Presence of anti-insulin natural autoantibodies in healthy cats and its interference with immunoassay for serum insulin concentrations

A substance interfering with the enzyme-linked immunosorbent assay (ELISA) for feline insulin concentration was investigated in healthy cats. An insulin-binding substance isolated from feline serum showed 2 bands at 25 and 50 kDa in SDS-PAGE, suggesting the presence of immunoglobulin G (IgG). Insulin-binding IgG from healthy cats indeed reduced insulin immunoreactivity in the ELISA for determining insulin concentration. The insulin-binding IgG was polyclonal/polyreactive and showed certain specificity, high affinity, and high binding capacity, which was evaluated by liquid-phase radioimmunoassay with Scatchard plot analysis. Epitope analysis revealed that the insulin-binding IgG showed significant binding at residues A1-5 and B20-30 of the insulin molecule. Removal of the antibodies from serum enabled the determination of serum insulin concentrations by ELISA. Our data indicated that serum from healthy cats contained substantial amounts of natural autoantibodies combined with insulin, and that the antibodies interfered with the heterologous immunoassay for serum insulin concentration.     

Analysis of a pair of END+ and END? viruses derived from the same bovine viral diarrhea virus stock reveals the amino acid determinants in Npro responsible for inhibition of type I interferon production

The Exaltation of Newcastle disease virus (END) phenomenon is induced by the inhibition of type I interferon in pestivirus-infected cells in vitro, via proteasomal degradation of cellular interferon regulatory factor (IRF)-3 with the property of the viral autoprotease protein N^pro. Reportedly, the amino acid residues in the zinc-binding TRASH motif of N^pro determine the difference in characteristics between END-phenomenon-positive (END⁺) and END-phenomenon-negative (END⁻) classical swine fever viruses (CSFVs). However, the basic mechanism underlying this function in bovine viral diarrhea virus (BVDV) has not been elucidated from the genomic differences between END⁺ and END⁻ viruses using reverse genetics till date. In the present study, comparison of complete genome sequences of a pair of END⁺ and END⁻ viruses isolated from the same virus stock revealed that there were only four amino acid substitutions (D136G, I2623V, D3148G and D3502Y) between two viruses. Based on these differences, viruses with and without mutations at these positions were generated using reverse genetics. The END assay, measurements of induced type I interferon and IRF-3 detection in cells infected with these viruses revealed that the aspartic acid at position 136 in the zinc-binding TRASH motif of N^pro was required to inhibit the production of type I interferon via the degradation of cellular IRF-3, consistently with CSFV.     

Characterization of peptides in ensiled alfalfa treated with different chemical additives

Effects of different chemical additives on peptide composition in ensiled alfalfa (Medicago sativa L.) were investigated by using gel filtration and determination of N characteristics. The alfalfa silages were prepared untreated (control) or with formic acid, formaldehyde or tannic acid as additives at ensiling. All additives reduced non‐protein N (NPN), ammonia nitrogen (NH₃‐N) and amino acid N (AA‐N) in the ensiled forage, and the most effective reduction of NPN and AA‐N was observed in the formaldehyde‐treated silages. Peptides in the control silage were mainly dipeptides to peptides with five amino acid residues. Most peptides in the formic acid‐treated silage contained 4–12 amino acid residues. Although most peptides in the formaldehyde‐treated silages contained 4–6 amino acid residues, there was a considerable proportion of peptides with 7–11 amino acid residues. Tannic acid had little effect on peptide size of ensiled alfalfa extract in which most peptides contained 5–6 amino acid residues. Peptide size in formic acid‐treated alfalfa silage was greater than that in the other treatments. Addition of formic acid and formaldehyde not only increased the peptide concentration in alfalfa silage, but enlarged the peptide size.     

Antibiotic resistance,antimicrobial residues,and bacterial community diversity in pasture-raised poultry,swine, and beef cattle manures

Animal manure can be a source of antibiotic-resistant genes (ARGs) and pharmaceutical residues; however, few studies have evaluated the presence of ARG in pasture-raised animal production systems. The objective of this study was to examine changes in microbiome diversity and the presence of antibiotic residues (ABRs) on three farms that contained a diverse range of animal species: pasture-raised poultry (broiler and layer), swine, and beef cattle. Total bacterial communities were determined using 16S rRNA microbiome analysis, while specific ARGs (sulfonamide [Sul; Sul1] and tetracycline [Tet; TetA]) were enumerated by qPCR (real-time PCR). Results indicated that the ARG abundances (Sul1 [P < 0.05] and TetA [P < 0.001]) were higher in layer hen manures (16.5 × 10⁻⁴ and 1.4 × 10⁻⁴ µg kg⁻¹, respectively) followed by broiler chickens (2.9 × 10⁻⁴ and 1.7 × 10⁻⁴ µg kg⁻¹, respectively), swine (0.22 × 10⁻⁴ and 0.20 × 10⁻⁴ µg kg⁻¹, respectively) and beef cattle (0.19 × 10⁻⁴ and 0.02 × 10⁻⁴ µg kg⁻¹, respectively). Average fecal TetA ABR tended to be greater (P = 0.09) for broiler chickens (11.4 µg kg⁻¹) than for other animal species (1.8 to 0.06 µg kg⁻¹), while chlortetracycline, lincomycin, and oxytetracycline ABRs were similar among animal species. Furthermore, fecal microbial richness and abundances differed significantly (P < 0.01) both among farms and specific species of animal. This study indicated that the microbial diversity, ABR, ARG concentrations, and types in feces varied from farm-to-farm and from animal species-to-animal species. Future studies are necessary to perform detailed investigations of the horizontal transfer mechanism of antibiotic-resistant microorganisms (ARMs) and ARG.     

鸳鸯鸭MHC-Ⅰ α链基因的克隆与分子进化分析

根据GenBank中登录（登录号：AB115244）的鸭MHC-Iα链序列设计并合成1对引物,采用RT-PCR技术从鸳鸯鸭脾脏组织中克隆出鸳鸯鸭MHC-Iα链基因,并进行T-A克隆和序列测定。结果表明,所获得的鸳鸯鸭MHC-Iα链基因长909 bp,编码303个氨基酸的多肽,含一个完整的鸳鸯鸭MHC-Iα链胞外区成熟肽基因。序列比较发现,鸳鸯鸭MHC-Iα链基因与GenBank登录的其它品种的鸭MHC-Iα链核苷酸同源性为89.4%～91.0%,与人和其他动物的MHC-Iα链胞外区成熟肽基因的氨基酸同源性为11.1%～79.3%,表明MHC-Iα链胞外区成熟肽基因存在着种的多样性,且亲缘关系越近,同源性越高。鸳鸯鸭MHC-Iα链胞外区成熟肽基因的成功克隆为进一步研究鸭MHC-I基因表达、生物学活性和应用奠定基础。     

Establishing conditions for the storage and elution of rabies virus RNA using FTA? cards

The Flinders Technology Associates filter paper cards (FTA^® cards) can be used to store nucleic acid from various samples and are easily portable. However, RNA is physicochemically unstable compared with DNA, and appropriate methods have not been established for storage and extraction of RNA from FTA^® cards. The present study investigated the optimum conditions for storage and elution of viral RNA (vRNA) using rabies virus (RABV) applied to FTA^® cards. When TE buffer was used, the elution rates of vRNA increased with the length of the elution time. When the cards were stored at −80°C or −20°C, vRNA was stable over 3 months. Degradation of vRNAs occurred following storage at 4°C and room temperature, suggesting that RNA should be extracted from cards as soon as possible if no freezer is available. When we tried to amplify vRNA from RABV-infected animal brains applied to FTA^® cards and stored at −80°C for 6 months, we did not detect any amplified products with the primer set for 964 bp of RABV N gene. However, we were able to detect amplified products by increasing the elution time of vRNA from FTA^® cards from 30 min to 24 hr or by changing the primer sets to amplify 290 bp of N gene. Thus, we recommend extending the elution time for damaged or low concentration samples in FTA^® cards.     

Identification of T-helper and linear B epitope in the hypervariable region of nucleocapsid protein of PPRV and its use in the development of specific antibodies to detect viral antigen

Peste des petits ruminants is a highly contagious viral disease of small ruminants making its diagnosis difficult from the similar symptoms of Rinderpest. Computer based prediction algorithms was applied to identify antigenic determinants on the nucleocapsid (N) protein of PPRV. Specificity and antigenicity of each peptide was evaluated by solid phase ELISA. Six specific peptide sequences were evaluated in multiple antigenic peptide (MAP) form and immune response was evaluated by supplementing universal T-helper epitope human IL-1beta peptide (VQGEESNDK, amino acids 163-171). Out of the six peptides 19mer sequence corresponding to 454-472 region of N protein of PPRV was found to be highly immunogenic and specific to PPRV. Evaluation of overlapping peptides differing in length for this 452-472 region, showed minimum length of 14 amino acid residues were required for the stable affinity binding of antigen-antibody. The results of immunization and indirect ELISA indicated the presence of T-helper epitope at the N-terminal end and linear B epitope at the C-terminal region of 454-472 19mer of nucleocapsid peptide of PPRV-nucleocapsid protein. The antipeptide antibodies developed against this region showed specificity to PPRV antigen differentiating it from RPV when used in indirect ELISA and western blot analysis.     

吕氏泰勒虫主要表面蛋白P32基因的克隆与序列分析

从吕氏泰勒虫宁县株中提取RNA,用RT-PCR方法扩增主要表面蛋白P32基因的cDNA片段,克隆后测定其核苷酸序列,并推导了相应的氨基酸序列。结果表明,扩增的P32基因长度为875个核苷酸,共编码284个氨基酸。核苷酸序列与已发表的牛的4种泰勒虫的主要表面蛋白基因相比较,其与瑟氏泰勒虫俄罗斯株、日本株、中国辽阳株的核苷酸和氨基酸序列相似性均在98.9%以上。其推导的氨基酸序列中有3个糖基化位点。     

Study of serum drug levels in calves following intramuscular administration of three tetracycline drug preparations

Three antibiotic formulations, oxytetracycline (A) in propylene glycol and oxytetracycline (B) in polyvinyl pyrrolidine and pyrrolidino-methyltetracycline in an oil suspension were given to calves by the intramuscular route. Only oxytetracycline (A) appeared to cause much pain after injection.

The half-time elimination (t_½cl) for oxytetracycline (A) was 14.000 ± 3.580 h for oxytetracycline (B) 10.290 ±5.159 h and for pyrrolidinomethyltetracycline 8.160 ± 0.920 h. The rate of elimination `beta slope” for oxytetracycline (A) was 0.052 ± 0.012 h⁻¹ for oxytetracycline (B) 0.077 ± 0.261 h⁻¹ and for pyrrolidinomethyltetracycline 0.086 ± 0.010 h⁻¹. The Y intercept of the “beta” elimination slope C_os (μg/mL) for oxytetracycline (A) was 2.490 ± 1.040, for oxytetracycline (B) 3.463 ± 1.874 and for pyrrolidinomethyltetracycline 2.852 ± 1.360.

Pyrrolidinomethyltetracycline appeared to have a two component elimination curve, however, only the “beta slope” was used for the calculations.

     

Heterologous Radioimmunoassay for Llama and Alpaca Luteinizing Hormone with a Monoclonal Antibody,an Equine Standard and a Human Tracer

A radioimmunoassay for llama and alpaca LH was developed using a human I¹²⁵LH tracer from a commercial kit, equine LH diluted in human LH free serum as standard, and a monoclonal antibody (518B7) specific for LH but with low species specificity. A 60-min delay in the addition of the tracer and overnight incubation gave a sensitivity of 0.8 μg L⁻¹. The intra-assay coefficient of variation was 37% at 1 μg L⁻¹, declined to 15% at 4 pg L⁻¹ and was below 6% for concentrations up to 32 μg L⁻¹. The inter-assay coefficients of variation for 3 control samples were 20% (2.8 μg L⁻¹), 16% (7.1 μg L⁻¹) and 9.8% (19 μg L⁻¹). In an attempt to increase sensitivity, all tubes were preincubated for 4 h at room temperature before adding the tracer, and the sample volume was increased from 50 μL to 100 μL· (in the standard curve the increased volume was compensated for by human LH free serum). With this protocol, the assay sensitivity was 0.5 μg L⁻¹. The assay was validated clinically and demonstrated increased concentrations of LH after mating in llamas and alpacas. Furthermore, the assay was used to monitor LH responses to a single dose of GnRH in llamas (adult males and females at different ages).     

Genetic variation and cross-reactivity of Clostridium septicum alpha-toxin

Clostridium septicum alpha-toxin genes were sequenced with the polymerase chain reaction (PCR) products amplified from DNAs of 25 C. septicum strains, and were classified into 10 patterns. Alpha-toxins were purified from the culture supernatant of four C. septicum strains (strains No. 44, Kagoshima 8, Mie and Tokachi) which were specially chosen from patterns of the deduced amino acid sequences. The molecular weights of the alpha-toxins were not different according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. However, the isoelectric points between the alpha-toxins of No. 44 and Tokachi strains differed markedly. Cross-neutralization tests were performed with purified alpha-toxins and antitoxins in mice and in Vero cells. Each antitoxin showed roughly the same titers against the four alpha-toxins in mice and completely identical titers against these in Vero cells. Calves immunized with toxoid prepared from the culture supernatant of No.44 strain were challenged by exposure to spores of Mie strain. The toxoid conferred protection against the challenge in calves. From these results, although genetic variation has been observed within the C. septicum alpha-toxin gene, C. septicum strains toxoid of strain No.44 induces protective immunity against exposure to C. septicum that produce other subtypes of alpha-toxin containing several different amino acid residues.     

Identification of the variant C of β-lactoglobulin in sheep using a polymerase chain reaction-restriction fragment length polymorphism method

β-Lactoglobulin (β-LG) is the major whey protein in the milk of ruminants and is able to bind and transport small hydrophobic molecules. However, its biological role is mainly unknown (G odovac -Z immermann 1988). Previously three genetic variants have been found in sheep: A, B and C. The genetic variants A and B differ at amino acid position 20, where variant A has a His and variant B has a Thr (K olde and B raunitzer 1983). The variant C is a subtype of variant A with a single amino acid exchange of Arg→Glu at position 148 (E rhardt et al. 1989). The genotype β-LG BB was found to be associated with higher milk yield, whereas genotypes AA and AB had a higher milk protein and casein content as well as yielding more curd (G arzon and M artinez 1992). No data is available concerning the relationship between the β-LG C allele and production traits or milk properties. Since DNA-based genotyping has already been performed for alleles A and B (S chlee et al. 1993), the aim of the present study was to develop a DNA-based method for identifying the β-LG C variant. However polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) typing has been established (P rinzenberg and E rhardt 1999) recently, and this study shows an alternative method to detect β-LG C.     

Binding characteristics of swine erythrocyte insulin receptors

Crossbred gilts (controls; n = 7) had 8.8 +/- 1.1% (mean +/- SEM) maximum binding of [125I]insulin to insulin receptors on erythrocytes. The number of insulin-binding sites per cell was 137 +/- 19, with a binding affinity ranging from 7.4 X 10(7)M-1 to 11.2 X 10(7)M-1 and mean of 8.8 X 10(7)M-1. Pregnant sows (n = 5) had a significant increase (P less than 0.01) in maximum binding due to an increase in number of receptor sites per cell. Lactating sows fed a high-fiber diet (n = 3) and a low-fiber diet (n = 4) did not develop a significant difference in maximum binding of insulin. Sows fed the low-fiber diet had a significantly higher number of binding sites and a significantly lower binding affinity than did sows fed a high-fiber diet. Receptor-binding affinity was lower in the low-fiber diet group than in cycling gilts, whereas data from sows fed the high-fiber diet did not differ from data for cycling gilts. Data from this study indicated that insulin receptors of swine erythrocytes have binding characteristics similar to those in other species. Pregnancy and diet will alter insulin receptor binding in swine.     

Direct contact and environmental contaminations are responsible for HEV transmission in pigs

Hepatitis E virus (HEV) can cause enterically-transmitted hepatitis in humans. The zoonotic nature of Hepatitis E infections has been established in industrialized areas and domestic pigs are considered as the main reservoir. The dynamics of transmission in pig herds therefore needs to be understood to reduce the prevalence of viremic pigs at slaughter and prevent contaminated pig products from entering the food chain. An experimental trial was carried out to study the main characteristics of HEV transmission between orally inoculated pigs and naïve animals. A mathematical model was used to investigate three transmission routes, namely direct contact between pigs and two environmental components to represent within-and between-group oro-fecal transmission. A large inter-individual variability was observed in response to infection with an average latent period lasting 6.9 days (5.8; 7.9) in inoculated animals and an average infectious period of 9.7 days (8.2; 11.2). Our results show that direct transmission alone, with a partial reproduction number of 1.41 (0.21; 3.02), can be considered as a factor of persistence of infection within a population. However, the quantity of virus present in the environment was found to play an essential role in the transmission process strongly influencing the probability of infection with a within pen transmission rate estimated to 2 ⋅ 10^− 6g ge^− 1d^− 1(1 ⋅ 10^− 7; 7 ⋅ 10^− 6). Between-pen environmental transmission occurred to a lesser extent (transmission rate: 7 ⋅ 10^− 8g ge^− 1d^− 1(5 ⋅ 10^− 9; 3 ⋅ 10^− 7) but could further generate a within-group process. The combination of these transmission routes could explain the persistence and high prevalence of HEV in pig populations.     

Endocytosis of Clostridium botulinum type B neurotoxin into rat brain synaptosomes

Clostridium botulinum type B neurotoxin cleaves VAMP (vesicle-associated membrane protein)/synaptobrevin into two fragments, which results in inhibition of neurotransmitter release. The induced fragment did not react to the antibody raised against the synthetic peptide of the amino-terminal 20 residues of VAMP-2, suggesting that the toxin treatment has caused antigenical alteration in the amino-terminal region of VAMP-2. In rat brain synaptosomes, type B neurotoxin was reduced presumably with sulfhydryls in the membrane and detected in the synaptic vesicle fraction which involved the degradation of VAMP-2 and the inhibition of neurotransmitter release. The light chain in a free form was present in the cytosol fraction. These findings suggest a possibility that type B neurotoxin endocytoses into synaptic vesicles by the recycling pathway and the light chain is penetrable through synaptic vesicle membrane. However, the amount of type B neurotoxin entrapped into synaptic vesicles appears to be extremely small, which may be attributed to a lower sensitivity of the toxin to brain synaptosomes than to peripheral nerve endings.     

No effect of exogenous melatonin on development of cryopreserved metaphase II oocytes in mouse

Background

This study was conducted to investigate effect of exogenous melatonin on the development of mouse mature oocytes after cryopreservation.

Results

First, mouse metaphase II (MII) oocytes were vitrified in the open-pulled straws (OPS). After warming, they were cultured for 1 h in M₂ medium containing melatonin at different concentrations (0, 10⁻⁹, 10⁻⁷, 10⁻⁵, 10⁻³ mol/L). Then the oocytes were used to detect reactive oxygen species (ROS) and glutathione (GSH) levels (fluorescence microscopy), and the developmental potential after parthenogenetic activation. The experimental results showed that the ROS level and cleavage rate in 10⁻³ mol/L melatonin group was significantly lower than that in melatonin-free group (control). The GSH levels and blastocyst rates in all melatonin-treated groups were similar to that in control. Based on the above results, we detected the expression of gene Hsp90aa1, Hsf1, Hspa1b, Nrf2 and Bcl-x1 with qRT-PCR in oocytes treated with 10⁻⁷, or 10⁻³ mol/L melatonin and untreated control. After warming and culture for 1 h, the oocytes showed higher Hsp90aa1 expression in 10⁻⁷ mol/L melatonin-treated group than in the control (P < 0.05); the Hsf1, Hsp90aa1 and Bcl-x1 expression were significantly decreased in 10⁻³ mol/L melatonin-treated group when compared to the control. Based on the above results and previous research, we detected the development of vitrified-warmed oocytes treated with either 10⁻⁷ or 0 mol/L melatonin by in vitro fertilization. No difference was observed between them.

Conclusions

Our results indicate that the supplementation of melatonin (10⁻⁹ to 10⁻³ mol/L) in culture medium and incubation for 1 h did not improve the subsequent developmental potential of vitrified-warmed mouse MII oocytes, even if there were alteration in gene expression.     

猪传染性胃肠炎华毒弱毒株纤突蛋白B、C抗原位点片段基因的克隆与序列分析

猪传染性胃肠炎华毒（TGEV H）是我国分离的代表毒株,H弱毒株是我国自行培育成功的具有良好免疫原性的疫苗株。应用RT－PCR方法扩增出1．3kb的目的片段,包括S基因的编码的B、C两个主要抗原位点的片段,目的片段与pUC19质粒载体进行连续,转化,鉴定后得到阳性重组质粒,命名为pTS1,将重组质粒插入的目的基因片段进行序列分析和比较,其结果表明TGEV H株和S基因5’端B、C位点片段1190bp的核苷酸序列与国外的TGEV毒株Miller、Purdue-115、FS772／70及台湾省TFI毒株序列均具有很高的同源性,达94．72以上。推导的氨基酸序列同源性为94．70％以上。编码抗原位点C的序列与其它毒株完全一致,但在B位点发生改变,即第97个氨基酸发生改变,由Trp突变为Ser。该核苷酸该片段全长1261bp,包括起始密码子ATG及基因间的共同序列ACTAAAC。本研究首次报道了国内的TGEV分离株－H弱毒株S基因的RT－PCR扩增及其编码的B、C抗原位点片段的分子克隆及序列分析,为进一步揭示TGEV强弱毒差别的分子机制、开展新型疫苗及进行分子诊断的研究奠定了重要基础。     

Purification of secretory immunoglobulin A from milk of the brushtail possum (Trichosurus vulpecula)

AIM: To identify and purify secretory immunoglobulin A (sIgA), a key effecter molecule in mucosal immune responses, from milk of the brushtail possum (Trichosurus vulpecula).

METHODS: Milk samples were collected from female possums with pouch young, and clarified by centrifugation and precipitation methods. The clarified fraction was purified by gel filtration and affinity chromatography to yield sIgA. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting techniques were used to assess the purity of the final product, and to identify the heavy (H) chain, light (L) chain and secretory component (SC) of possum sIgA.

RESULTS: Immunoblotting, using antibodies raised against cloned possum sIgA SC and H-chain, and a synthetic peptide fragment of the H-chain, confirmed the identity of the purified protein. The N-terminal amino acid sequence of purified possum sIgA showed strong homology to reported sequences of H-chain variable regions of marsupial immunoglobulins.

CONCLUSIONS: Milk was shown to be a convenient source of mucosal secretion containing sIgA, and a process involving 2 precipitation and 2 chromatography steps produced purified sIgA. This IgA preparation will prove useful for the generation of sIgA-specific immunological reagents for measurement of immune responses in the development of mucosal-based vaccines for biological control of possums.