Assay of protein A in Staphylococcus hyicus subsp. hyicus by ELISA and immunoelectron microscopy

The presence and quantity of protein A in Staphylococcus hyicus subsp. hyicus isolates were examined by an enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy. Cell-bound protein A was demonstrated in 45 (94%) of 48 isolates from diseased pigs and in 113 (86%) of 132 isolates from healthy pigs by ELISA using peroxidase-conjugated rabbit antibody, but was not found in isolates from chickens and cows. Most of these swine isolates contained about 100 to 300 ng of cell-bound protein A/ml. Extracellular protein A was not detected in any isolates from pigs, chickens or cows. In the immunoelectron microscopy assay, swine isolates were labeled with goat anti-mouse IgG conjugated to colloidal gold particles, but chicken and cow isolates were not labeled.     

Staphylococcosis of turkeys. 2. Assay of protein A levels of staphylococci isolated from turkeys

Ninety-eight Staphylococcus aureus isolates and 227 coagulase-negative staphylococcal isolates from turkeys were assayed for protein A content. The amount of protein A of each isolate was quantitated using a direct enzyme-linked immunosorbent assay. Of the S. aureus isolates, 83% possessed some protein A, whereas only 13% of the coagulase-negative staphylococci contained some protein A. No correlation was seen between protein A content and ability to adhere to turkey cells. No differences in virulence were seen between isolates of S. aureus possessing high or low levels of protein A; however, an isolate with no protein A was avirulent.     

Protein A in Staphylococcus intermedius isolates from dogs and cats

The presence and quantity of extracellular and cell-bound protein A of Staphylococcus intermedius isolates from dogs and cats were determined, using an enzyme-linked immunoglobulin-binding assay. Horseradish peroxidase-conjugated rabbit anti-bovine immunoglobulin G purified by affinity chromatography was reacted with whole cell and supernatant fractions of S intermedius (n = 139), a protein A-producing strain of S aureus, and a protein A-deficient strain of S epidermidis. Extracellular protein A was found in 118 (84.9%) of 139 isolates of S intermedius. Most (69/118; 58.5%) of these isolates produced greater than 0.2 micrograms of extracellular protein A/ml. Cell-bound protein A was found in 6 (4.3%) of 139 isolates. Only 1 of these isolates contained cell-bound protein A exclusively. The other 5 isolates produced significantly greater amounts of extracellular protein A than cell-bound protein A. Additionally, greater than 96% of extracellular protein A could be removed from supernatants by adsorption with agarose gel containing immunoglobulin G.     

A comparison of extracted proteins of isolates of Dermatophilus congolensis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting.

Antigenic diversity within a collection of 18 isolates of Dermatophilus congolensis from different Continents was examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blotting with sera from cattle with clinical dermatophilosis using whole cell extracts obtained by three methods and one extract of extracellular products of D. congolensis. One of the methods involving the release of a lysostaphin-solubilized protein (LSP) of whole cells of D. congolensis revealed a number of discrete and easily-identifiable bands in SDS-PAGE which were found suitable for characterizing protein patterns and was, therefore, subsequently used for a comparative analysis of the proteins of all the D. congolensis isolates. Six electropherotypes (ET) of D. congolensis were identified among the 18 isolates using the protein profiles based on the presence of four protein bands at Molecular weights (MW) 62, 28, 17.4 and 16.4 kDa. The ETs were found among isolates from different animal species and from different sources with ET1 consisting of three bovine and two equine isolates; ET2, two bovine and three ovine isolates; ET3, two bovine isolates; ET4, two bovine isolates; ET5, one bovine and one ovine isolates and ET6, two bovine isolates. Immunoblotting of the extracts of D. congolensis isolates with sera from cattle with clinical dermatophilosis infection demonstrated protein bands of MW ranging from 9 kDa to 188 kDa. Sera from chronic dermatophilosis infection demonstrated a 28 kDa protein which was immunodominant in the LSP extracts of all the 18 isolates of D. congolensis tested while sera from mild infections demonstrated mainly the 62 kDa protein in the same extracts. However, many protein bands were demonstrated in surface membrane (TSMP) and extracellular protein extracts with sera from only mildly infected animals. The protein patterns observed in all isolates of D. congolensis revealed global antigenic similarities and distinct differences among isolates which could not be associated with either geographic, climatic or host factors. Also sera from infected animals from endemic regions of dermatophilosis could not differentiate isolates of D. congolensis. This suggests the possibility that such sera must have come from animals that had been infected by a multitude of D. congolensis strains present in the herd environment and strains an animal could have come across during the 'ritual' annual cross-country migration of the cattle herds.     

Characterization of swimming motility and identification of flagellar proteins in Salmonella pullorum isolates

OBJECTIVE: To identify swimming motility in Salmonella pullorum isolates and to characterize the flagellar proteins produced by motile isolates. SAMPLE POPULATION: 30 S pullorum isolates and isolates of 7 other Salmonella sp. PROCEDURE: Salmonella pullorum isolates were inoculated into high motility medium to evaluate swimming motility. Putative flagellar proteins were purified from the organisms and analyzed by means of gel electrophoresis and western blotting procedures, using various antisera specific for flagellar proteins. Antisera shown to be reactive with putative flagellar proteins were incorporated into the growth medium to examine their effects on motility of the isolates. RESULTS: All S pullorum isolates had evidence of swimming motility. Two putative flagellar proteins were purified from 2 of the S pullorum isolates: a 60 to 62 kd protein shown to react with antiserum specific for type y flagellar protein, and a 58 to 59 kd protein shown to react with antiserum specific for type d flagellar protein and with antibody reactive to a highly conserved flagellar epitope found on various Enterobacteriaceae. Antiserum specific for type d flagellar protein inhibited swimming motility of S pullorum isolates, but antiserum specific for type y flagellar protein did not. CONCLUSIONS: Results suggest that S pullorum isolates can be induced to manifest swimming motility when grown on medium with a low agar concentration and possess a 58 to 59 kd protein of d serotype and a second protein of 60 to 62 kd that also may be a flagellar protein.     

9株绵羊肺炎支原体内蒙古分离株P113基因克隆及蛋白序列分析

[目的]克隆绵羊肺炎支原体内蒙古分离株P113基因,比对、分析不同菌株P113蛋白序列,为研究绵羊肺炎支原体P113蛋白的生物学功能提供参考。[方法]设计绵羊肺炎支原体P113基因特异性引物,采用PCR方法扩增9株绵羊肺炎支原体内蒙古分离株的P113基因片段;对获得的9株绵羊肺炎支原体P113基因片段进行测序分析,将DNA序列翻译为氨基酸序列,对不同菌株的P113氨基酸序列进行比对。[结果]不同分离株P113基因片段扩增产物大小不同。氨基酸序列分析显示,C末端序列重复区域长度存在差异,以KKAEGA（S）QNQG为主要重复序列单元。不同菌株重复序列单元数量不同,NM01-MO株和CK-MO株的重复序列单元数量最多,为16个;LK-MO株重复序列单元数量最少,为3个;多数菌株之间重复序列单元数量差异较大。[结论]绵羊肺炎支原体内蒙古分离株P113氨基酸序列的C末端重复序列单元数量不同,这些不同数量的重复序列影响P113蛋白结构,进而可能影响其生物学功能。     

Identification of variations in SzP proteins of Streptococcus equi subspecies zooepidemicus and the relationship between protein variants and clinical signs of infection in horses

OBJECTIVE: To determine whether previously unidentified variations of the SzP protein of Streptococcus equi subsp zooepidemicus were present in horses with various clinical signs of infection and whether any relationship could be identified between SzP protein variants and naturally occurring clinical conditions. SAMPLE POPULATION: 23 isolates of S equi subsp zooepidemicus were recovered from specimens of horses with various clinical conditions and used as a representative population of isolates for evaluation of different SzP protein variants. PROCEDURE: Genetic heterogeneity of the isolates was demonstrated by repetitive extragenic palindromic-polymerase chain reaction analysis. The SzP gene was sequenced and the presumed protein sequence determined for each isolate. Characteristics of the SzP proteins were compared among the isolates and in relation to the clinical conditions of horses from which they were recovered. RESULTS: The signal peptide types, number of proline-glutamic acid-proline-lysine repeats, and anchor sequences were consistent with those previously described for the SzP protein. Many of the isolates clustered with 5 previously described types on the basis of the hypervariable region of the SzP protein. One additional variant, which represented 8 of the isolates, was identified. Particular motifs in the hypervariable region accounted for many of the differences among hypervariable types. CONCLUSIONS AND CLINICAL RELEVANCE: The SzP protein appears to be limited to a selected number of types. Variations in the SzP protein are frequently determined on the basis of different motifs rather than random amino acid substitutions. There does not appear to be any association of SzP protein variations and clinical manifestations of infection in horses.     

Variation of the agr locus in Staphylococcus aureus isolates from cows with mastitis

Staphylococcus aureus isolates from mastitic cow's milk were examined for production of alpha-hemolysin and protein A and their accessory gene regulator (agr locus) was analyzed. An inverse relationship between alpha-hemolysin and protein A production was found in most of the 76 isolates, suggesting that the isolates tested may be classified into group I (high alpha-hemolysin/low protein A), II (low alpha-hemolysin/high protein A), or III (low alpha-hemolysin/low protein A). The agr locus, which consists of hld, agrB, agrD, agrC, and agrA, was detected in most of the 78 isolates including two reference strains (Wood 46 and Cowan I) by polymerase chain reaction (PCR). When the PCR products for agr locus of 22 isolates from groups I and II were digested with restriction enzyme MboI, seven bands of the expected lengths were recognized in strain Wood 46, but not in the other isolates tested. Nucleotide sequence analysis of PCR products from six isolates revealed that the agr locus sequence of strain Wood 46 corresponded to that of the published sequence data, but the other five isolates from groups I and II diverged at agrB and agrD sequences and thus the deduced amino acid sequences. These variations of agr locus in S. aureus bovine isolates differed from those reported by Ji et al. [Science 276 (1997) 2027].     

Outer membrane protein profiles of Yersinia ruckeri

The outer membrane protein (OMP) profiles of 135 isolates of Yersinia ruckeri, obtained from nine European countries (100 isolates), North America (23 isolates), Australia (six isolates) and South Africa (two isolates), and including four reference strains, were examined by SDS-PAGE. Outer membranes were isolated by selective solubilisation of the cytoplasmic membrane with 0.5% (w/v) sodium N-lauroyl sarcosinate (Sarkosyl). Outer membrane proteins were stable after in vitro passage and there was no variation in OMP profiles due to colony selection. With the exception of a 39.5 kDa peptidoglycan-associated protein there was also no variation at different stages of the growth cycle. The 39.5 kDa protein was not produced during logarithmic growth phase but increased in abundance as the stationary phase progressed. Interstrain variation occurred in the possession of a 36.5 or 38 kDa heat-modifiable protein and in the possession of peptidoglycan-associated proteins in the molecular weight range 36.5 to 40.5 kDa. Based on variation of these proteins five OMP-types, designated OMP-types 1-5, were identified among the 135 isolates examined. Outer membrane protein analysis was demonstrated to be useful in epidemiological studies of Y. ruckeri.     

Characterisation of the outer membrane protein antigens of British field isolates of Moraxella bovis

Outer membranes of a single isolate of Moraxella bovis were prepared and their purity evaluated by a comparison of the iodinated proteins from whole cells and on outer membranes. The protein patterns of the outer membranes of 10 isolates were examined by SDS-polyacrylamide gel electrophoresis, and the antigenic relationship between proteins of different isolates determined by immunoblotting, using antiserum produced against the outer membrane of the single isolate of M. bovis. The overall protein pattern of the different isolates was similar although not identical, but, significantly, only three separate immunogenic proteins were common to all the isolates under examination.     

Protein variability among Mycoplasma hyopneumoniae isolates

Sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to study the protein variability of Mycoplasma hyopneumoniae isolates. Fifty-six M. hyopneumoniae isolates from 6 different countries and 37 different herds were used. From eight herds, more than one isolate was available. All SDS-PAGE patterns of isolates originating from different herds were clearly divergent. Intra-species protein variability was quantified using the reference strain J and seven field strains all obtained from different herds and classified according to virulence. Between the field strains, a variability of 25% was found, while the culture-adapted strain J was clearly divergent and showed 30% variability with the field strains. No clustering according to virulence was obtained, but a protein band of about 181 kDa was present in the two highly virulent isolates whereas this protein band was absent in the moderately and low virulent isolates. Protein patterns of isolates derived from different animals from the same herd, were identical or differed in only a few protein bands. This study clearly indicates that, in agreement with previous studies on genomic diversity of M. hyopneumoniae isolates, proteomic variability within the species is high. Our study did not find clear evidence that more than one M. hyopneumoniae isolate circulates within a herd at a specific time point. The minor differences found between M. hyopneumoniae isolates from the same herd might reflect the organism's ability to alter its proteomic expression profile under field conditions.     

2株鸽源新城疫病毒的毒力鉴定和基因型分析

2008年从临床鸽病例中分离到2株新城疫病毒毒株,经测定,鸡胚平均死亡时间(MDT)分别为48h和46h,8周龄SPF鸡静脉接种指数(IVPI)分别为2.88和2.91,一日龄雏鸡脑内接种指数(IPCI)分别为1.88和1.89,按照OIE推荐的标准均为强毒株。RT-PCR扩增出融合蛋白(F)基因片段,测序,推导的氨基酸序列中蛋白酶裂解位点附近的氨基酸序列均符合强毒特征序列。根据F蛋白的部分基因序列绘制的系统进化发生树和F基因片段中3种限制性内切酶(RE)位点分布判定这2个毒株均为基因Ⅶc型。     

Genomic sequences of low-virulence avian paramyxovirus-1 (Newcastle disease virus) isolates obtained from live-bird markets in North America not related to commonly utilized commercial vaccine strains

Avian paramyxovirus 1 (APMV-1), also referred to as Newcastle disease virus (NDV), variants of low virulence were isolated from chickens, ducks and other unidentified species found in live-bird markets of the northeastern United States. These isolates were characterized as APMV-1 by the hemagglutination-inhibition (HI) assay utilizing NDV-specific polyclonal antisera. However, the isolates failed to react with a monoclonal antibody that has specificity for a wide variety of APMV-1 isolates. Although only highly virulent isolates require reporting to international regulatory agencies, the ability to correctly identify APMV-1 types is important for control and regulatory purposes. Protein gel patterns of the purified isolates resembled previously reported APMV-1 and anti-NDV polyclonal sera recognized the viral proteins. For three isolates oligonucleotide primers specific for the nucleoprotein, fusion protein and polymerase genes of NDV were utilized to synthesize cDNA using viral RNA as a template. Approximately 12kb of the genome was subsequently sequenced for the three isolates that included the nucleoprotein, phosphoprotein, matrix protein, fusion (F) protein, hemagglutinin-neuraminidase protein genes and a 5' portion of the polymerase gene. The isolates had an F protein cleavage site sequence of ERQER/LVG indicating low-virulence viruses that phylogenetically separated with other unique NDV isolates designated as a lineage 6 genotype. Additionally, a four amino acid insert was detected in the predicted phosphoprotein which complies with the "rule of six" among paramyxoviruses. These APMV-1 genotypes have not been previously reported in North America and further substantiate the heterogeneous genetic nature of these commercially important pathogens found worldwide.     

Comparison and partial characterization of the protein profiles and outer membrane antigens of Actinobacillus species isolated from ram lambs with epididymitis

Three monoclonal antibodies (LG17, LG30, LG33) were used in the indirect fluorescent antibody test, the ELISA, and the immunoelectrotransfer blot technique to identify group-specific and strain-specific epitopes on the outer membranes of Actinobacillus seminis, A actinomycetemcomitans, and 17 field isolates of Actinobacillus spp. The field isolates had been obtained by bacteriologic culture of specimens from ram lambs with epididymitis. Only antibody LG33 consistently had specificity for an outer membrane epitope shared by most of the bacterial isolates tested. Staining of polyacrylamide gels with periodic acid-Schiff reagent, Sudan black B, and Coomassie brilliant blue R250 indicated that target antigens for antibodies LG17 and LG33 contained carbohydrate and lipoprotein components, respectively. The chemical composition of the LG30 target antigen was not determined because of its instability after exposure to sodium dodecyl sulfate. Discontinuous-gradient polyacrylamide gel electrophoresis in sodium dodecyl sulfate and spectrophotometric scans of the gels were used to analyze n-octyl-beta-D-glucopyranoside protein extracts from A seminis, A actinomycetemcomitans, and 13 representative field isolates of Actinobacillus spp. Bacterial isolates could be grouped according to their protein profiles. The first group consisted of A seminis, A actinomycetemcomitans, and 7 field isolates of Actinobacillus spp, all of which shared common protein bands with molecular masses of approximately 94 kilodaltons (kD), 64 kD, 60 kD, 52 kD, 44 kD, and 26 kD. The second group was composed of 6 field isolates, each with unique protein profiles; isolates had relatively few protein bands in common. These data suggested that members of the genus Actinobacillus cultured from ram lambs with epididymitis probably include a number of various strains.     

Characterization of pigeon-origin Newcastle disease virus isolated in China

Fourteen pigeon-origin Newcastle disease virus (NDV) isolates were obtained from sick pigeons in China between 1996 and 2005. The mean death time (MDT) of embryonated eggs and the intracerebral pathogenicity indices (ICPI) were tested to determine the virulence of the field isolates. The result indicated that most isolates were proved to be mesogenic (MDT 60-90 hr and ICPI > 1.2). The main function regions of F protein gene of the isolates were amplified and sequenced for phylogenetic and residue substitutive analysis. The fusion protein cleavage site sequences of most isolates had multiple basic amino acids R/KRQKRF at positions 112-116 and a phenyl alanine at position 117, characteristic of velogenic isolates. In the phylogenetic tree, the majority of the isolates were clustered into a single genetic lineage, termed genotype VIb, and were typical pigeon paramyxovirus type 1, whereas a small number of recent isolates (three strains) were grouped into genotype VIId, a predominant genotype responsible for most Newcastle disease outbreaks in chickens and geese since the end of last century. One isolate, PK9901, was proved to be a lentogenic strain, of genotype II NDV, to which the vaccine strain La Sota belongs.     

Electrophoretic profiles of Pasteurella multocida isolates from animals with hemorrhagic septicemia.

We determined that the protein profiles of 14 isolates from animals with hemorrhagic septicemia were relatively homogeneous and could be placed in 2 distinct groups on the basis of their country of origin. Such differences correlated with the serotypic properties of the individual isolates; hemorrhagic septicemia isolates of Asian and North American origin (Carter B) had a major protein band with an apparent molecular mass of 32 kDa, whereas those of African origins (Carter E) had a major protein band with an apparent molecular mass of 37 kDa. The possession of a major 32-kDa protein band appeared to be unique to Carter B isolates, suggesting that electrophoresis may be a useful nonserologic technique for the identification of organisms of this serotype. Other major bands with apparent molecular masses of 27, 45, and 47 kDa were shared by all strains, regardless of their serotype. The lipopolysaccharides were of low molecular mass and relatively uniform from 1 isolate to the next.     

Epidemiology of ovine Campylobacter infection determined by numerical analysis of electrophoretic protein profiles

The relationship of 50 Campylobacter strains isolated from aborted ovine foetuses, and the faeces of sheep, cattle and chickens were determined by numerical analysis of electrophoretic (SDS-PAGE) protein profiles. Comparison of protein patterns by numerical methods revealed differences between C. fetus ssp. fetus, C. jejuni, and C. coli strains as well as heterogeneity among isolates from different outbreaks. Isolates from each farm produced a distinct cluster and flocks from different locations were found to be infected with relatively different strains. In most cases, protein patterns of ovine foetal isolates were very similar to those of ovine faecal isolates. Ovine isolates of C. fetus ssp. fetus, C. jejuni and C. coli gave similar protein patterns to the corresponding Campylobacter species isolated from cattle or chicken, on the same farm. Thus, it was concluded that certain protein types of ovine Campylobacter strains were more likely associated with local areas, and Campylobacter strains causing ovine abortions are distributed in the environment more widely than assumed to date.     

Surveillance and characterization of Newcastle disease viruses isolated from northern pintail (Anas acuta) in Japan during 2006-09

A total of 38 Newcastle disease virus (NDV) isolates were obtained from 6060 fecal samples from northern pintail (Anas acuta) ducks collected in the Tohoku district in Japan during 2006-09. One isolate from each sampling location and date was selected for a total of 38 isolates, then 15 of these were characterized for their pathogenicity by mean death time of minimum lethal dose (MDT/MLD) using chicken embryos and by plaque formation on chicken embryo fibroblasts. Furthermore, nine isolates were randomly selected from these 15 isolates, and the fusion protein genes were sequenced to characterize amino acid sequences around the cleavage site. All 15 were confirmed to be nonvirulent by MDT/MLD test, and nine isolates were also confirmed as nonvirulent by the cleavage site of the fusion protein 112G/E-K/R-Q-G/E-R*L117 that was specific for nonvirulent NDVs. The characteristics of nine isolates identified by phylogenic analysis of the fusion protein gene indicated that the isolates belong to genotype I or II. In addition, we also isolated 68 avian influenza viruses and 28 other hemagglutinating viruses. Our data indicate that northern pintails are subclinically infected by, perpetuate, and distribute NDV along with different subtypes of avian influenza viruses and other hemagglutinating viruses during their migrations across vast areas over the Northern Hemisphere to Japan.     

Analysis of protein compositions and surface protein epitopes of Anaplasma centrale and Anaplasma marginale.

Protein composition was compared and epitopes were analyzed among the isolates of Anaplasma centrale and A. marginale by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting using bovine antisera and monoclonal antibodies, and enzyme-linked immunosorbent assay. Common and unique proteins were found among the isolates. All isolates tested had a major surface protein with an apparent molecular weight of 38 to 40 kilodalton which had slight molecular size variations between species. This protein was also a dominant immunogen to the host. At least two species-common epitopes, one of which might contain carbohydrate(s), were present on the major surface protein. One species-specific epitope was identified on the major surface protein of A. marginale isolates.     

Outer membrane protein profiles of Edwardsiella ictaluri from fish.

Outer membrane proteins (OMP) prepared with sodium N-lauroyl sarcocinate (SLS) from 33 Edwardsiella ictaluri isolates from fish were examined by electrophoresis. Twenty-eight isolates from channel catfish (Ictalurus punctatus) had similar OMP profiles. Ten bands (71 kilodaltons [kD] to 19.5 kD) were identified in all isolates from channel catfish. One major 35-kD protein comprised most of the protein content of the outer membrane of isolates from channel catfish. Differences existed among isolates in the amount of protein within minor OMP bands. Edwardsiella ictaluri ATCC 33202 contained larger quantities of the 38.5- and 37-kD proteins than did the other isolates. Outer membrane protein profiles of E ictaluri derived from Bengal danio (Danio devario) and walking catfish (Clarias batrachus) were identical to OMP profiles of isolates from channel catfish. In contrast, OMP profiles from single isolates from green knife fish (Eigemannia virescens) and white catfish (Ictalurus catus) were different. Variations in incubation time, SLS extraction time, SLS extraction number, and in vivo and in vitro passage had no effect on the OMP profile of E ictaluri ATCC 33202. An increase in duration of sample solubilization did affect the OMP profile of E ictaluri ATCC 33202 by decreasing the amount of protein in 52-, 46-, and 43.5-kD bands. Accompanying the decrease were increased staining intensity in the 31.5- and 28.5-kD bands and the appearance of 4 new bands (34, 33, 25.5, and 22.5 kD). Edwardsiella ictaluri, a gram-negative bacterium in the family Enterobacteriaceae, is the cause of enteric septicemia of catfish.(ABSTRACT TRUNCATED AT 250 WORDS)