Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of chicken anaemia virus.

AIM: Chicken anaemia virus (CAV) causes an economically important viral disease in chickens worldwide. The main aim of this study was to establish a rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) assay for detecting CAV infection. METHODS AND RESULTS: A set of four specific LAMP primers were designed based on the nucleotide sequence of the CAV VP2 gene, which encodes a nonstructural protein. These were used for the amplification of a specific target region of the VP2 gene. LAMP amplicons were successfully amplified and detected by DNA electrophoresis and by direct naked eye SYBR Green I visualization. A sensitivity test systematically demonstrated that the LAMP assay was superior to a conventional PCR assay with a minimum concentration limit of 100 fg compared to 10 ng for the conventional PCR. The specificity of the LAMP assay for CAV detection is consistent with conventional PCR. Using this established LAMP assay, infected and uninfected clinical samples obtained from an experimental farm were fully verified. CONCLUSIONS: A novel nucleic acid-based approach of LAMP assay was successfully developed for detecting CAV infection. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, these results indicate that the developed LAMP assay herein for CAV detection is a time-effective, simple, sensitive and specific test that can be used as an alternative approach in the future for large-scaled diagnosis on the farm of CAV infection.

Epidemiology and molecular characterization of chicken anaemia virus from commercial and native chickens in Taiwan.

Chicken infectious anaemia (CIA) is a disease with a highly economic impact in the poultry industry. The infected chickens are characterized by aplastic anaemia and extreme immunosuppression, followed by the increased susceptibility to secondary infectious pathogens and suboptimal immune responses for vaccination. Commercially available CIA vaccines are routinely used in the breeders in Taiwan to protect their progeny with maternal-derived antibodies. However, CIA cases still occur in the field and little is known about the genetic characteristics of Taiwanese chicken anaemia viruses (CAVs). In this study, CAV DNA was detected in 72 of 137 flocks collected during 2010-2015. Among the PCR-positive samples, the coding regions of 51 CAVs were sequenced. Phylogenetic analysis of the VP1 gene revealed that, although most of Taiwanese CAVs belonged to genotypes II and III, some isolates were clustered into a novel genotype (genotype IV). Moreover, a Taiwanese isolate in this novel genotype IV appeared to be derived from a recombination event between genotypes II and III viruses. Five Taiwanese CAV isolates were highly similar to the vaccine strains, 26P4 or Del-Ros. Taken together, these results indicate that the sequences of CAVs in Taiwan are variable, and inter-genotypic recombination had occurred between viruses of different genotypes. Moreover, vaccine-like strains might induce clinical signs of CIA in chickens. Our findings could be useful for understanding the evolution of CAVs and development of a better control strategy for CIA.

Cloning and nucleotide sequencing of the second internal transcribed spacer of ribosomal DNA for three species of Eimeria from chickens in Taiwan.

Coccidiosis of chickens caused by protozoan parasites of the genus Eimeria (Coccidia: Eimeriidae) is an enteric disease that results in great economic losses throughout the world, including Taiwan. Using polymerase chain reaction (PCR) with primers specific for the second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA), three species of Eimeria, E. tenella, E. maxima, and E. acervulina have been successfully characterised from chickens in Taiwan. The sizes of PCR products from various isolates representing these three species were between 370 and 580 base pairs (bp). After cloning and sequencing of the PCR products, high nucleotide sequence identity (96.8-100%) was observed within a species. In addition, ITS-2 nucleotide sequences for E. tenella had higher homology (98.5-99.3%) than E. maxima (81.6-96.5%) when compared with appropriate sequences deposited in GenBank. To our knowledge, this is the first report of a 412-bp ITS-2 sequence for E. acervulina from chickens.

Salmonellae and campylobacters in household and stray dogs in northern Taiwan.

Rectal swabs were collected from 437 household and 491 stray dogs in northern Taiwan from May 2003 to June 2005 to investigate the prevalence and antimicrobial susceptibilities of salmonellae and campylobacters. The results revealed that 2.1% of household dogs and 6.3% of stray dogs were positive for salmonellae, with Salmonella Duesseldorf being the most dominant serotype in both. Additionally, 2.7% of the household dogs and 23.8% of the stray dogs were positive for campylobacters. Campylobacter jejuni was the most prevalent species (86.8%), followed by C. upsaliensis (9.3%) and C. coli (3.9%). Both salmonella and campylobacter isolation rates from the stray dogs were significantly higher than those from the household dogs (p < 0.01). The susceptibility of 33 C. jejuni isolates to eight antimicrobials was studied by the E-test. A high rate of resistance was observed to azithromycin (93.9%), clindamycin (87.9%), erythromycin (81.8%), tetracycline (78.8%), chloramphenicol (69.7%), nalidixic acid (51.5%), gentamicin (33.3%), and ciprofloxacin (18.2%). The susceptibility of 40 Salmonella isolates to 15 antimicrobials was also studied by the disc-diffusion method. All the Salmonella isolates were susceptible to ciprofloxacin and ceftriaxone. Resistance was observed most frequently to tetracycline (77.5%), chloramphenicol (52.5%), and ampicillin (50%).

Study of gammopathies with immunofixation electrophoresis.

Immunofixation electrophoresis and immunoelectrophoresis were compared in 60 samples (51 sera and 9 urines) containing apparently homogenous bands, detected by electrophoresis. To determine the correlation with clinical findings, the patients were divided into three groups: (1) symptomatic with monoclonal immunoelectrophoretic patterns; (2) asymptomatic with monoclonal immunoelectrophoretic patterns; (3) asymptomatic with polyclonal immunoelectrophoretic patterns. The results from immunoelectrophoresis and immunofixation electrophoresis were consistent with each other in all cases of Groups I and III in terms of clonality (i.e., whether monoclonal or polyclonal) and immunoglobulin class; whereas in Group II, which was composed of asymptomatic patients, two sera and three urines were identified to have monoclonal changes by immunoelectrophoresis but polyclonal changes by immunofixation electrophoresis. It is recommended that immunoelectrophoresis still be used for routine clinical service, but supplemented by immunofixation electrophoresis in equivocal cases, namely: (1) light-chain changes masked by an umbrella effect of immunoglobulin G (IgG); (2) abnormal bands located in atypical areas or overlapped with a normal serum protein; (3) a normal-looking "free" light chain present in the urine, mimicking Bence Jones protein; (4) controversial cases of biclonal gammopathy; (5) mini-monoclonal or oligoclonal protein bands; (6) immune complexes.

Synthesis of immunoglobulin within the central nervous system in multiple sclerosis and other neurological diseases. Detection by analysis of CSF/serum IgG ratio.

This study included 49 patients with clinical multiple sclerosis (MS), 105 patients with other neurological diseases (OND), and 30 controls. It compared seven assays for CNS IgG synthesis with the oligoclonal banding (agarose electrophoresis) method. A newly developed assay which determined the differences between the measured and calculated CSF/serum IgG ratio (M-C value), using albumin as a reference protein, was particularly sensitive to the diagnosis of MS. In 40/46 (87%) of patients with MS, the M-C value was 0.001 or more, while oligoclonal banding was found in CSF of 38/49 (78%). In the 30 controls, the M-C value was invariably less than 0.001 and oligoclonal banding was not found. In patients with OND, 26/104 (25%) had an M-C value of 0.001 of greater while 11/105 (11%) had oligoclonal banding in CSF. The M-C value also offers a convenient means of quantifying CNS IgG synthesis during disease activity of treatment. It is concluded that the combined use of the oligoclonal banding method and the M-C value determination gives the greatest predictive value for the diagnosis of MS.

B-haplotype control of CD4/CD8 subsets and TCR V beta usage in chicken T lymphocytes.

The major histocompatibility (B) complex of the chicken contains genes similar to Class I (B-F) and Class II (B-L beta) genes in mammals, as well as a highly-polymorphic gene family (B-G) whose exact function is not known. Specific B-haplotypes are strongly associated with resistance to a number of infectious diseases, and with immune responses to soluble and cellular antigens. In mammals, Class I and Class II molecules control development of the T cell repertoire, including selection of CD4+ and CD8+ T cells. One study of chickens reported that low CD4:CD8 ratio was associated with the B4 haplotype, which shares expressed B-F/B-L genes with the B13 haplotype. In studies reported here, chickens of two haplotypes carried in the Auburn R line, B302 and B305 (which is B13-related), were evaluated for percentages of T cells expressing the CD4, CD8, CD3, TCR1, TCR2 and TCR3 antigens in peripheral blood lymphocytes (PBL), thymus, and spleen. These two haplotypes were chosen for comparison because they differ in resistance to Marek's disease (MD) and are closely-related in B-F and B-L genes by restriction fragment length polymorphism analyses. Homozygous birds of each B haplotype were produced from crosses of (B302 x B305)F1 sires and dams. PBL, thymocytes, and splenocytes from B302 homozygotes had higher CD4:CD8 ratios than B305 homozygotes. However, CD4:CD8 ratio differences could not be attributed to haplotype-controlled differences in V beta usage within CD4/CD8 subsets, as has been described for certain V beta families in mice and humans. These results indicate that thymic selection events involving CD4 and CD8 subsets and TCR V beta usage are controlled by a gene or genes closely-linked to the B-complex, which may or may not be Class I or Class II genes.

Misdiagnosis of acute myocardial infarction.

A total of 979 cardiac profiles were reviewed. Seventeen cases were found to have elevated CK-BB by electrophoresis and were misidentified by the immunoinhibition/immunoprecipitation technique as elevated creatine phosphokinase (CK-MB). Eleven of the 17 cases also had elevated lactate dehydrogenase (LD) LD-5/LD-1 ratio; five cases were motor vehicle accident (MVA), four cases were prostatic carcinoma (PC), and one case each of breast carcinoma and coronary heart disease. One case of PC and one of MVA with a preliminary clinical diagnosis of acute myocardial infarction (AMI) were presented. Our findings underscore the importance of electrophoretic confirmation of the presence of CK-MB when detected by a quantitative technique. Clinicians should consider the possibilities of PC or other cancers when elevated "CK-MB" is present in conjunction with a raised LD-5/LD-1 ratio in patients who fail to show clear-cut clinical evidence of AMI. The mechanism of elevated CK-BB and LD-5/LD-1 ratio in PC patients are discussed.

Production and diagnostic application of a purified, E. coli-expressed, serological-specific chicken anaemia virus antigen VP3.

The aim of this study was to evaluate the production of chicken anaemia virus VP3 protein in different Escherichia coli strains and to address the diagnostic application of purified E. coli-expressed VP3 protein for the detection of chicken anaemia virus (CAV) infection and the development of an ELISA kit. Three E. coli strains, BL21, BL21 codonplus RP and BL21 pLysS, each harbouring a VP3 protein expressing plasmid, were investigated after induction to produce recombinant VP3 protein. After isopropyl-ß-D-thiogalactoside (IPTG) induction, VP3 protein was successfully expressed in all three E. coli strains. The BL21 pLysS strain gave the best performance in terms of protein productivity and growth profile. In addition, the optimal culture temperature and IPTG concentration were found to be 0.25 mM and 20 °C, respectively. Using Ni-NTA-purified VP3 protein as an ELISA coating antigen, the purified VP3 was shown to be highly antigenic and able to discriminate sera from chickens infected with CAV from those that were uninfected during an evaluation of CAV infection serodiagnosis. A VP3-based ELISA demonstrated 100% (6/6 x 100%) specificity and sensitivities of 91.3% (21/23 x 100%) and 82.6% (19/23 x 100%) using cut-off values of the mean plus 2 SD and the mean plus 3 SD, respectively.

Unusual pox lesions found in Chinese jungle mynahs (Acridotheres cristatellus).

Pox lesions involving feathered and unfeathered skin, the oral cavity and the uropygial gland were found in Chinese jungle mynahs. Characteristic intracytoplasmic inclusions were detected in the proliferative cells of all lesions. Ultrastructurally, the virus particles consisted of a convoluted outer membrane enclosing lateral bodies and a biconcave central core, typical for poxvirus. The nucleotide sequences of the amplicon obtained with a set of primers for the 4b core protein of fowl poxvirus revealed that the mynah poxvirus was phylogenetically related to wood pigeon poxvirus. This is the first report of poxvirus infection affecting the uropygial gland.

Multiple myeloma protein with three light chains.

A case of immunoglobulin G (kappa) myeloma showed, in addition to the monoclonal IgG(kappa) arc, two kappa chains in the serum. The urine specimen contained 7.75 g of kappa chains per liter. The electrophoretically fast-moving kappa chain in serum was shown by immunoelectrophoresis and Ouchterlony immunodiffusion to be a complex of kappa chains and alpha 1-antitrypsin. This complex, which was detected only transiently in the patient's blood, was composed of a monomeric kappa chain bound to the antitrypsin by a disulfide bond. The predisposing factor for the formation of this complex is unclear, but patients showing this complex usually have kappa type myeloma protein and excrete kappa chain in urine at more than 1 g/L. The relationship between chemotherapy and formation of the kappa chain-antitrypsin complex needs further investigation.