this study provide powerful data for the study of physiological functions of submaxillary gland in ruminants and also provide important clues for the study of adaptive physiological mechanisms in plateau organisms.

Chicken telomerase reverse transcriptase promotes the tumorigenicity of avian leukosis virus subgroup J by regulating the Wnt/ß-catenin signaling pathway.

This research aimed to analyze the regulatory effect of chicken telomerase reverse transcriptase (chTERT) on the Wnt/ß-catenin signaling pathway and its effect on the tumorigenicity of avian leukosis virus subgroup J (ALV-J) through in vivo experiments. The chTERT eukaryotic expression plasmid and its recombinant lentivirus particles were constructed for in vivo transfection of chTERT to analyze the effect of chTERT continuously overexpressed in chickens on the tumorigenicity of ALV-J. During 156 days of the artificial ALV-J tumor-inducing process, 7 solid tumors developed in 3 chickens in the chTERT-overexpression group (n = 26*2) and no tumors developed in the control group (n = 26*2). Another 18 tumors induced by ALV-J were confirmed and collected from breeding poultry farms. And we confirmed that chTERT was significantly highly expressed in ALV-J tumors. The ELISA data suggested that the protein levels of ß-catenin and c-Myc in the chicken plasma of the chTERT-overexpressing group with ALV-J infected were consistently and significantly higher than those of the control group. Compared with that of the tumor-adjacent tissues, the activity of the Wnt/ß-catenin signaling pathway and expression of the c-Myc was significantly increased in ALV-J tumors. And the percentage of apoptosis in ALV-J tumors significantly lower than that in tumor-adjacent tissues. Immunohistochemistry, Western blot and RT-qPCR suggested that the replication level of ALV-J in tumors was significantly higher than that in tumor-adjacent tissues. This study suggests that chTERT plays a critical role in the tumorigenicity of ALV-J by enhancing the Wnt/ß-catenin signaling pathway, which will contribute to further elucidating the tumor-inducing mechanism of ALV-J.

Silencing myostatin increases area fraction of smooth muscle in the corpus cavernosum of pigs.

Myostatin (MSTN), an inhibitor of skeletal muscle growth, is also expressed in penile smooth muscle; however, it is unclear whether MSTN plays an inhibitory role in penile smooth muscle growth. We investigated the role of MSTN in the smooth muscle of the penile corpus cavernosum of pigs using MSTN homozygous mutant knockout (KO) and wild type (WT) pigs (n = 4 in each group). The mean of area fraction (%) of smooth muscle in the penile corpus cavernosum was 65.9 % ± 1.79 in the KO and approximately 41.7 % ± 5.39 in the WT (P < 0.001). KO pigs showed significantly increased expression of smooth muscle-specific genes, including smooth muscle protein 22 (TAGLN) (6.62-fold), smooth muscle myosin heavy chain (MYH11) (2.41-fold), myocardin (MYOCD) (3.05-fold), and serum response factor (SRF) (4.95-fold), and decreased expression of vimentin (VIM) (1.36-fold). Immunofluorescence staining and Western blotting showed smooth muscle-specific expression of α-smooth muscle actin (SMA) and calponin was higher in KO pigs (P < 0.05) than in WT pigs. KO pigs had less fat deposition inside the corpus cavernosum, and showed downregulation of adiponectin (ADIPOQ) and fatty acid synthase (FASN) (2.5-fold and 1.9-fold loss, respectively). In vitro experiments showed MSTN interference promoted corporal smooth muscle cell growth and expression of smooth muscle-specific markers, whereas it downregulated the expression of fat-specific genes, ADIPOQ and FASN. MSTN inhibition could promote smooth muscle growth and decrease fat deposition in the corpus cavernosum. MSTN, thus, could be a possible target for the treatment of smooth muscle dystrophy-related disorders such as erectile dysfunction.

Development and evaluation of an indirect ELISA for detection of Teladorsagia circumcincta infection in sheep.

BACKGROUND: The gastrointestinal helminth, Teladorsagia circumcincta, is one of the major health risks and production-limiting diseases in small ruminant populations, particularly in temperate regions. With the increasing importance of disease management and recruited anthelmintic resistant types, accurate approaches are needed for the diagnosis of the infection in the host. Due to uncertain results using faecal examinations, the ELISA method was indicated for the detection of nematode antigenic materials. Despite some promising results, problems were described in terms of test specificity and cross-reactions. Therefore, this study aimed to evaluate the IgG response to worm somatic and excretory/secretory (ES) products using western blot analysis and an indirect ELISA for the detection of T. circumcincta infection in sheep. RESULTS: Based on the immuno-reactivity analysis, immunogenic fractions with molecular weights (MWs) of approximately 60, 75 and 100 kDa were detected in somatic content and two antigens of about 63 and 75 kDa in ES material. Accordingly, a specific product at 75 kDa had the strongest reaction and appeared as the most common antigenic protein. In ELISA, all the sera from the infected sheep revealed the OD rates above the calculated cut-off value with about two-fold greater average. Negative control samples were also specifically recognized with the mean OD rate of about 1/3 of the estimated cut-off value. The cross-reaction test, using rabbit anti-T. circumcincta IgG, did not show reactivity with the ES antigens of other prevalent nematodes including Haemonchus contortus, Protostrongylus rufescens and Marshallagia marshalli. In contrast, a strong positive reaction was observed with the somatic antigens of M. marshalli. CONCLUSIONS: The results of this study indicated that the indirect ELISA method using the ES content enables distinguishing the T. circumcincta infected sheep with high specificity. Those antigenic ES peptides with 63 and particularly 75 kDa MWs should be further investigated due to the potential for serological diagnostic methods and immunoprotective targets in the host.

Leishmania tarentolae and Leishmania infantum in humans, dogs and cats in the Pelagie archipelago, southern Italy.

Visceral leishmaniasis (VL) caused by Leishmania infantum is endemic in the Mediterranean basin with most of the infected human patients remaining asymptomatic. Recently, the saurian-associated Leishmania tarentolae was detected in human blood donors and in sheltered dogs. The circulation of L. infantum and L. tarentolae was investigated in humans, dogs and cats living in the Pelagie islands (Sicily, Italy) by multiple serological and molecular testing. Human serum samples (n = 346) were tested to assess the exposure to L. infantum by immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) and to L. tarentolae by IFAT. Meanwhile, sera from dogs (n = 149) and cats (n = 32) were tested for both Leishmania species by IFAT and all blood samples, including those of humans, by specific sets of real time-PCR for L. infantum and L. tarentolae. The agreement between serological tests performed for human samples, and between serological and molecular diagnostic techniques for both human and animal samples were also assessed. Overall, 41 human samples (11.8%, 95% CI: 8.9-15.7) were positive to L. infantum (5.2%, 95% CI: 3.3-8.1), L. tarentolae (5.2%, 95% CI: 3.3-8.1) and to both species (1.4%, 95% CI: 0.6-3.3) by serology and/or molecular tests. A good agreement among the serological tests was determined. Both Leishmania spp. were serologically and/or molecularly detected in 39.6% dogs and 43.7% cats. In addition to L. infantum, also L. tarentolae circulates in human and animal populations, raising relevant public health implications. Further studies should investigate the potential beneficial effects of L. tarentolae in the protection against L. infantum infection.

Second passage of chronic wasting disease of mule deer to sheep by intracranial inoculation compared to classical scrapie.

The origin of chronic wasting disease (CWD) in cervids is unclear. One hypothesis suggests that CWD originated from scrapie in sheep. We compared the disease phenotype of sheep-adapted CWD to classical scrapie in sheep. We inoculated sheep intracranially with brain homogenate from first-passage mule deer CWD in sheep (sCWDmd). The attack rate in second-passage sheep was 100% (12 of 12). Sheep had prominent lymphoid accumulations of PrPSc reminiscent of classical scrapie. The pattern and distribution of PrPSc in the brains of sheep with CWDmd was similar to scrapie strain 13-7 but different from scrapie strain x124. The western blot glycoprofiles of sCWDmd were indistinguishable from scrapie strain 13-7; however, independent of sheep genotype, glycoprofiles of sCWDmd were different than x124. When sheep genotypes were evaluated individually, there was considerable overlap in the glycoprofiles that precluded significant discrimination between sheep CWD and scrapie strains. Our data suggest that the phenotype of CWD in sheep is indistinguishable from some strains of scrapie in sheep. Given our results, current detection techniques would be unlikely to distinguish CWD in sheep from scrapie in sheep if cross-species transmission occurred naturally. It is unknown if sheep are naturally vulnerable to CWD; however, the susceptibility of sheep after intracranial inoculation and lymphoid accumulation indicates that the species barrier is not absolute.

Designing of Western Blot Technique for Glanders Diagnosing in Iran.

Burkholderia mallei is the etiologic agent of glanders. It is difficult to diagnose this zoonotic disease in its early stages. Some methods such as the complement fixation test (CFT) cause some problems for veterinary authorities and financial losses to animal owners due to false-positive results. The mallein test requires appropriate laboratory equipment and skilled personnel. To quickly and accurately diagnose the disease, especially in areas where animals cannot be kept, new methods (such as the Western blot test [WBT]) should be used to identify the disease. This study designed and optimized the Western blot (immunoblot) test using sera from 84 glanderous equids, and the sensitivity and specificity of ELISA and CFT were compared with the WBT. ELISA tests are based on B. mallei antigens whereas a purified lipopolysaccharide-containing B. mallei antigen is used in the WBT. The sensitivity and specificity of the tests were estimated using the cut-off values recommended by the test developers. The WBT and ELISA were significantly more specific than the CFT. The ELISA based on B. mallei antigens was significantly less sensitive than the CFT. Given their comparable sensitivities and specificities, the CFT (95.7%, 98.5%), the WBT (95%, 100%) and the ELISA (85%, 100%) should be further developed. The CFT is still the prescribed technique for serological investigation of equids for trade purposes to certify individual animals without glanders. Therefore, more efforts should be made to further improve and optimize the WBT and ELISA tests.

Fibrinogen heterogeneity in horses.

BACKGROUND: Fibrinogen heterogeneity has been observed in humans and can influence fibrinogen measurements when using the modified Clauss assay. We hypothesized that fibrinogen heterogeneity also exists in horses. OBJECTIVES: To determine whether fibrinogen heterogeneity exists in horses. ANIMALS: Five clinically healthy horses from the university equine teaching herd. METHODS: Presumed fibrinogen was purified from pooled citrated plasma and electrophoresis performed. The purified protein was subjected to Western blotting using sheep antiserum against human fibrinogen, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Gel electrophoresis of nonreduced equine purified protein yielded 2 protein bands (approximately 377 and 318 kDa) that corresponded with the molecular weights of human high molecular weight fibrinogen and low molecular weight fibrinogen fractions, respectively. Electrophoretograms of reduced purified protein, Western blots, and LC-MS/MS supported that the purified nonreduced protein bands were fibrinogen. CONCLUSION: Fibrinogen heterogeneity exists in horses.

Canine osteosarcoma checkpoint expression correlates with metastasis and T-cell infiltrate.

BACKGROUND: Immune-targeted therapies are being successfully implemented into cancer clinical practice. In particular checkpoint inhibitors are employed to modulate the immune microenvironment of solid tumors. We sought to determine the expression of PD-L1, HVEM, and B7H3 in human and canine osteosarcoma, and correlate expression with clinical features and tumor infiltrating lymphocytes in naturally-occurring canine osteosarcoma. METHODS: Flow cytometry was used to measure ligand surface expression of five human and three canine cell lines. Immunohistochemistry was utilized for expression of ligands and lymphocyte markers in thirty-seven treatment-naïve canine osteosarcoma patients. RESULTS: All cell lines expressed all three ligands at variable levels in both species. Metastatic lesions were associated with higher expression of all three ligands in patient tumor samples. PD-L1 expression strongly correlated with B7H3 and HVEM expression, while HVEM and B7H3 were weakly correlated. Whereas peritumoral T-cell expression positively correlated with PD-L1 and HVEM tumor expression, the presence of T-cells intratumorally were rare. Furthermore, intratumor penetration by T-cells was greatest in metastatic lesions, despite log-fold increases in peritumoral T-cells. In summary, PD-L1, HVEM, and B7H3 are expressed in osteosarcoma, with metastatic disease lesions expressing higher levels. We show for the first time that these ligands expressed on osteosarcoma cells positively correlate with each other and the presence of peritumoral T cell infiltration. Furthermore, osteosarcoma appears to be an intratumoral immune desert with significant resistance to effector T cells. Multiple agents targeting checkpoints are in clinical practice, and may have immune modulating benefit in osteosarcoma.

Seropositivity for Trypanosoma cruzi and Leishmania mexicana in dogs from a metropolitan region of Central Mexico.

Trypanosoma cruzi and Leishmania mexicana are parasites of humans and other mammals, causing American Trypanosomiasis and Cutaneous Leishmaniasis, respectively. Domestic dogs are considered key hosts for these parasites in the domicile and peridomicile cycles of transmission, due to their abundance and contact with human population. In Mexico, there are few studies that involve the study of infection with these parasites in dogs, and have only been carried out mainly in the endemic areas for these diseases. In the state of Querétaro (Mexico), infections with both parasites have been reported for dogs only from rural areas, with no records for the metropolitan zone. We analyzed the seropositivity to T. cruzi and L. mexicana in dogs from localities within of the metropolitan zone of Querétaro City in order to determine if these animals are exposed to these parasites and thus, could be an important part of the transmission cycle of these trypanosomatids in a densely populated urban region within the state of Querétaro, Mexico. Serum samples were collected from 303 dogs housed in the Animal Control centers of the municipalities of Querétaro and El Marques, analyzed by indirect ELISA and Western Blot using as an antigen the Iron Superoxide Dismutase (FeSODe) of the parasites. From the total serum samples, we detected 10.2% of seropositivity for T. cruzi and 2.9% for L. mexicana. Our results represent the first evidence of infection with T. cruzi in domestic dogs from the Metropolitan Zone of Querétaro, and the first record for L. mexicana in Central Mexico. Ongoing investigations seek to confirm the circulation of these parasites in the area to evaluate the risk associated to the human population.

Mycoplasma bovis mbfN Encodes a Novel LRR Lipoprotein That Undergoes Proteolytic Processing and Binds Host Extracellular Matrix Components.

Mycoplasma bovis causes serious infections in ruminants, leading to huge economic losses. Lipoproteins are key components of the mycoplasma membrane and are believed to function in nutrient acquisition, adherence, enzymatic interactions with the host, and induction of the host's immune response to infection. Many genes of M. bovis have not been assigned functions, in part because of their low sequence similarity with other bacteria, making it difficult to extrapolate gene functions. This study examined functions of a surface-localized leucine-rich repeat (LRR) lipoprotein encoded by mbfN of M. bovis PG45. Homologs of MbfN were detected as 48-kDa peptides by Western blotting in all the strains of M. bovis included in this study, with the predicted 70-kDa full-length polypeptide detected in some strains. Sequence analysis of the gene revealed the absence in some strains of a region encoding the carboxyl-terminal 147 amino acids found in strain PG45, which could account for the variation detected by immunoblotting. In silico analysis of MbfN suggested that it may have an adhesion-related function. In vitro binding assays confirmed MbfN to be a fibronectin and heparin-binding protein. Disruption of mbfN in M. bovis PG45 significantly reduced (P = 0.033) the adherence of M. bovis PG45 to MDBK cells in vitro, demonstrating the role of MbfN as an adhesin.IMPORTANCE Experimental validation of the putative functions of genes in M. bovis will advance our understanding of the basic biology of this economically important pathogen and is crucial in developing prevention strategies. This study demonstrated the extracellular matrix binding ability of a novel immunogenic lipoprotein of M. bovis, and the role of this protein in adhesion by M. bovis suggests that it could play a role in virulence.

Antiviral effects of Bovine antimicrobial peptide against TGEV in vivo and in vitro.

BACKGROUND: In suckling piglets, transmissible gastroenteritis virus (TGEV) causes lethal diarrhea accompanied by high infection and mortality rates, leading to considerable economic losses. This study explored methods of preventing or inhibiting their production. Bovine antimicrobial peptide-13 (APB-13) has antibacterial, antiviral, and immune functions. OBJECTIVES: This study analyzed the efficacy of APB-13 against TGEV through in vivo and in vitro experiments. METHODS: The effects of APB-13 toxicity and virus inhibition rate on swine testicular (ST) cells were detected using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT). The impact of APB-13 on virus replication was examined through the 50% tissue culture infective dose (TCID50). The mRNA and protein levels were investigated by real-time quantitative polymerase chain reaction and western blot (WB). Tissue sections were used to detect intestinal morphological development. RESULTS: The safe and effective concentration range of APB-13 on ST cells ranged from 0 to 62.5 µg/mL, and the highest viral inhibitory rate of APB-13 was 74.1%. The log10TCID50 of 62.5 µg/mL APB-13 was 3.63 lower than that of the virus control. The mRNA and protein expression at 62.5 µg/mL APB-13 was significantly lower than that of the virus control at 24 hpi. Piglets in the APB-13 group showed significantly lower viral shedding than that in the virus control group, and the pathological tissue sections of the jejunum morphology revealed significant differences between the groups. CONCLUSIONS: APB-13 exhibited good antiviral effects on TGEV in vivo and in vitro.

Immunoglobulin E and immunoglobulin G cross-reactive allergens and epitopes between cow milk αS1-casein and soybean proteins.

Some infants allergic to cow milk-based formula are also sensitive to soybean-based formula. This paper aimed to explore the association of IgE and IgG cross-reactivity between αS1-casein in cow milk (CM) and soybean proteins. The IgE and IgG cross-reactive allergens and epitopes were identified using sera from infants allergic to CM or mice monoclonal antibodies. The AA sequence alignment was performed using bioinformatics software. Finally, the digestion and heating stability of the cross-reactive allergen were explored by sodium dodecyl sulfate (SDS)-PAGE and Western blotting. The results showed that the IgE and IgG cross-reactive allergen was α subunit of ß-conglycinin named Gly m Bd 60K. The IgE and IgG epitopes were the sequences at AA 319-341 and AA 164-182. No intact Gly m Bd 60K allergen could be observed after 2 min in simulated gastric fluid by SDS-PAGE. Heating did not change IgE and IgG cross-reactivity by Western blotting. Therefore, the existence of cross-reactivity between CM αS1-casein and soybean proteins possibly contributes to the frequently observed cosensitization for these allergens in cow milk-allergic patients. The same IgE- and IgG-binding epitopes of cross-reactive allergens may provide important information for elucidation of the association between IgG and IgE antibody generation.

Response of selenoproteins gene expression profile to mercuric chloride exposure in chicken kidney.

Kidney is a primary target organ for mercuric chloride (HgCl2) toxicity. Selenium (Se) can exert antagonistic effect on heavy metals-induced organ toxicity by regulating the expression of selenoproteins. The objective of this study was to investigate the effect of HgCl2 on the gene expression of selenoproteins in chicken kidney. Sixty male Hyline brown chickens were randomly and evenly divided into two groups. After acclimatization for one week, chickens were provided with the standard diet as well as non-treated water (CON group), and standard diet as well as HgCl2-treated water (250 ppm, HgCl2 group). After seven weeks, kidney tissues were collected to examine the mRNA expression levels of 25 selenoproteins genes and protein expression levels of 4 selenoproteins. Moreover, correlation analysis and principal component analysis (PCA) were used to analyze the expression patterns of 25 selenoproteins. The results showed that HgCl2 exposure significantly decreased the mRNA expression of Glutathione peroxidase 1 (GPX1), GPX4, Thioredoxin reductase 2 (TXNRD2), Iodothyronine deiodinase 1 (DIO1), Methionine-Rsulfoxide reductase 1 (SELR), 15-kDa selenoprotein (SEP15), selenoprotein I (SELI), SELK, SELM, SELN, SELP, SELS, SELT, SELW, and SEPHS2. Meanwhile, HgCl2 exposure significantly increased the mRNA expression of GPX3, TXNRD1, and SELU. Western blot analysis showed that the expression levels of GPX3, TXNRD1, SELK, and SELN were concordant with these mRNA expression levels. Analysis results of selenoproteins expression patterns showed that HgCl2-induced the main disorder expression of selenoproteins with antioxidant activity and endoplasmic reticulum resident selenoproteins. In conclusion, selenoproteins respond to HgCl2 exposure in a characteristic manner in chicken kidney.

Carnitine palmitoyltransferase 1 A expression profile in canine mammary tumors.

Genetic alterations and/or epigenetic modifications occur frequently in the majority of cancer cells. In addition to playing a crucial role as promoters of tumorigenesis, these processes can also generate metabolic pathways that are different from those in normal cells. Besides the Warburg effect, an alteration in lipid metabolism is also found in cancer cells. Thus, elucidation of the regulators involved in this metabolic reprogramming might provide tools for diagnosis, prognosis, and ultimately treatment of canine mammary tumours (CMTs) in particular. One such regulator is carnitine palmitoyltransferase 1A (CPT1A), which is involved in transportation of long-chain fatty acids into the mitochondrial matrix for beta-oxidation, thereby providing an alternative pathway for the generation of energy for tumour growth and development. In this study, the canine cell lines MDCK, CMT-U309, CMT-U27, and P114 were used as in vitro models for western blot and quantitative real-time polymerase chain reaction (qRT-PCR) analyses. Furthermore, western blot and immunohistochemistry were carried out to evaluate CPT1A protein expression in the CMT specimens. The CPT1A protein and mRNA expression levels were increased in the CMT cell lines relative to their levels in normal epithelial cells. Moreover, increased CPT1A expression levels were found in the CMT tissues, being inversely correlated with the tumour differentiation grade. However, additional studies are required to further specify the role of CPT1A in CMTs.

Application of the Leishmania infantum 21-kDa recombinant protein for the development of an immunochromatographic test.

BACKGROUND: Visceral leishmaniasis (VL), caused by Leishmania infantum, is a systemic parasitic disease and presents a global health problem which can be fatal if not diagnosed and treated. Dogs are the main hosts and provide reservoirs for the transmission of the disease to humans. METHODS: In this study, the gene encoding a 21-kDa protein was cloned and expressed as a fusion protein in Escherichia coli strain BL21 (DE3) for developing a rapid immunochromatographic test (ICT) to identify infected dogs. The expression of the recombinant 21-kDa protein (r21) was investigated using SDS-PAGE and Western blot methods. The purified r21-kDa protein was spotted onto ICT strips and tested by sera from experimentally infected, naturally infected and uninfected dogs. RESULTS: The SDS-PAGE and Western blot methods showed the successful expression of r21-kDa protein. The ICT strip test revealed that the r21-kDa protein was detected by the sera of experimentally and naturally infected dogs. The specificity tests also confirmed no cross-reactivity with animals infected with Trypanosoma cruzi, Toxoplasma gondii and Ehrlichia canis. CONCLUSIONS: Based on these findings, the new r21-kDa protein may be a suitable target for developing a new simple, specific and rapid serological method to detect VL in infected dogs.

Isolation and characterization of mammalian orthoreoviruses using a cell line resistant to sapelovirus infection.

Porcine sapelovirus (PSV) is a causative agent of acute diarrhoea, pneumonia and reproductive disorders in swine. Since PSV infection interrupts the growth of other viruses due to its high replication capability in cell culture, the prevention of PSV replication is a keystone to the isolation of non-PSV agents from PSV-contaminated samples. In the present study, we established the PSV infection-resistant cell line N1380 and isolated three mammalian orthoreoviruses (MRV) strains, sR1521, sR1677 and sR1590, from swine in Taiwan. These Taiwanese isolates induced an extensive cytopathic effect in N1380 cells upon infection. The complete and empty virus particles were purified from the cell culture supernatants. Next-generation sequencing analyses revealed that the complete virus particles contained 10 segments, including 3 large (L1, L2 and L3), 3 medium (M1, M2 and M3) and 4 small (S1, S2, S3 and S4) segments. In contrast, the empty virus particles without genome were non-infectious. Phylogenetic analyses revealed that the Taiwanese strains belong to serotype 2 MRV (MRV2). We established an ELISA for the detection of IgG antibody against MRV2 by using the empty virus particles as the antigen. A total of 540 swine and 95 wild boar serum samples were collected in Japan, and the positive rates were 100% and 52.6%, respectively. These results demonstrated that MRV infection occurred frequently in both swine and wild boar in Japan. We established a cell line that is efficient for the isolation of MRV, and the ELISA based on the naturally occurring empty particles would be of great value for the surveillance of MRV-related diseases.