Counter current line absorption immunoelectrophoresis in an alternative diagnostic screening test to counter current immunoelectrophoresis in Aleutian disease (AD) eradication programs

Counter current immunoelectrophoresis (CCIE) is the diagnostic method used in the ongoing Aleutian disease virus eradication program on Danish mink farms. There has been an increasing demand for an alternative diagnostic test especially to evaluate suspected false positive CCIE reactions. We compared test results of a number of negative and positive mink sera in indirect counter current immunoelectrophoresis (ICCIE), counter current line absorption immunoelectrophoresis (CCLAIE) and radio immuno assay (RIA) with test results from counter current immunoelectrophoresis and found that counter current line absorption immunoelectrophoresis is the best alternative diagnostic screening test to counter current immunoelectrophoresis for Aleutian disease eradication programs. Not only proved the CCLAIE test to be useful for evaluation of doubtfully positive CCIE reactions, but it was found to have a higher sensitivity than the CCIE test.     

Comparison of three immunodiagnostic tests for experimental Heterophyes heterophyes infection in dogs

The aim of this study was to compare the performance of three in-house diagnostic tests, i.e. counter current immunoelectrophoresis (CCIE), intradermal (ID) and indirect fluorescent immunoassay (IFI), for the diagnosis of Heterophyes infection. One hundred and twenty puppies were randomly divided into eight groups (n=15/group). Heterophyes heterophyes infections were established in these puppies by administering a dose of 50 H. heterophyes encysted metacercariae/puppy by gavage. Forty puppies of similar age and sex were divided into eight groups, of five puppies each and were used as negative controls. Sera pooled from separate infected and uninfected groups were tested against H. heterophyes antigens, weekly for 8 weeks post-infection (PI). The ID assay detected infected puppies sooner than any of the serological tests. Sero-conversion was first detected 2 weeks PI by ID assay and 1 week later by CCIE and IFI assays. ID test performed well till the end of the experiment (sensitivity and specificity: 100% and 90%, respectively). Both IFI and CCIE assays had a sensitivity of 40% and 20%, respectively for early detection of antibody, which was less sensitive than ID but both assays were more specific (100%) than the ID assay. The lowest agreement was between ID and IFI tests (40.3%), whilst the highest was observed between CCIE and IFI tests (67.2%). Of the three evaluated methods, the ID test could be recommended for scientific and laboratory diagnosis of heterophyosis in naturally infected animals. However, since none of the investigated method are optimal (i.e, offers 100% specificity and sensitivity), the choice of test employed must depend on the aim of the study.     

A unified serotyping scheme for Moraxella bovis.

Fifty-three Australian, seven British, two American and two New Zealand isolates of Moraxella bovis were classified into seven serogroups on the basis of their variable fimbrial (pilus) antigens using whole cell slide agglutination (SA), enzyme-linked immunosorbent assays (ELISA) and tandem-crossed immunoelectrophoresis (TCIE). Although results of serogroup classification by SA and ELISA were identical in 68.7% of isolates, it was found necessary to resolve the discrepancies between the two systems using TCIE. Results suggest that world-wide variation in the potentially host-protective fimbrial antigens of M. bovis may be relatively limited. It is proposed that the previous numerical classifications of British and Australian serogroups are appropriately amalgamated as a result of this latest study and are designated as serogroups A to G inclusive. A protocol for the further serotyping of fresh, fimbriate isolates of M. bovis is suggested.     

Serogroups of Campylobacter jejuni from man and animals

A total of 186 campylobacter strains from aborted calf and sheep fetuses, from scouring dogs, rabbits and man, and from retailed poultry were isolated and examined biochemically and serologically for heat stable antigens. Immune sera were produced in rabbits against Penner reference strains from 1 to 60, and against two field isolates. Out of 186 biochemically tested strains 179 (96.2%) proved C. jejuni and only 6 (3.2%) C. coli. One strain has been identified as C. laridis. In cattle and sheep 3.2 and 21.7% respectively of all campylobacter abortions were due to C. jejuni infection. The same agent caused 12.7% of diarrhoea of dogs. The campylobacter infection rate of freshly slaughtered and dressed chicken varied between 25 and 64.3%. Out of the serologically examined 140 C. jejuni strains 118 (84.3%) could be assigned to 16 Penner serogroups and 13 (9.3%) to 2 further serogroups. Serogroups 8 (31.4%), 1 (19.3%) and 2 (12.1%) occurred most frequently. The human isolates represented the widest serotype distribution, as 32 tested strains belonged to 12 serogroups. All those serogroups which caused abortion or diarrhoea in animals or were isolated from poultry carcases were isolated also from man with diarrhoea, but some serogroups were found only in man.     

DNA homology studies of leptospires of serogroups Sejroe and Pomona from cattle and swine

Hybridization studies of chromosomal DNA from leptospiral strains representing Leptospira interrogans, serogroups Sejroe and Pomona from cattle and swine were performed to determine the degree of homology among their DNA sequences. Chromosomal DNA isolated from leptospires of the Sejroe and Pomona serogroups was radiolabeled and used to probe DNA isolated from other strains in these serogroups. Serovars hardjo (hardjoprajitno), hardjo (hardjo-bovis), balcanica (1627 Burgas), pomona (pomona), and kennewicki (LT-1026) were probed to determine the degree of homology among their chromosomes. Serovars pomona and kennewicki were homologous to each other. They also had a high degree of homology with hardjo (hardjoprajitno) and, to a lesser extent, with hardjo (hardjo-bovis) strains. However, hardjoprajitno and hardjo-bovis had little homology to each other. Serovar balcanica had a high degree of homology with hardjo-bovis isolates, but little homology with hardjoprajitno. Although serologically indistinguishable, the reference strain hardjoprajitno was genetically dissimilar to hardjo-bovis strains isolated from North American cattle.     

Extensive diversity in New Zealand Dichelobacter nodosus strains from infected sheep and goats

Footrot is a contagious bacterial disease of ruminants spread by the Gram-negative, anaerobic organism, Dichelobacter nodosus. It is endemic in New Zealand and throughout sheep and goat farming regions of the world. Using the polymerase chain reaction (PCR) to amplify fragments of the fimbrial gene (fimA), D. nodosus was detected in 14 hoof scrapings, sampled from six farming regions within New Zealand. DNA sequencing revealed 15 strains covering eight serogroups on the New Zealand farms. The predominant serogroup was B which contained six strains, followed by serogroups F, H and G. No strains from serogroups D and I were detected in this investigation. Eleven out of the 15 D. nodosus strains had fimbriae sequences different to those previously reported and the presence of multiple strains on a single hoof was common (86% samples). Individual sheep from the same farm, or the same paddock, were often infected by a different range of strains, which suggests a host role in mediating footrot infection.     

肉仔鸡病原大肠杆菌地方分离株的血清型鉴定

从河北的三河、滦南、乐亭、卢龙及昌黎等地 2 9个养鸡场 (户 )分离鉴定了 82株病原大肠杆菌 ,用 54种常见动物病原大肠杆菌单价 O抗血清进行了血清型鉴定。结果 82株分离菌有65株被鉴定出了相应 O血清群 (占鉴定菌株的79.2 6%) ,分属 6种不同血清型 ,其中以 O78和O45两种为优势血清型 (占定型菌株的64.61 %) ,其余 4种 O血清群占定型菌株的 3 5.3 9%,尚有 1 7株未能定型 (占鉴定菌株的 2 0 .74%)     

Escherichia coli strains from edema disease: O serogroups, and genes for Shiga toxin, enterotoxins, and F18 fimbriae

The objectives of the research were to determine the presence of the gene sequences for Shiga Toxin 2e (Stx2e), enterotoxins (ST-I, ST-II and LT-I), and F18 fimbriae in 144 Escherichia coli strains isolated from pigs with edema disease; to assess the ability of stx2e(+) strains to produce Stx2e; and to determine the O serogroups of the E. coli strains. Presence of the genes was determined by polymerase chain reaction (PCR), production of Stx2e was assessed by cytotoxicity for Vero and Hela cells, O serogroups were identified by agglutination with specific antisera. Of the 144 strains tested, 99 were stx2e(+) by PCR, but only 45 of these were Stx2e(+) in the cell culture assays. Among the 99 stx2e(+) strains, PCR detected the genes for F18ab, ST-I, ST-II, LT-I in 76, 40, 31 and 16 strains, respectively. Forty-one of the 99 sxt2e(+) strains belonged to O group 139; the rest did not belong to the classical edema disease O serogroups. It is likely that the enterotoxins, whose genes were detected at high frequency, are responsible for diarrhea seem in pigs with edema disease in Brazil.     

Serotyping of O and pilus antigens of Escherichia coli strains isolated from chickens with coli-septicemia.

One hundred and fifty one Escherichia coli strains were isolated from broiler chickens with coli-septicemia in Aichi (63 strains), Shizuoka (58 strains), and Kagoshima (30 strains) prefectures from 1980 to 1987, and their O and pilus antigens were serologically typed. One hundred and twenty five strains (82.8%) were typed into 23 O serogroups, and twenty six strains (17.2%) remained untypable. The predominant O serogroups were O2 (35 strains, 23.2%) and O78 (24 strains, 15.9%). Distribution of O serogroup was different, depending on prefectures where they are isolated. In total, 109 strains (72.2%) possessed Type 1 and/or Fmsha pili (Type 1; 41 strains, Fmsha; 22 strains, and Type 1 and Fmsha; 46 strains), and 42 strains (27.2%) were non-piliated. All the strains lacked K88, K99, 987P, F41, and Att25 pili. The ratios of piliated strains to non-piliated ones were almost the same among the three prefectures. Strains possessing Type 1 pili showed variety of O antigens, but most of the strains with Fmsha pili belonged to O2 serogroup.     

Prevalence of fimbrial antigens and enterotoxins in nonclassical serogroups of Escherichia coli isolated from newborn pigs with diarrhea

Ninety-nine nonclassical serogroups isolated from newborn pigs with neonatal diarrhea were tested for fimbrial antigens F4(K88), F5(K99), F6(987P), F41, and F165, and for heat-labile enterotoxin, heat-stable enterotoxin a (STa), heat-stable enterotoxin b, verocytotoxin, and cytolethal-distending toxin. Thirty-two strains, belonging mostly to serogroups O64:K"V142,", O9:K103, and O20:K101, were F5-positive and usually produced STa, although 5 strains produced only heat-stable enterotoxin b. Fifteen strains, belonging mostly to serogroups O64:K"V142" and O20:K101, were F41 positive and usually produced STa. Twenty-three stains, belonging mostly to serogroups O64:K"V142," O9:K103, and O20:K101, were F6-positive and usually produced STa. Several strains produced more than one fimbrial antigen, either F5 and F41, F5 and F6, F6 and F41, F6 and F165, or F5, F6, F41, and F165. None of the strains produced heat-labile enterotoxin, verocytotoxin, or cytolethal-distending toxin. The indirect immunofluorescence test was much more sensitive than was the slide agglutination test for the detection of each of the fimbrial antigens F5, F6, F41, and F165 on strains grown in vitro. The production of F6 by certain strains for which only a low proportion of cells were F6-positive in vitro, as demonstrated by immunofluorescence, was confirmed by experimental infection of newborn pigs.     

Prevalence of serogroups and virulence factors of Escherichia coli strains isolated from pigs with postweaning diarrhoea in eastern China

This study was undertaken to determine the present distribution of serogroups, hemolytic activity and virulence factors among Escherichia coli strains isolated from pigs with postweaning diarrhoea from eight provinces in eastern China. Two hundred and fifteen E. coli isolates were serogrouped with O-antisera, investigated for hemolytic activity, assessed for F4, F5, F6, F18 and F41 fimbrial antigens by monoclonal antibodies and detected for genes of enterotoxins and shiga-toxin-two-variant (Stx2e) by a multiplex polymerase chain reaction (PCR). Among these E. coli isolates, 140 were determined to be placed in serogroups, 52 were unable to be serogrouped and the rest 23 auto-agglutinated. These isolates distributed in 45 serogroups and 64.3% (90/140) belonged to 12 O serogroups: O8, O9, O11, O20, O32, O91, O93, O101, O107, O115, O116 and O131. Hemolytic activity was detected in 11.6% (25/215) of all isolates. Several uncommon O serogroups were discovered in this study. Agglutination tests showed that 50.2% (108/215) of these isolates were positive for one or more of the five fimbrial antigens. Seventy-two E. coli strains expressed single fimbria and 36 strains expressed two or more fimbriae. Among these 215 E. coli isolates, strains expressing F18, F4, F6, F6 + F18 or F5 + F41 occurred more frequently. PCR analysis showed that 60.5% (130/215) of the isolates only harboured the gene of estI (STI) while 6.0% (13/215) strains possessed the genes of stx2e, estI and estII and 5.6% (12/215) of strains had the genes of estI/estII. Of all these isolates, 107 (49.8%) were negative for the fimbrial antigens examined. The fimbria-negative isolates usually possessed genetic determinant of estI (78, 72.9%).     

Streptoccal infections in pigs. 2. Serological and biochemical examinations.

A total of 2,275 strains of haemolytic streptococci (HS) from 1,338 post mortem examined sucking pigs were studied. The serological examination revealed that 43.9 per cent of the strains belonged to group-C and 33.7 per cent belonged to group-L (Table I). Other serogroups amounted to 8.0 per cent and 13.8 per cent of the strains were non-typable. The distribution of serogroups in relation to different organs is presented in Table II. The fermentation pattern of 115 group-C strains against trehalose, sorbitol and lactose was determined (Table III). All but one of the strains appeared to be S. equisimilis. The ability of 127 group-L strains to ferment lactose, hippurate, sorbitol and glycerol is shown in Table IV. Eleven different fermentation patterns were encountered. An antimicrobial resistance pattern was determined for 69 strains (Table V). All the strains, except one, were sensitive to penicillin and chloramphenicol. Only two strains were sensitive to streptomycin. None of the isolated serogroups or biochemical types could be related to a particular pathological lesion or to a particular herd or group of sows within the herd. By comparing the fermentation patterns of the group-L strains isolated from pigs dead with HS infection with those of previously group-L strains from healthy swine, different predominant patterns were found. Possibilities of using these differences in biochemical reactions in separating pathogenic and saprophytic strains are discussed.     

不同地区101个禽源性大肠杆菌分离株的致病性试验

从国内１８个省、市、自治区大肠杆菌病疑似病禽中分离并鉴定出大肠杆菌５９５株,鉴定出其中４４０个分离株的血清型。采用Ｒｏｓｅｎｂｅｒｇ氏报道的方法,测定了来自不同地区的６０个优势血清型分离株对ＳＰＦ鸡、３０个其他常见血清型及１１个国内少见血清型分离株对普通易感鸡的致病性。结果表明：所测定的１０１个分离株均为致病株,且９０％以上属高、中度致病株。     

Reactivity of sera from sheep immunised with individual outer membrane proteins of Bacteroides nodosus against heterologous bacterial strains

In order to identify those bacterial antigens which might be involved in immunity against ovine footrot, antisera were raised in sheep to 6 proteins in the outer membrane complex (OMC) of one strain of Bacteroides nodosus. Examination of the specificity of these antisera by Western blotting, crossed immunoelectrophoresis (XIEP) and IEP, revealed that they recognized the homologous OMC protein, but did not precipitate either undenatured pili or OMC, nor could they agglutinate the homologous bacteria. In contrast, anti-OMC and anti-pili sera could precipitate OMC or pili respectively, and agglutinate whole bacteria. Subsequent analysis of these sera against 5 strains of B. nodosus from different serogroups revealed that Proteins 1, 3 and 4 had a similar antigenic structure in all strains examined. The reactivity of anti-pili sera was restricted to homologous bacteria whereas anti-pilin sera (raised against denatured pili) also reacted with pilin from 2 of 3 heterologous strains. However, none of the patterns of staining or absorption of any of these sera matched the spectrum of cross-protection afforded by vaccination of sheep with B. nodosus strain 198 cells. The results question the role of individual OMC proteins in cross-protective immunity and may imply that interactions between several bacterial components are involved in the phenomenon.     

Serogroups of Campylobacter fetus and Campylobacter jejuni isolated in cases of ovine abortion

Of 38 aborted ovine fetuses from 23 sheep flocks 29 C. fetus subsp. fetus and 22 C. jejuni were isolated and examined biochemically and serologically for heat-stable antigens. Serologic examinations were carried out by passive haemagglutination test. In case of C. fetus subsp. fetus strains alkaline antigen extraction was used. Antisera to two serogroups of C. fetus and to Penner serotype reference strains 1 to 60 were produced in rabbits. Abortion was caused in 18 (78.3%) flocks by C. fetus subsp. fetus and in 5 (21.7%) flocks by C. jejuni. Six C. fetus subsp. fetus strains grew well at both 43 and 25 degrees C. With one exception all C. fetus subsp. fetus were resistant, whereas all 29 C. fetus subsp. venerealis strains were sensitive to 30 micrograms/ml cefoxitin and cefamandole. These two cephalosporins can be used to differentiate the two subspecies of C. fetus. Passive haemagglutination test using alkaline antigen extraction is a proper method for the examination of heat-stable antigens of both C. fetus subspecies. Out of 24 C. fetus subsp. fetus strains 13 belonged to serogroup A(01), and 11 to serogroup B(02). C. jejuni strains examined belonged to Penner serogroup 1 (6 strains), to serogroup 5 (4 strains) and to serogroup 8 (4 strains).     

Applicability of a multiplex PCR to detect O26, O45, O103, O111, O121, O145, and O157 serogroups of Escherichia coli in cattle feces

Shiga toxin-producing Escherichia coli (STEC), particularly O157, are major food borne pathogens. Non-O157 STEC, particularly O26, O45, O103, O111, O121, and O145, have also been recognized as a major public health concern. Unlike O157, detection procedures for non-O157 have not been fully developed. Our objective was to develop a multiplex PCR to distinguish O157 and the 'top six' non-O157 serogroups (O26, O45, O103, O111, O121, and O145) and evaluate the applicability of the multiplex PCR to detect the seven serogroups of E. coli in cattle feces. Published sequences of O-specific antigen coding genes, rfbE (O157) and wzx and wbqE-F (non-O157), were analyzed to design serogroup-specific primers. The specificity of amplifications was confirmed with 138 known STEC strains and the reaction yielded the expected amplicons for each serogroup. In feces spiked with pooled 7 STEC strains, the sensitivity of the detection was 4.1 × 10(5)CFU/g before enrichment and 2.3 × 10(2) after 6h enrichment in E. coli broth. Additionally, 216 fecal samples from cattle were collected and tested by multiplex PCR and cultural methods. The multiplex PCR revealed a high prevalence of all seven serogroups (178 [O26], 108 [O45], 149 [O103], 30 [O111], 103 [O121], 5 [O145], and 160 [O157]) of 216 samples in fecal samples. Cultural procedures identified 33.1% (53/160) and 35.5% (11/31) of PCR-positive samples for E. coli O157 and non-O157 serogroups, respectively. Samples that were culture-positive were all positive by the multiplex PCR. The multiplex PCR can be used to identify serogroups of putative STEC isolates.     

Characterisation and quantitation of pilus antigens of Moraxella bovis by ELISA

Hyperimmune serums raised in rabbits to purified pili from 9 Australian and 2 American strains of Moraxella bovis from infectious bovine keratoconjunctivitis (IBK) affected herds were used to study the degree of binding between combinations of antigen and antiserum in a conventional enzyme linked immunosorbent assay (ELISA). With the aid of appropriate absorption tests major antigenic differences among pili were found permitting 6 distinct serogroups to be recognised. Further, production of specific antiserums to representative strains of each serogroup in goats facilitated the development of a double antibody sandwich ELISA which could be used to quantitate pilus expression of a given strain of M. bovis, or to differentiate pilus serogroups of 22 strains of M. bovis obtained from a total of 12 Australian herds. Most isolates were found to belong to serogroups designated IV and V. One strain from the United States of America showed total homology with Australian serogroup IV while the other showed some cross-reactivity with serogroups V and VI.     

Serogrouping,phylotyping, and virulence genotyping of commensal and avian pathogenic Escherichia coli isolated from broilers in Hamedan,Iran

In the present study, 100 Avian-Pathogenic Escherichia coli (APEC) isolates from colibacillosis-suspected broilers and 100 Avian Faecal Escherichia coli (AFEC) isolates from healthy broilers in Iran were examined by PCR for confirmation of their serogroups and phylogenetic background, and their association with ten virulence-associated genes (VAG) including fimC, iutA, chuA, sitA, iss, cvaA/B, hylA, stx1, stx2, and yjaA. Serogroups O78, O1, O2 and O18 were the prominent strains including 54 % of the APEC and 23 % of the AFEC strains. At phylotyping, the majority of APEC strains belonged to phylogenetic group E (22 %) while for the AFEC strains, half of the isolates were not assigned to any group but the predominant phylogroup was E (27 %). Virulence genotyping, revealed that the predominant VAGs were iutA (97 %), fimC (87 %) and iss (84 %) among APEC strains, and fimC (95 %), iss (93 %) and sitA (87 %) in AFEC strains. This is the first time that phylogroup E is described as predominant phylogroup among APEC strains also, this is the first report on the presence of the stx1 gene in APEC strains isolated from broilers in Iran. The results of the present study indicate that VAGs are more prevalent in APEC strains belonging to O2 and O78 serogroups, also phylogroups E and D have more frequency of VAGs than other phylogroups. Therefore, the APEC strains belonging to O2 and O78 serogroups and phylogroups E and D probably have more pathogenicity to broilers.     

Prevalence of thermophilic campylobacters in crows (Corvus levaillantii, Corvus corone) and serogroups of the isolates

A total of 500 fecal droppings of crows collected from a seashore of an ocean bay and from a cemetery on a hill surrounded by a forest were examined for thermophilic campylobacters, and the Skirrow's biovars and Penner's serogroups of the isolates were determined. The organisms were isolated from 169 (62.6%) of 270 seashore crow samples and 106 (46.1%) of 230 cemetery crow samples. During the investigation period from May 1986 to April 1987, the monthly isolation rate of thermophilic campylobacters in the seashore crow varied from 32.0 to 85.0%. C. jejuni, C. coli, and C. laridis were isolated from 150, 21 and 14 samples, respectively. In case of the cemetery crow, the monthly isolation rate varied from 20.0 to 75.0%, and C. jejuni, C. coli, and C. laridis were detected from 80, 12 and 16 samples, respectively. Among 192 strains of C. jejuni selected from 98 seashore and 57 cemetery crow samples, 106 (93.0%) of 114 seashore crow strains and 69 (88.5%) of 78 cemetery crow strains were identified as Skirrow's biovar I. Of 192 strains of C. jejuni serogrouped, 169 strains were classified into 20 serogroups. The Penner's serogroup 2, one of common serogroups among poultry and human isolates in Japan, was the most predominant in crow strains.     

Serotypes,phage types and virulence genes of shiga-producing Escherichia coli isolated from sheep in Spain

PROBLEM ADDRESSED: Shiga toxin-producing Escherichia coli (STEC), have emerged as food poisoning pathogens which can cause severe diseases in humans. OBJECTIVE: The aim of this study was to determinate the serotypes and virulence genes of STEC strains isolated from sheep in Spain, with the purpose of determining whether sheep represent a potential source of STEC pathogenic for humans. METHODS AND APPROACH: Faecal swabs obtained from 697 healthy lambs on 35 flocks in Spain during the years 2000 and 2001 were examined for STEC using phenotypic (Vero cells) and genotypic (PCR) methods. RESULTS: STEC O157:H7 strains were isolated from seven (1%) animals in six flocks, whereas non-O157 STEC strains were isolated from 246 (35%) lambs in 33 flocks. A total of 253 ovine STEC strains were identified in this study. PCR showed that 110 (43%) strains carried stx(1) genes, 10 (4%) possessed stx(2) genes and 133 (53%) both stx(1) and stx(2). Enterohaemolysin (ehxA) and intimin (eae) virulence genes were detected in 120 (47%) and in 9 (4%) of the STEC strains. STEC strains belonged to 22 O serogroups and 44 O:H serotypes. However, 70% were of one of these six serogroups (O6, O91, O117, O128, O146, O166) and 71% belonged to only nine serotypes (O6:H10, O76:H19, O91:H-, O117:H-, O128:H-, O128:H2, O146:H21, O157:H7, O166:H28). A total of 10 new O:H serotypes not previously reported in STEC strains were found in this study. Seven strains of serotype O157:H7 possessed intimin type gamma1, and two strains of serotype O156:H- had the new intimin zeta. STEC O157:H7 strains were phage types 54 (four strains), 34 (two strains) and 14 (one strain). CONCLUSIONS: This study confirms that healthy sheep are a major reservoir of STEC pathogenic for humans. However, because the eae gene is present only in a very small proportion of ovine non-O157 STEC, most ovine strains may be less pathogenic.