Neospora caninum antigens recognized by mouse IgG at different stages of infection including recrudescence

Western blotting was performed to analyze Neospora caninum tachyzoite antigens recognized by mouse IgG at different stages of infection including recrudescence. At the early stage of infection, a 36-38 kDa antigen was clearly recognized by the mouse antisera. After day 48 postinoculation, the signal of the 36-38 kDa antigen gradually weakened. Meanwhile, a 43 kDa antigen was intensely and continuously recognized from 48 to 125 days postinoculation. This 43 kDa antigen was clearly detectable with the antisera from the mice under immunosuppression. Sera from naturally infected cattle strongly reacted with the 43 kDa antigen. Therefore, the 43 kDa antigen may be useful for immunological reactions to detect infected animals except in the early stage of the infection.     

不同禾本科牧草种子萌发及幼苗耐盐性鉴定

采用培养皿纸上发芽法和盆栽幼苗培育法,在NaCl和Na2SO4复盐溶液7个盐浓度梯度下(0.4%~1.6%),比较分析了5种禾本科牧草种子的相对发芽率、相对发芽势、胚根胚芽比、发芽指数、简化活力指数;在NaCl和Na2SO4复盐溶液5个盐浓度梯度下(0.3%~1.5%),比较分析了5种禾本科牧草幼苗的MDA含量和游离脯氨酸含量的变化。运用隶属函数评价、分析盐胁迫对不同禾本科牧草种子萌发及幼苗的影响,为牧草耐盐性鉴定、耐盐育种和盐碱地牧草栽培提供理论参考。研究结果表明,5种禾本科牧草在萌发期和苗期耐盐能力排序依次为:垂穗披碱草细茎冰草、老芒麦扁穗冰草无芒雀麦。     

Identification of new morphological and life-cycle stages of Cochlosoma anatis and experimental transmission using pseudocyst

Cochlosoma anatis is a flagellated intestinal parasite that infects a variety of avian species. C. anatis infections have been associated with decreased weight gain and increased morbidity and mortality. Conditions favoring the growth of this organism in birds are current pathogenic intestinal infections and/or young age. There is little data describing the life cycle of this parasite. In this study, electron microscopy images are presented that document longitudinal binary fission of the trophozoite stage and outline the events of pseudocyst formation, which includes a rounding stage. Evidence provided here indicates that the pseudocyst stage may be a mechanism for transmission of this organism. The observations reported here provide additional evidence of homology between Cochlosoma and members of the trichomonad order.     

Determination of whole blood cholinesterase in different animal species using specific substrates

Whole blood cholinesterase was measured using acetyl-, butyryl- and propionylthiocholine as substrates in 10 healthy adult dogs, cats, horses, pigs, goats, sheep and cows, in order to determine and characterise the cholinesterase activity in whole blood of the main domestic animals. An in vitro exposure test with two anticholinesterase compounds, the organophosphate insecticide coumaphos and the carbamate insecticide imidocarb, was also performed. In whole blood of ruminants and pigs, acetylthiocholine yielded the highest cholinesterase activity and other substrates were poorly hydrolysed; in dogs and cats, although acetylthiocholine showed the highest cholinesterase activity, butyryl- and propionylthiocholine also produced high cholinesterase values; in horses, propionylthiocholine was the substrate that yielded the highest cholinesterase activity, closely followed by butyrylthiocholine. All within- and between-run coefficients of variation observed in whole blood samples were less than 5 and 7 per cent, respectively, except when butyrylthiocholine was used as substrate in ruminant blood samples. Butyryl- and propionylthiocholine were the substrates that yielded higher inhibitions after coumaphos exposure, whereas the use of acetylthiocholine showed the highest cholinesterase inhibition after imidocarb exposure. The use of at least two substrates (acetyl and butyrylthiocholine) is recommended for whole blood cholinesterase analyses in domestic animals since it will allow monitoring of both acetyl- and butyrylcholinesterase activities, respectively, and a more accurate detection of exposure to anticholinesterase compounds. However, acetylthiocholine could be used as a unique substrate for whole blood cholinesterase determination in porcine and ruminant samples since butyrylcholinesterase activity is very low in these species. Additionally, propionylthiocholine could be used as an alternative substrate to butyrylthiocholine in horse whole blood samples.     

Evaluation of the test for detecting trypanosoma circulating antigens using monoclonal antibodies. Experimental and natural infections]

An antigen detection ELISA for the diagnosis of trypanosomes was recently proposed by Nantulya and Lindqvist (1989). Based on species-specific monoclonal antibodies, this test could be used to diagnose a current infection and to identify the causing trypanosomes. The test was evaluated at CRTA during experimental infections in small ruminants and with sera from naturally infected cattle, thanks to reagents supplied by the International Laboratory for Research on Animal Diseases (ILRAD). Sera from cattle sampled in France were also tested. Cattle sera from France gave optical densities (OD) from 0.007 to 0.009 with three monoclonal antibodies against T. congolense, T. vivax and T. brucei. These OD values were well below 0.050, which is considered as a positive threshold OD reading. In the small ruminant experimental infections, the sensitivity of the test was 63.2% for T. congolense-infected animals and 9.9% for T. vivax-infected animals. The sensitivity of parasitological tests was 55.1 and 48.6%, respectively. The combination of the antigen- and parasite-detection tests increased the sensitivity to 82.4 and 52.8%, respectively. Means of OD values, with the naturally infected cattle sera, were 0.116 +/- 0.030 for T. congolense, and 0.011 +/- 0.028 for T. vivax-infected animals. Sixteen out of 20 T. congolense-infected sera (sensitivity of 80%) and one out of 20 T. vivax-infected sera (sensitivity of 5%) gave an OD value exceeding 0.050. The determination of a threshold OD reading lower than 0.050 would greatly improve the sensitivity of the test. This determination could either be done by studying the preinfection sera or a local population of animals living in an area free from trypanosomosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential expression of surface membrane antigens on bovine monocytes activated with recombinant cytokines and during Trypanosoma congolense infection

The expression of surface membrane antigens on peripheral blood monocytes (PBM) of cattle of the Boran and N'Dama breeds activated with recombinant cytokines (TNF-alpha and IFN-gamma) and during experimental infection with Trypanosoma congolense was investigated using monoclonal antibodies (MoAbs) and fluorescein-activated cell sorter (FACS). The surface antigens investigated were C3bi receptor, major histocompartibility (MHC) II complex (Ia antigen) and two monocyte/macrophage (Mphi) differentiation antigens. The study revealed that both cytokines caused the enhancement of the expression of all the PBM surface antigens studied. rBolFN-gammaat low concentrations was more efficient in causing the activation of PBM. While the PBM of Boran cattle were more significantly activated to express the C3bi receptor vis-à-vis the Ia antigen than N'Dama cattle, the reverse was the case with the PBM of N'Dama cattle which expressed more Ia antigens than Boran PBM. Similar results were observed during T. congolense infection in the two breeds of cattle. The significantly higher expression of C3bi receptor and correspondingly lower Ia antigen expression by the PBM of Boran cattle, both during trypanosomosis and in vitro may be responsible for the higher rate of erythrocyte phagocytosis, hence the development of more severe anaemia by Boran cattle during trypanosomosis than N'Dama. In addition, the expression of significantly higher numbers of Ia antigen by N'Dama Mphi, hence are more able to process, present and initiate better trypanosome antigen-specific immune response than Boran cattle during infection. These two attributes are known genetic characteristics of trypanotolerance in cattle.     

不同物种Agouti基因编码区生物信息学分析

为了研究不同物种Agouti基因的遗传多样性,试验采用生物信息学方法比较分析了山羊、绵羊、牛、髯羊、野猪、马、犬、狒狒、黑猩猩、人、兔、家猫、鼠和猕猴Agouti基因编码区(CDS)的遗传多样性。结果表明:来自14个物种的77条基因序列中检测到93个多态位点,共生成30种单倍型,物种间及物种内Agouti基因序列编码区存在较丰富的遗传多样性。     

Identification of highly specific and cross-reactive antigens of Leishmania species by antibodies from Leishmania (Leishmania) chagasi naturally infected dogs

The Leishmania species present a genetic homology that ranges from 69 to 90%. Because of this homology, heterologous antigens have been used in the immunodiagnosis and vaccine development against Leishmania infections. In the current work, we describe the identification of species-specific and cross-reactive antigens among several New World Leishmania species, using symptomatic and asymptomatic naturally Leishmania chagasi-infected dog sera. Soluble antigens from five strains of New World Leishmania were separated by electrophoresis in SDS-PAGE and immunoblotted. Different proteins were uniquely recognized in the L. chagasi panel by either symptomatic or asymptomatic dog sera suggesting their use as markers for the progression of disease and diagnosis of the initial (sub-clinical) phase of the infection. Cross-reactive antigens were identified using heterologous antigenic panels (L. amazonensis strains PH8 and BH6, L. guyanensis and L. braziliensis). L. guyanensis panel showed the highest cross-reactivity against L. chagasi specific antibodies, suggesting that proteins from this extract might be suitable for the diagnosis of visceral canine leishmaniasis. Interestingly, the 51 and 97 kDa proteins of Leishmania were widely recognized (77.8% to 100%) among all antigenic panels tested, supporting their potential use for immunodiagnosis. Finally, we identified several leishmanial antigens that might be useful for routine diagnosis and seroepidemiological studies of the visceral canine leishmaniasis.     

Comparison of different Fasciola hepatica antigens and their use to detect infection in sheep by an enzyme immunoassay

Soluble antigens of adult Fasciola hepatica were extracted from homogenized parasites and purified with gel chromatography on Sephadex G-200. Six peaks were obtained, and the second highest in molecular weight showed highest correspondence in a microplate enzyme immuno assay with parasite metabolite antigens. Optimal coating antigen concentration was 5 micrograms/ml and sample dilution 1:20. Total running time of assay was 3 hours. Positive and negative sheep sera was obtained from a group of experimentally infected tracer lambs.     

Evaluation of relationship among three purified antigens from Pasteurella multocida strain P-1059 and of their protective capacities in turkeys

Three antigens were prepared from Pasteurella multocida strain P-1059, and their immunogenicity and antigenic relationships were investigated. The 3 antigens were a soluble antigen purified from a 2.5% NaCl extract (2.5S), a similar antigen purified from an extract in 0.3% formalin solution containing 0.85% NaCl (FS), and lipopolysaccharide (LPS). The antigens were treated with various chemicals and enzymes to study their antigenic and immunogenic determinants. Antigenic analyses with ELISA inhibition tests indicated that 2.5S and FS were similar LPS-protein complex antigens. The 2.5S and FS antigens induced protective immunity in turkeys with high antibody titers against LPS antigen. Although LPS was a component of 2.5S and FS, LPS itself was poorly immunogenic in turkeys. The antigenicity of protein compounds in 2.5S was deteriorated by protease treatment, which, however, did not significantly diminish the protective immunogenicity. Treatment of 2.5S with sodium periodate, altering its carbohydrate moieties, decreased its immunogenicity. The immunogenicity of 2.5S also was abolished by phenol-water treatment, owing to dissociation of the LPS-protein complex. These findings suggest that a certain form of LPS-protein complex is essential for the induction of immunity against the P multocida infection in turkeys.     

Distribution and genetic variability among Campylobacter spp. isolates from different animal species and humans in Switzerland

In Switzerland, a national database with 1028 Campylobacter isolates from poultry, pigs, cats, dogs, cattle, humans, zoo animals and water has been created. The database contains the genetic fingerprint and background information of each Campylobacter isolate. Dominant species could be identified in the different sources with a majority of Campylobacter jejuni in poultry (73%), humans (79%), cattle (95%), zoo animals (40%) and water (100%), of Campylobacter coli in pigs (72%), and of Campylobacter upsaliensis/helveticus in cats and dogs (55%). The comparison of three genotyping methods, amplified fragment length polymorphism (AFLP), pulsed field gel electrophoresis and restriction fragment length polymorphism, revealed that AFLP allows discrimination between the different Campylobacter species and is the most appropriate method to distinguish specific strains within the same species. Genotyping analysis demonstrated that the Campylobacter population is heterogeneous among the different sources and that no dominant clone is spread in the country. Genotyping and the resulting database are useful tools to trace back future Campylobacter infections.     

不同季节和种鸡日龄对种蛋孵化率的影响

夏季和冬季出雏的二批罗曼种鸡饲养和孵化测定结果表明，不同季节对种蛋的受精率和孵化率有一定的影响；而不同的种鸡日龄对种蛋的受精率和孵化率也有较大的影响；季节和种鸡日龄的双重影响对受精率和孵化率的影响更明显。     

Nucleotides in sow colostrum and milk at different stages of lactation

An experiment was conducted with the objective of measuring the concentrations of total milk solids (TMS), CP, and 5'monophosphate nucleotides in sow colostrum and milk. Twelve multiparous sows (Landrace x Yorkshire x Duroc) were used. Litter size was standardized at 11 piglets for all sows at farrowing. Sows were fed an 18.45% CP corn-soybean meal-based diet throughout lactation. The experimental period was the initial 28 d of lactation, with colostrum collected within 12 h of farrowing and milk collected on d 3, 7, 14, 21, and 28. Colostrum and milk samples were analyzed for TMS, CP, adenosine 5'monophosphate (5'AMP), cytidine 5'monophosphate (5'CMP), guanosine 5'monophosphate (5'GMP), inosine 5'monophosphate (5'IMP), and uridine 5'monophosphate (5'UMP). Total milk solids decreased (P < 0.05) from 26.7% on d 0 to 23.1% on d 3. The TMS further decreased (P < 0.05) to 19.3% on d 7, but remained relatively constant thereafter at 18.2, 18.8, and 19.2% on d 14, 21, and 28, respectively. The concentration of CP decreased from 16.6% in colostrum to 7.7, 6.2, 5.5, 5.7, and 6.3% in milk collected on d 3, 7, 14, 21, and 28, respectively (linear and quadratic effect; P < 0.05). Concentrations of 5'AMP, 5'CMP, 5'GMP, and 5'IMP increased from d 0 to d 3 and d 7, and then decreased during the remaining lactation period (quadratic effect; P < 0.05). The concentration of 5'UMP decreased from d 0 to 28 of lactation (linear and quadratic effects; P < 0.05). In colostrum, 5'UMP represented 98% of all 5'monophosphate nucleotides, and in milk, 5'UMP accounted for 86 to 90% of all nucleotides, regardless of day of lactation. The results of this experiment suggest that the concentrations of TMS and CP in sow mammary secretions changed during the first week of lactation, but were constant thereafter. Likewise, the concentrations of 5'monophosphate nucleotides changed during the initial week postpartum, but during the last 2 wk of a 4-wk lactation period, the concentrations were constant.     

High antigenic cross-reaction among the bacterial species responsible for diseases of cultured freshwater fishes and strategies to overcome it for specific serodiagnosis

Antigenic sharing among the most commonly bacterial pathogens such as Aeromonas hydrophila, Edwardsiella tarda and Pseudomonas fluorescens of Indian major carps has been studied using immunological reactions such as cross-agglutination, disc diffusion and indirect enzyme linked immunosorbent assay (ELISA). The data were analysed using statistical analysis (SAS), version 6.12. The results showed high antigenic similarities among the bacterial whole cells, whole cell lysates, somatic 'O' antigens, lipopolysaccharides (LPS) and extracellular products (ECP). However, few or no similarities were observed in ECP components of <20kD. The present study indicates a need to develop differential diagnostic methods based on serology by choosing the highly specific less cross-reactive ECP antigen.     

Functional and structural comparison of cytokines in different species

As the number of recombinant cytokines increases, so does our knowledge of their structure and function in different species. The biological cross-reactivity of cytokines from one species on cells from a different species has been reported on in the literature but this information is scattered over many publications and some of it has not yet been published. Comparing sequence information combined with three-dimensional and receptor-binding information (i.e. biological cross-reactivity) in different species provides insight into the underlying rules governing cross-reactivity and conservation. It was observed that there is quite a strict threshold of 60% amino acid identity above which cytokines tend to cross-react. Below this threshold few cytokines cross-react on cells from a different species. When comparing frequencies of reported species cross-reactivities between cytokines belonging to different cytokine-folding families it is obvious that not all cytokines within these folding families are equally cross-reactive. The underlying reason for these differences may lay in the ability of certain folding families to accumulate more mutations and still produce a protein, which is able to fold in the desired tridimensional structure. For example, cytokines belonging to the short 4-alpha-helix bundle can accumulate mutations in the 4-alpha-helices and large loops connecting the 4-alpha-helices and are the least cross-reactive. In contrast, cytokines belonging to the beta-sheet based folds (beta-trefoil and beta-sandwich) are the most cross-reactive and also the most conserved cytokines amongst the different species studied.     

Production and characterization of monoclonal antibodies to serotype specific antigens of Haemophilus pleuropneumoniae serotype 2

Mouse monoclonal antibodies were produced to Haemophilus pleuropneumoniae bacteria of the most common serotype 2. 11 hybridoma cultures were recovered that produced antibodies with moderate to strong reactivity to the antigen. 10 of these antibodies were specific to isolated capsular antigens from H. pleuropneumoniae of serotype 2, while one antibody reacted with capsular antigens from bacteria of all 8 serotypes. One hybridoma producing antibodies with a titre of 1:1000 was cloned and the antibody specificity studied further. The binding of this antibody (1F3) to whole bacteria, and capsular extracts isolated at different temperatures indicate that the antibody is specific for a thermostable polysaccharide antigen present in the cellular capsule of H. pleuropneumoniae of serotype 2.     

间接酶免疫组化检测DPV在感染鸭体内细胞定位的研究和应用

以超速离心浓缩结合蔗糖密度梯度离心法提纯的鸭瘟病毒（DPV）作为抗原免疫家兔制备兔抗DPV血清,用饱和硫酸铵沉淀结合DEAE-Sephedax-A-50离子交换柱层析提纯兔抗DPV IgG,建立了检测甲醛固定鸭体组织石蜡切片上DPV抗原的间接酶免疫组化法。该法能特异地检测出DPV感染鸭组织中的DPV而对鸭疫里默氏菌、鸭源多杀性巴氏杆菌（5∶A）、鸭源大肠杆菌（O1）感染致死的鸭组织以及正常鸭组织呈阴性反应,检测DPV感染死亡鸭的肝、十二指肠、脑、脾和胸腺的结果与PCR检测结果相同。其敏感性高于原位杂交。对DPV CH强毒株人工感染致死鸭的检测结果表明,该法可特异检测到肝、肺、肾、脑、十二指肠、空肠、回肠、直肠、法氏囊、脾脏、腺胃以及食管中的DPV。DPV主要分布于这些器官的上皮细胞和巨噬细胞。对1992-2004年经10%福尔马林保存的鸭瘟临床病例的肝脏检测结果均为阳性。该法可用于DPV感染鸭的诊断和定位检测,也可用于对甲醛固定组织进行回顾性诊断检测。