Induction of early immunopotentiation to Fimbriae of Salmonella Enteritidis (SE) by administering thymulin and zinc to SE-vaccinated chicken breeders: relationship to protection

The purpose of this study is to attempt the induction of early immunopotentiation of antibodies specific to fimbriae of Salmonella enterica serovar Enteritidis (SE), by administering thymulin and zinc to SE-vaccinated chicken breeders, and the improvement of protection against a controlled-live challenge by SE. The first two groups of breeders were administered subcutaneously at 15 and 19 weeks of age a killed SE vaccine. Breeders of the third and fourth groups were left unvaccinated. Breeders of the first group, immunopotentiated by thymulin and zinc, were able to induce the earliest antibodies in their pooled sera at 2 weeks post the first SE-vaccination, specific to fimbriae (approximately 21 KDa) of SE. However, the second group that was only vaccinated with the same SE-vaccine produced specific antibodies to fimbriae at 3 weeks following the second vaccination (22 weeks of age). Breeders of the third group, that were neither SE-vaccinated nor immunopotentiated by thymulin and zinc, but were challenged by live SE at 22 weeks of age, were able to show specific antibodies to fimbriae at 3 weeks post challenge (25 weeks of age). The fourth group that was deprived of SE-vaccination, immunopotentiators, and challenge didn't show any background antibodies specific to SE-fimbriae. The presence of the earliest antibody-immunopotentiation to fimbriae of SE in breeders of the first group, administered thymulin and zinc, was associated with the lowest frequency of SE-infected ceca (10%) among the challenged groups. In addition, breeders of the first group were the only challenged birds resulting in absence of SE infection in their cecal tonsils. The first group-vaccinated, immunopotentiated, and challenged, and the second group-vaccinated and challenged only resulted in breeders with absence of SE infection in their oviducts and spleens. In conclusion, immunopotentiation of chicken breeders by thymulin and zinc induces the earliest specific antibodies to fimbriae of SE associated with the lowest frequency of SE-infected ceca, and absence of SE infection from cecal tonsils, oviducts and spleens.     

Comparison of Immunosuppression in Dry and Lactating Awassi Ewes due to Water Deprivation Stress

In seminomadic farming practice, dry and lactating ewes are exposed to different degrees of water deprivation, leading to stress followed by various disease outbreaks. This study compares quantitatively the immunosuppression to Salmonella Enteritidis (SE) fimbriae (14 and 21 kDa) and other major polypeptides (28.9, 37.7, 42.9, 68.0, 92.6 and 96.8 kDa) in water-deprived dry and lactating ewes. Sixteen dry and lactating multiparous Awassi ewes were divided into four treatment groups (A, A', B and B'). Ewes in groups A and B were lactating, whereas ewes in groups A' and B' were dry. All ewes were administered a killed SE vaccine, subcutaneously in the neck, at the initiation of the experiment. The water availability for ewes in groups B (lactating) and B' (dry) was ad libitum, while that for ewes in groups A (lactating) and A' (dry) was once every 4 days. A serum sample was collected from the jugular vein of each ewe at zero time (initiation of the experiment, when SE bacterin was delivered) and at 2, 9, 12, 15 and 18 days post SE vaccination. The percentage reduction in the level of humoral antibody response to polypeptides of > or = 21 kDa was more apparent in water-deprived lactating ewes of group A between 9 and 18 days post initiation of thirst. In this period, immunosuppression to polypeptides > or = 21 kDa was present in 14 out of 16 observations in group A (water-deprived lactating), with significant immunosuppression in 9 observations in relation to the respective control (p<0.05), while it was present in only 4 out of 16 observations in group A' (water-deprived dry), with significant immunosuppression in 2 observations (p <0.05). In conclusion, immunosuppression to polypeptides of > or =21 kDa is more significant in lactating water-deprived ewes in the period 9-18 days post initiation of thirst, a result that will influence our future sheep welfare awareness programmes targeting an elimination of the practice of water deprivation in seminomadic sheep farming.     

Identification of two highly performing Leishmania infantum antigens for serodiagnosis of canine leishmaniosis

In the aim of improving serodiagnosis of canine leishmaniosis, we analysed the humoral immune response of dog against Leishmania infantum parasite. The antigenic reaction of L. infantum polypeptides with sera from 31 dogs with parasitologically confirmed leishmaniosis was studied by using the immunoblot technique. Electrophoretic profile of the parasite extract showed more than 50 polypeptides, with molecular weights ranging from 12 to 170 kDa. Among these polypeptides, 37 antigen components, ranging from 14 to 91 kDa, were recognised by antibodies of L. infantum infected dogs. Three polypeptides (14, 16 and 76 kDa) reacted with all of the 31 serum samples. The other most frequently recognised antigens were those of 29.5, 32, 46, 59 and 66 kDa with a sensitivity of 87.1%, 93.6%, 96.8%, 87.1% and 80.6%, respectively. The 14 and 16 kDa bands were the most intense and remained detectable until a serum dilution of 1:6400. No reaction of these two major antigens was observed with sera collected from 50 Leishmania-free dogs, living in the leishmaniosis-free region of Rabat in Morocco, whereas the crude antigen used in IFAT or ELISA lead to three false positive results. Four antigen components of 29, 41, 55, and 70 kDa were recognised by some sera samples from negative controls. These results demonstrated the potential interest of the fractions of 14 and 16 kDa in immunodiagnosis of canine leishmaniosis.     

Immunodominant antigens of zoospores from ovine isolates of Dermatophilus congolensis.

Zoospores of Dermatophilus congolensis were analysed by SDS-PAGE and western blotting. The electrophoretic profiles of zoospores from 13 isolates of D. congolensis were similar but not identical when stained with Coomassie blue or silver. Immunodominant polypeptides with apparent molecular masses of 76 and 31 kDa were identified in western blots of 13 of 13 and 12 of 13 isolates respectively of D. congolensis reacted with hyperimmune, ovine, antizoospore sera. Identical immunodominant polypeptides were observed in western blots reacted with sera obtained from naturally infected sheep. Initial characterisation of the 76 and 31 kDa polypeptides indicated that they were probably surface exposed because (i) antibodies eluted from the surface of live zoospores after adsorption of hyperimmune antizoospore serum, reacted principally against the 76 and 31 kDa subunit polypeptides in western blots, (ii) adsorption of hyperimmune antizoospore serum with live zoospores resulted in significant diminution of reactivity against both the 76 and 31 kDa polypeptides in western blots, (iii) indirect fluorescent immunostaining of zoospores with antiserum prepared against gel-purified 76 kDa polypeptide, resulted in intense staining of the zoospore outer coat. Immuno-gold electron microscopy of negatively stained zoospores with antiserum prepared against gel-purified 31 kDa polypeptide identified this antigen as a flagella subunit.     

Partial Antigenic Characterization of Buffalopox Virus

The present study was undertaken to antigenically characterize the buffalopox virus (BPV). Six monoclonal antibodies (MAbs) against the BP4 strain of BPV have been produced and characterized. All six MAbs appeared to be specific to BPV, as none of them showed cross-reactivity with other poxviruses in antigen capture ELISA. Only two MAbs (20AB8 and 20CD11) bound significantly with different BPV isolates in antigen capture ELISA, whereas the remaining four MAbs bound weakly with the BPV. In Western blot analysis with purified BPV-BP4, the rabbit hyperimmune serum against purified BPV-BP4 reacted with 15 immunodominant polypeptides (100 kDa to 25 kDa), whereas two MAbs (21CB6, 21DB11) reacted with 42 kDa and 45 kDa polypeptides, respectively. However, three MAbs (20AB8, 20CD11, 21CB5) reacted with three degraded polypeptides (100 kDa, 40 kDa and 87 kDa) of BPV-BP4. In radioimmunoprecipitation assay (RIPA) with the rabbit hyperimmune serum to BPV-BP4, three virus specific polypeptides (69 kDa, 34 kDa, 32 kDa) were recognized in BPV-BP4, whereas two polypeptides (69 kDa, 34 kDa) were recognized in other BPV isolates (BPV-Bly, BPV-Vij96, BPV-Vij97). In virus neutralization test, none of the six MAbs tested showed any significant neutralizing ability to infection with different BPV isolates. However, the hyperimmune serum showed weak neutralizing ability to BPV infection.     

Prevalence and characteristics of necrotoxigenic Escherichia coli (NTEC) strains isolated from diarrhoeic dairy calves.

Fecal samples from 246, 1-90-days old diarrhoeic dairy calves in 72 herds were screened for the presence of cytotoxic necrotizing factors (CNF)-producing Escherichia coli (NTEC). NTEC were detected by tissue culture assays and PCR in 39 (15.8%) of the diarrheic calves, and the majority of these animals (34 of 39, ca. 87.2%) were infected by NTEC producing CNF2. Calves were grouped according to their age (1-7 days, 8-14 days, 15-21 days, 22-30 days and 31-90 days) and analyses of prevalence were done by the Mantel-Haenzsel chi2-test for trend. A significant age-associated increase in the prevalence of NTEC producing CNF2 (p<0.0001) was found. Eighty-one (8.4%) of the 958 E. coli isolates from the 246 diarrheic calves were positive for CNF in the tissue culture assays. These strains were analyzed by PCR and this technique showed that three (3.7%) strains were CNF1-positive and 75 (92.6%) were CNF2-positive. Moreover, three of the strains positive in the tissue culture assays were negative by PCR. These strains were subsequently assayed in several biological tests (rabbit skin test, mouse intraperitoneal test and mouse footpad test) which showed that they were really NTEC, probably producing CNF2, but with some different properties to classical strains producing CNF2. NTEC strains producing CNF2 belonged to different serogroups (O2, O7, O9, O14, O15, O41, O43, O45, O55, O76, O86, O88, O109, O115, O123, O128, O153 and O159) than strains producing CNF1 (O11 and O32) or PCR-negative strains (O111). Moreover, a strong association between CNF2 and F17 fimbriae was found (78.6% of CNF2-positive strains were F17-positive, whereas only 22.9% of CNF2-negative strains were F17-positive).     

Pathogenic characteristics of Escherichia coli strains isolated from newborn piglets with diarrhea in Brazil

Ninety-one Escherichia coli isolates obtained from diarrheic and normal feces of newborn piglets (0-11 days of age) from three states of Brazil were assessed for phenotypic and genotypic characteristics associated with pathogenic processes. These isolates expressed fimbriae F18ac and type 1, but not fimbriae K88, K99, 987P or F41. Genes for toxins (LT-I, STa, SLT-I, SLT-II, SLT-IIv) either individually or combined were found to be present in most of the diarrheic strains (65.7%) and in 42.8% of the non-diarrheic ones. The eaeA gene was present in 25.7% of the diarrheic isolates and in 9.5% of the non-diarrheic ones. Colicin, hemolysin and aerobactin were also found to be produced by some strains from both sources. Because of the great variety of biological characteristics associated with different illness processes, we suggest that, in Brazil, pigs may act as a reservoir for transmission of Escherichia coli strains to other animals.     

Identification and characterization of corpuscular, soluble and secreted antigens of a Venezuelan isolate of Anaplasma marginale

Anaplasma marginale is the etiological agent of anaplasmosis, a tick-transmitted disease with an important economic impact that affects cattle throughout the world. Although, North American isolates of A. marginale and their antigens have been extensively studied, relatively little information is available on the antigenic composition of South American isolates. The characterization of diverse geographical isolates of A. marginale will result in a thorough antigenic profile and may lead to the identification of additional diagnostic and immunoprophylactic tools. Short-term cultures of a Venezuelan isolate (Ta) of A. marginale were maintained for up to 13 days in vitro. During that period, the A. marginale remained viable and were propagated in the bovine erythrocyte culture system. During the initial days of culture, cell division and reinvasion were evidenced by a significant rise in parasitemia up to a 50%. A. marginale antigens were identified by metabolic labeling with (35S) methionine, followed by fractionation and immunoprecipitation with homologous and heterologous bovine sera. This yielded a complete antigenic set for the Ta isolate of A. marginale, including soluble, secreted and corpuscular polypeptide antigens. Fifteen immunodominant polypeptides were recognized by the bovine sera in the soluble and corpuscular fractions with relative molecular weights of 200, 150, 100-110, 86, 60, 50, 47, 40, 37, 33, 31, 25, 23, 19 and 16kDa. Seven polypeptides were present in the exoantigen fraction. The 31 and 19kDa antigens were recognized by the ANAR76A1 and ANAF16C1 monoclonal antibodies, respectively which are specific for MSP-4 and MSP-5 from North American isolates of A. marginale. Metabolic labeling with (14C) glucosamine prior to immunoprecipitation with bovine sera allowed the identification of glycoprotein antigens of 200, 100-150, 60, 55, 50, 45-43, 37, 33, 31, 22, 19 and 16kDa in the soluble fraction.     

Neutralizing and Complement-fixing Monoclonal Antibodies as an Aid to the Diagnosis of Equine Herpesvirus-1 Infection

One complement-fixing (C-MAb) and three complement-dependent neutralizing monoclonal antibodies (N-MAbs) were raised against Hisar-90-7 equine herpesvirus-1 (EHV-1) strain. The target antigen of the C-MAb (2A5) and two of the N-MAbs (1H6, 9C4) was identified as a 140 kDa polypeptide in Western blotting. The target antigen of N-MAb (9C6) could not be identified. Purified polypeptides of five EHV-1 strains isolated from different regions and at different times gave intense bands at 140 kDa when reacted with N-MAb (1H6) in Western blots. In sandwich ELISA, all four MAbs captured the viral antigen from clinical materials, giving a reliable and rapid diagnosis of EHV-1 infection in equines.     

Pathogenicity of Rhodococcus equi expressing a virulence-associated 20 kDa protein (VapB) in foals

Rhodococcus equi strains of intermediate virulence (IMV) for mice possess a 20kDa protein designated Virulence Associated Protein B (VapB) and a virulence plasmid of 79-100kb, and can be recovered from the submaxillary lymph nodes of pigs. The pathogenicity of such R. equi strains for foals is unknown. In this study, two foals, 42 and 43 days of age, were infected intratracheally with 10(6) and 10(9) cells of R. equi IMV strain A5, respectively. The foal infected with 10(9) cells of strain A5 became clinically ill, with the onset of illness (pyrexia and depression) occurring 21 days after inoculation. R. equi was isolated from the feces and tracheal washings of the foal from 14 to 28 days after inoculation. The foal infected with 10(6) cells of A5 showed no clinical signs, and no R. equi was isolated from any of the samples of feces or tracheal washings during the 28 days of observation. Two foals of 45 and 50 days of age were infected with 10(5) or 10(6) of virulent R. equi ATCC 33701 having 15-17kDa surface proteins designated VapA. Both exhibited severe clinical signs (pyrexia, depression and anorexia) at 12 and 13 days after inoculation. Histopathological examination revealed that strain A5 caused focal granulomatous pneumonia in the foals. R. equi IMV strain A5 was isolated from lung lesions of both foals and from the contents of the intestinal tracts of the foal infected with 10(9) bacteria. These results suggest that IMV R. equi having VapB is less virulent than virulent R. equi having VapA in foals. This finding supports our previous results on the pathogenicities of R. equi strains having these virulence-associated antigens assessed by mouse pathogenicity tests.     

Identification of the antigenic components of the virulent Mycoplasma gallisepticum (R) in chickens: their role in differentiation from the vaccine strain (F)

The antibody response to different proteins of Mycoplasma gallisepticum (MG) was studied in chickens experimentally infected with virulent MG R strain. The chickens were challenged at 8 weeks of age by the intranasal route. Each cockerel received 1.3 X 10(6) colony-forming units (CFU). MG strains (R and F) were banded by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The banding pattern was distinctively different between the two strains in the range of 92.5 to 200 kilodaltons (kD). Chicken sera collected at different times following challenge were analyzed by Western blot to determine the patterns of antibodies raised to specific MG proteins (R versus F strains). Early in infection (2 weeks postchallenge), antibodies to 60-kD and 75-kD polypeptides of MG R strain were produced. Subsequently (greater than or equal to 4 weeks postchallenge), antibodies recognized a larger number of MG antigens in both strains. The immunoblot patterns remained the same in the period 8-11 weeks postinfection in each of the two strains; however, the patterns were different when the two strains were compared. The early response recognized the 75-kD protein in the R strain while it recognized the 80-kD protein in the F strain. The late response recognized the 130-kD protein and the protein slightly heavier than 200 kD in the R strain. These two bands did not appear in the immunoblot performed against the F strain of MG. Electroeluted protein of MG R strain, namely adhesin (75 kD), showed a hemagglutination activity (HA) on chicken red blood cells. With the appearance of antibodies specific to the 60-kD and 75-kD polypeptides, there was a significant rise in hemagglutination-inhibition geometric mean titer of chicken sera.     

Distribution of virulence genes in Escherichia coli strains isolated from diarrhoeic piglets in the Slovak Republic

Ninety-two Escherichia coli isolates from 14 to 28-day-old piglets that died because of diarrhoea were examined for genes for fimbriae (F4, F5, F6, F18 and F41), enterotoxins (STa, STb and LT), verotoxin (VT2e or Stx2e) and enteroaggregative heat-stable enterotoxin 1 (EAST1) by polymerase chain reaction. Twenty-two strains (24%) carried a gene for F4, whereas genes for F18, F6 and F5 + F41 were detected in 10.8, 3.3 and 1.1% of strains respectively. Genes for STb, LT, STa and Stx2e were detected in 40.2, 26.1, 14.1 and 1.1% of strains respectively. The astA gene was detected in 49 (53.3%) isolates, 35 of which also carried genes for enterotoxins and/or fimbriae. The major genotypes reached at (in decreasing order of prevalence) were F4/STb/LT/EAST1, F18/STa/STb/EAST1, STb/EAST1, F6/STa/STb/EAST1 and F18/STb/EAST1.     

鸡致病性大肠杆菌菌毛分型的初步研究

以鸡致病性大肠杆菌F1菌毛特异性单抗1F4－1、2C3、3H3和F11菌毛特异性单抗FA1、FB11作为检测试剂，将111株已知O抗原的鸡致病性大肠杆菌经MD液体培养基连续传代培养后，通过直接玻板凝集法对各菌毛进行初步分型。结果发现F1、F11菌毛与O78、O1及O2三种优势O抗原型菌株之间存在较为明显的相关性，即致病性大肠杆菌主要集中在上。F1、F11菌毛在这三种O抗原型上的总检出率分别为95．6％、75．4％及73．3％。另外，在所检测的111株鸡致病性大肠杆菌中，只表达F1菌毛的大肠杆菌占菌杆总数的33．3％。只表达F11菌毛的大肠杆菌占菌株总数的8．1％，两者都表达的占菌株总数的36％，F1、F11菌毛的总检出率为78．3％。     

The F4 fimbrial antigen of Escherichia coli and its receptors

F4 or K88 fimbriae are long filamentous polymeric surface proteins of enterotoxigenic Escherichia coli (ETEC), consisting of so-called major (FaeG) and minor (FaeF, FaeH, FaeC, and probably FaeI) subunits. Several serotypes of F4 have been described, namely F4ab, F4ac, and F4ad. The F4 fimbriae allow the microorganisms to adhere to F4-specific receptors present on brush borders of villous enterocytes and consequently to colonize the small intestine. Such ETEC infections are responsible for diarrhea and mortality in neonatal and recently weaned pigs. In this review emphasis is put on the morphology, genetic configuration, and biosynthesis of F4 fimbriae. Furthermore, the localization of the different a, b, c, and d epitopes, and the localization of the receptor binding site on the FaeG major subunit of F4 get ample attention. Subsequently, the F4-specific receptors are discussed. When the three variants of F4 (F4ab, F4ac, and F4ad) are considered, six porcine phenotypes can be distinguished with regard to the brush border adhesiveness: phenotype A binds all three variants, phenotype B binds F4ab and F4ac, phenotype C binds F4ab and F4ad, phenotype D binds F4ad, phenotype E binds none of the variants, and phenotype F binds F4ab. The following receptor model is described: receptor bcd is found in phenotype A pigs, receptor bc is found in phenotype A and B pigs, receptor d is found in phenotype C and D pigs, and receptor b is found in phenotype F pigs. Furthermore, the characterization of the different receptors is described in which the bcd receptor is proposed as collection of glycoproteins with molecular masses ranging from 45 to 70 kDa, the bc receptor as two glycoproteins with molecular masses of 210 an 240 kDa, respectively, the b receptor as a glycoprotein of 74 kDa, and the d receptor as a glycosphingolipid with unknown molecular mass. Finally, the importance of F4 fimbriae and their receptors in the study of mucosal immunity in pigs is discussed.     

Characterization of fowl adenovirus serotype-4 associated with hydropericardium syndrome in chicken

The polypeptides of three fowl adenovirus-4 (FAV-4) field isolates of hydropericardium syndrome from various geographical areas of the country and the standard FAV-1 (CELO virus) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and analysed by protein immunoblotting with polyclonal antibodies to FAV-4 and FAV-1. Protein profile analysis of FAV-4 isolates revealed similarity of all the eight polypeptides with molecular weight ranging from 20 to 107 kDa but differed from CELO, particularly in their 24.2 kDa protein. Subsequent immunoblotting showed relatedness of at least five protein fractions of FAV-4 to CELO virus.     

Identification of immunoreactive polypeptides of Babesia equi parasite during immunization

This study was carried out to identify immunoreactive polypeptides in Babesia equi merozoite antigen. Three fractions of killed B. equi merozoite antigen viz.; whole merozoite (WM), cell membrane (CM) and high speed supernatant (HSS) antigens were prepared from the parasite infected erythrocytes. These antigenic preparations along with ghost antigen from non-infected erythrocytes were fractionated on 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with sera showing high antibody titres. On SDS-PAGE, 16 polypeptides with molecular weight (Mr) in the range of 112-17kDa were obtained from the WM and CM antigens. But only six polypeptides were detected (96.5-28kDa) in the HSS antigen. On immunoblotting with high titred serum collected from donkeys following two immunizations with a killed B. equi merozoite immunogen, 11 polypeptides were observed in the WM and CM antigens (Mr 112-18kDa). Of these, four polypeptides (Mr 112, 45, 33 and 18kDa) were identified as most immunoreactive. Besides these, a 28kDa was observed as strong immunoreactive protein in WM and CM antigens. The HSS antigen showed only six polypeptides and one peptide (28kDa) was identified as immunoreactive. When high titred serum collected from immunized donkeys following challenge with B. equi infected blood and was used for immunoblotting, the protein profile of WM and CM antigens remained the same. However, three additional polypeptides (Mr 81, 54.5 and 39kDa) were detected in HSS antigen.     

Serum antibody responses of cats to soluble whole cell antigens and isolated fimbriae of feline Porphyromonas salivosa (macacae) and associations with periodontal disease

The whole cell soluble antigens of two strains (NCTC 11632 and VPB 3313) of feline Porphyromonas salivosa (macacae) were analyzed by Western blotting using serum taken from 40 domestic cats with various grades of periodontal disease. Nine strongly immunogenic protein bands (66, 52, 42, 29, 27, 23, 22, 21 and 19kDa) were selected from both strains for further study. Both strains showed a significant association between overall periodontal grade and serum responses to the 66 and 21kDa bands with significant responses across both strains to all other bands except the 52kDa band. Similarly, both strains showed a significant association between the total colony forming units and serum responses to the 66 and 42kDa bands with significant responses across both strains to all other bands except the 19kDa band. When sera from 25 of these cats were tested by Western blotting against the isolated fimbriae of VPB 3313, there was a significant association between the grade of response of cats to the 42kDa fimbrial preparation and (1) the total reactivity of the mouth (the sum of the responses to all individual whole cell antigens), (2) the total colony forming units of P. salivosa (macacae) at the premolar site, and (3) to their responsiveness to the 42kDa band in the soluble whole cell antigen preparations. These findings suggest that P. salivosa (macacae) is a strong immunogen in the mouths of cats and those cats with more severe periodontal disease have a greater serum antibody reactivity to various soluble whole cell antigens, specifically including the fimbriae of this organism, than those with less severe periodontal disease. Overall, the findings suggest that this organism may be a contributor to periodontal disease in cats.     

Active oral immunization of suckling piglets to prevent colonization after weaning by enterotoxigenic Escherichia coli with fimbriae F18

Immunoprophylaxis of porcine oedema disease and post-weaning diarrhoea caused by strains of Escherichia coli expressing fimbriae F18 is an unsolved problem. The study was designed to examine whether vaccination with a live F18ac vaccine of unweaned pigs born to sows with F18ac antibody in the colostrum requires preformed fimbriae in the vaccine, and whether protection against the heterologous fimbrial variant F18ab is induced as well. Genetically susceptible pigs were vaccinated orally on three consecutive days, beginning 10 days before weaning with 10(11) CFU of an F18ac culture. Challenge with a dose of 10(7) CFU of E. coli F18 on three consecutive days was initiated 9 or 11 days after weaning. Eighteen pigs given the fimbriated F18ac vaccine and challenged with a strain of the homologous fimbrial variant were protected against colonization; mean faecal viable counts of the challenge strain were >3 log10 lower than those from the 17 non-vaccinated control pigs. The vaccinated pigs developed a significant rise of F18ac IgA serum antibodies. The 23 pigs which had received the non-fimbriated vaccine showed no significant protection and exhibited much lower serum F18ac IgA ELISA reactivities. Eighteen pigs vaccinated with the fimbriated F18ac and challenged with an F18ab strain had faecal viable counts nearly as high as those from 16 non-vaccinated control pigs. It is concluded that only oral vaccines having preformed fimbriae induce protection limited to the homologous fimbrial variant.     

Serum antibodies against particular antigens of Borrelia burgdorferi sensu stricto and their potential in the diagnosis of canine Lyme borreliosis

Dog sera (n = 118) were tested for antibodies recognizing Borrelia (B.) burgdorferi sensu stricto strain B31 (ATCC 35210) antigens. In total, 18 of the dog sera gave positive results in a whole cell sonicate ELISA (WCS ELISA). These positive sera were further evaluated by immunoblot assay, utilizing a whole bacterial lysate as antigens. 94.4% (17 of 18) of the dog sera reacted with immunodominant antigens at 20-22 kDa (protein C, pC), 31 kDa (outer surface protein A, OspA), 34 kDa (outer surface protein B, OspB), 41 kDa (flagellin), 60 kDa ("common antigen"), and/or 100 kDa (presumably p100). Sera recognizing pC (20-22 kDa) and antigens > 94 kDa always detected the highest number of antigen bands, indicating the specificity of those antigens in serological diagnosis. The results clearly demonstrate that the WCS ELISA is a useful tool for testing sera of dogs for antibodies against B. burgdorferi. However, positive results should be confirmed by immunoblot, using WCS as antigen. According to the presented data, we recommend criteria for B. burgdorferi immunoblots using dog sera as follows: sera have to be considered as positive if they detect the 41 kDa flagellin, and two of the 5 immunodominant antigens, namely > 94 kDa (presumably p100), 60 kDa ("common antigen"), 34 kDa and 29-31 kDa (OspB and OspA, respectively) and 20-22 kDa (pC). If sera only recognize the 41 kDa flagellin, this result is equivocal, requiring testing a second serum sample 4 to 8 weeks later.     

Humoral immune response in hens naturally infected with Salmonella Enteritidis against outer membrane proteins and other surface structural antigens

A simple procedure for obtaining surface exposed antigens of Salmonella Enteritidis is described. A heat treatment of whole bacteria in saline solution induced the release of small membrane vesicles containing outer membrane components as well as surface appendage components, such as fimbriae and flagellin. The characterization of the structural components of this extract, called HE, was established by SDS-PAGE and immunoblotting using polyclonal and monoclonal specific antibodies. Five major groups of proteins were identified: flagellin, porins, OmpA, SEF21 and SEF14 fimbriae. The immunogenicity of these proteins was studied by immunoblotting with serum samples from naturally infected hens. Flagellin, porins, OmpA, SEF14 and SEF21 fimbriae were immunogenic in the S. Enteritidis infected hens (frequency of reactants: 47.3, 97.3, 64.7, 50.0 and 60.8%, respectively); porins also reacted with sera from non infected hens (66.7%). The immunogenicity of these antigens in infected birds provide promise that they may serve as components of an effective subcellular vaccine for poultry salmonellosis.