Immune response and antigen recognition in non-pregnant ewes experimentally infected with Neospora caninum tachyzoites

The cellular and humoral responses as well as the antigen recognition during the acute stage of a Neospora caninum (NC) infection were investigated in non-pregnant ewes. The experimentally infected ewes developed specific lymphoproliferative and humoral responses within 2 weeks post-infection (PI). The magnitude of the cellular response showed large variations between animals. A significant decrease in the proliferative response to Con A mitogen and N. caninum, Toxoplasma gondii (TG) antigens was recorded on day 21 post-infection (PI). The humoral response and the pattern of antigen recognition were similar among infected ewes. Proteins of 44, 42, 40, 39 and 28 kDa were intensively recognized by the infected animals during the experiment. The 42 and 28 kDa antigens should be considered as useful for the diagnostic of N. caninum infection, as the intensity of recognition infection of the other antigens had decreased markedly 8 weeks post-infection. For some antigens a sequential recognition was recorded. The 59, 54 and 38-37 kDa proteins were frequently recognized by infected sera during the first weeks of the infection, but recognition of these antigens was absent or rare at the end of the experiment. These antigens could be related to the acute stage of the infection.     

家蚕微孢子原虫(Nosema bombycis)在Antheraea eucalypti细胞中的增殖

家蚕微孢子原虫（ Ｎｏｓｅ ｍ ａ ｂｏ ｍ ｂｙｃｉｓ） 经 Ｋ Ｏ Ｈ 处理后,接种于 Ａｎｔｈｅｒａｅａ ｅｕｃａｌｙｐｔｉ 细胞系,调查其生活史。 Ｋ Ｏ Ｈ 处理的孢子发芽率约为４４３ ％ 、 Ａｎｔｈｅｒａｅａ ｅｕｃａｌｙｐｔｉ 细胞的初期感染率约为３０ ％ ,接种３６ｈ 后,裂殖子数量急速增加,７２ｈ 达到最高峰。接种４８ｈ 后可观察到短极丝孢子发芽后形成的空孢壳和二次感染体。感染 Ｎｏｓｅｍ ａ ｂｏ ｍｂｙｃｉｓ ４８ｈ 前的家蚕血淋巴对 Ｂｍ Ｎ 细胞无感染性,而感染７２ｈ 的血淋巴则表现出感染能力。     

Invasion of avian encephalomyelitis virus from the gastrointestinal tract to the central nervous system in young chickens

Invasion sites of avian encephalomyelitis virus in the internal organs of orally infected chicks were determined by the immunofluorescent method. The invasion began when the epithelium tunica mucosae of the duodenum (together with the proventriculus, jejunum, or cecum in certain birds killed at postinoculation day 1) became test-positive. Viremia persisted for more than 5 days in the early stage of infection, then the pancreas was rapidly infected, followed by the liver, kidney, and spleen. Subsequently, the virus spread to the CNS. Rapid infection of the duodenum and pancreas was clearly observable.     

A 38-kDa protein from Babesia gibsoni and its antibody response in an experimentally infected dog

A cDNA encoding the Babesia bovis 12D3 antigen homologue was obtained by immunoscreening the expression library prepared from Babesia gibsoni merozoite mRNA. The complete nucleotide sequence of the gene was 1406 bp. Computer analysis suggested that the sequence contains an open reading frame of 1052 bp encoding an expected protein with a molecular weight of 36kDa. Based on homology analysis, this putative protein was designated as the B. gibsoni 12D3 antigen (Bg12D3). The Bg12D3 gene was expressed in the Escherichia coli BL21 strain, and the chronically infected dog serum reacted with the recombinant protein. The antiserum against the recombinant Bg12D3 protein can recognize a 38-kDa native protein, which is consistent with its expected size. Moreover, the purified recombinant proteins were used as the antigen to detect the antibody response in an experimentally infected dog by the enzyme-linked immunosorbent assay (ELISA). Our results indicated that the Bg12D3 protein was recognized by the host immune system and that it induced an antibody response in chronic B. gibsoni infection. These results allowed us to identify a new member of the 12D3 antigens and its characteristic immune response in canine B. gibsoni infection.     

28 kDa Fasciola gigantica cysteine proteinase in the diagnosis of prepatent ovine fasciolosis

Coprological confirmation of ovine fasciolosis in the field, prior to out breaks of the disease and/or strategic antifluke medication, seem to be of little consequence. Efforts are, therefore, being made to evolve a putative antigen specific to serodiagnostic test for early diagnosis during prepatency. In the present investigation, 28 kDa cysteine proteinase was used in ELI SA and Western blot to detect Fasciola gigantica antibodies and further Dipstick-ELISA was developed for field application, using known positive monospecific sera from experimentally infected sheep with 100 F. gigantica metacercariae. Isolation of 28 kDa cysteine proteinase was achieved from bubalian origin flukes. The specific antigen, recognised homologous antifluke antibodies by Western blot as early as 2nd week post-infection (wpi) with 100% sensitivity, in sera samples of sheep harbouring 38 flukes and by 10th wpi in sheep harbouring 3-8 flukes. All sheep were found positive for the infection when ELISA and/or Dipstick-ELISA was applied from 4th wpi. In pooled sera of infected sheep, these were positive during 4th wpi.     

Pathogenesis of Marek's disease virus-induced local lesions. 1. Lesion characterization and cell line establishment

Subcutaneous (wing-web) or intramuscular inoculation of chickens with allogeneic normal or Marek's disease virus (MDV)-infected chicken kidney cells induced local lesions visible by 3-4 days postinoculation (PI). Lesions were slightly larger (P less than 0.05) in infected than uninfected chickens 5 and 8 days PI. They persisted and grew past 9 days PI only when infected. Infiltrating lymphocytes in infected and uninfected early lesions were similar; they included B-cells and also T-cells with and without Ia antigen. Up to 42% of lymphocytes from infected or uninfected lesions had the surface antigen MATSA. At 3 to 6 days PI, infected lesions contained lymphocytes with viral internal antigen, especially in Ia-bearing cells and MATSA-bearing cells, but thereafter infection was latent. Cells harvested daily from local lesions induced with allogeneic MDV-infected cells were cultured; MD tumor cell lines were established from lesions as early as 4 days PI, with a total success rate of about 50% thereafter. Either transformed tumor cells were already present during the early cytolytic infection period or else appropriate target cells were present that became infected in vivo and/or in vitro and then became transformed in vitro.     

Serum antibody response and Cryptosporidium parvum oocyst antigens recognized by sera from naturally infected sheep.

The response of specific serum immunoglobulins (IgG, IgM and IgA) and the major antigens of Cryptosporidium parvum recognized by these isotypes were investigated by using enzyme-linked immunosorbent assay and immunoblot techniques in lambs and ewes naturally infected throughout an outbreak of cryptosporidiosis. Serum samples were collected from 20 lambs the first day they showed diarrhoea (D1), and Days 11 and 22, in addition to single serum samples from 17 of their dams. Serum anti-C. parvum IgG, IgM and/or IgA antibodies were detected in lambs as early as Day 1. Levels of IgM antibodies remained steady from D1 to D11 and increased at D22, whereas the IgG response decreased from D1 to D11 and subsequently increased. In contrast, IgA antibodies rapidly fell from D1 and all lambs were seronegative at D11 and D22. The highest levels of specific antibodies were detected in sera from ewes. In fact, all ewes were seropositives for IgM and IgA isotypes and most (16/17) showed positive levels of IgG. Four protein fractions (37-39, 42-48, 51-57 and 60-69 kDa) were the most frequently recognized by IgG and IgM from lamb sera. A low molecular weight fraction (12-14 kDa) reacting with IgG and IgA in most lamb sera was scarcely recognized by IgM and three broad bands were frequently recognized by IgA antibodies (23-25, 51-57 and 90-95 kDa). The recognition pattern of 23-25 kDa peptides by IgA from lamb sera clearly increased with the age. Peptides of 42-48, 51-57, 60-69 and 71-78 kDa were most frequently recognized by IgG and IgM from ewe sera. In relation to IgA antibodies from ewe sera, a frequent immunoreactivity was found with proteins in the intervals between 12 and 22 kDa as well as between 32 and 34 kDa and practically all sera reacted with fractions from 42 to 95 kDa.     

Antibody response in sheep experimentally infected with different small ruminant lentivirus genotypes

Two groups of sheep were experimentally infected by intratracheal route with two small ruminant lentivirus (SRLV) isolates belonging to different genotypes (It-561 genotype A3 and It-Pi1 genotype B2). Seroconversion was evaluated using recombinant homologous and heterologous matrix protein/capsid antigen fusion protein. Results clearly indicate that seroconversion against homologous antigen was detected well in advance as regards heterologous antigen in both groups, although the advantage of using homologous antigen was less evident in detecting seroconversion against the caprine arthritis encephalitis virus (CAEV)-like strain, compared with the maedi-visna virus (MVV)-like infection. Commercially available ELISAs detect CAEV-like seroconversion earlier than MVV-like infection suggesting a closer relationship between CAEV-like isolate and the antigen used in the latter ELISA tests. Seven recombinant subunits developed from matrix protein and capsid antigen of strain K1514 (prototype A1) were used to better define the antibody response in sheep infected with It-561 isolate. Two animals clearly reacted against type specific epitopes in the early stage of infection. This study highlights the relative insensitivity of gag encoded cross-reacting epitopes during the early stage of infection and suggests the development of novel diagnostic tests based on both genotype specific antigens.     

Detection of circulating 54 kDa antigen in sera of bovine calves experimentally infected with F. gigantica

The antibody response and circulating antigen levels in bovine calves, infected experimentally with Fasciola gigantica, were monitored using enzyme-linked immunoelectrotransfer blot (EITB) and sandwich ELISA, respectively. By EITB, the infected calves' sera recognized the polypeptides in the range of 54-58 kDa as early as 2 weeks post-infection. By 12th week post-infection, the lower two polypeptides of 12 and 8 kDa had disappeared. In sandwich ELISA, the circulating 54 kDa and whole worm antigen of F. gigantica were detected in the sera samples of infected calves as early as 2 weeks post-infection and persisted until the end of experiment (26th week PI). The 54 kDa antigen of F. gigantica appears to be specific and possesses promising immunodiagnostic potential for early prepatent diagnosis of bovine fasciolosis.     

Borna disease virus-specific antigens. II. The soluble antigen is a protein complex.

Borna disease virus-specific soluble antigen from persistently infected rat brains was purified to homogeneity using preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The soluble antigen is a complex of three proteins with apparent molecular weights of 35 kDa, 38 kDa and 24 kDa. The 35/38 kDa antigen double band was separated into its two components. The 24 kDa protein has no common epitopes with the 35/38 kDa protein.     

Polypeptides associated with Pasteurella multocida infection in rabbits.

Polypeptides from whole cell preparations of Pasteurella multocida serotypes A:12 and A:3 were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose paper. Antigens were detected by immunoblot analysis, using sera from 3 groups of rabbits. Sera were obtained from rabbits inoculated intranasally with P multocida serotype A:12 or A:3, from rabbits maintained in a rabbitry with enzootic P multocida A:12 infection, and from rabbits maintained in a rabbitry with enzootic P multocida A:3 infection. Immunoblot analyses of pre- and postinoculation sera from experimentally infected rabbits, using serotype A:12 antigen, revealed 3 polypeptides with approximate molecular mass of 28, 30, and 37 kDa that consistently detected antibodies after P multocida-induced infection. Sera from rabbits naturally infected with either serotype, tested against serotype A:12 and A:3 antigens, detected the same polypeptides in both serotypes. Thus, immunologic reactivity to these polypeptides may be useful for serologic detection of P multocida infection.     

Isolation and purification of a protective protein antigen of Erysipelothrix rhusiopathiae

The rapid growth and high survival rate of Erysipelothrix rhusiopathiae was determined using a culture of the bacterium in tryptic soy broth supplemented with 0.3% Tris-hydroxymethyl aminomethane and 0.1% Tween 80 (TT-TS broth). High concentrations of 64, 66 and 43 kDa proteins, which are associated with protection against E. rhusiopathiae infection in mice, were obtained by alkaline treatment of whole cells using 0.05-1 N NaOH. The supernatant of alkaline treated cells (alkaline extract; AE) was stable at alkaline or neutral pH. However, aggregates appeared at neutral pH in the absence of sodium dodecyl sulphate (SDS). A high yield of 64, 66 and 43 kDa proteins was obtained from strain Agata (serovar 5). The proteins were eluted from gel bands following SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the AE from strain Agata and designated P64 and P43. The amounts of P64 and P43 isolated were 0.7 and 0.3 mg/16 g of wet bacteria, respectively. In a mouse protection test, 50% protective doses (PD50) of P64 and P43 were 0.58 and 0.63 microgram, respectively. Upon Western blotting of the AE, both anti-P64 and anti-P43 antibodies reacted with the 64 and 43 kDa proteins. From these results, it is suggested that P64 is the most effective protective antigen and that P43 (43 kDa protein) is a degradation product of P64. Therefore, the 64 kDa structural proteins are associated with the induction of a protective activity against E. rhusiopathiae infection in mice.     

Oocysts, IgG levels and immunoblot patterns determined for Cryptosporidium parvum in bovine examined during a visit to a farm (northeastern Spain)

Single fecal and serum samples were individually collected from 101 bovines selected at random during a visit to a farm in northeastern Spain (Group I, 26 animals aged 2-36 days; Group II, 34 animals aged 1.5-4.5 months; Group III, 41 animals aged 20-24 months). Testing for the presence of Cryptosporidium parvum oocysts in feces (Monofluo Kit Cryptosporidium, Diagnostics Pasteur, France) indicated that 26% animals were infected (81% of Group I, 15% of Group II and 0% of Group III). Serological testing (ELISA for detection of specific anti-C. parvum IgG) indicated that 59% animals were seropositive (12% of Group I, 74% of Group II and 78% of Group III). Immunoblotting results indicate that cattle sera recognize C. parvum antigens of widely varying molecular weights and that the number of antigens recognized increases with age. Immunoblots revealed that some of the sera belonging to the Group I reacted with protein fractions between 15 and 20 kDa but none recognized the 21-23 kDa antigen. Only few sera in the Group II recognized the protein fraction between 15 and 20 kDa. The recognition of 21-23 kDa fraction was observed by four sera from uninfected and seropositive animals. Sera from all the seronegative Group II animals recognized few antigens and always with molecular weight greater than 50 kDa. Serum samples from both seropositive and seronegative animals belonging to the Group III recognized antigens with molecular weight ranging 15-20 kDa. Surprisingly, the protein fractions between 21 and 28 kDa reacted with approximately 30% of the sera from seropositive animals and only one of the nine sera from seronegative animals. The recognition of 42-46 kDa antigens increased with the age and only reacted with the sera from uninfected animals.     

Evaluation of cercarial antigen for the serodiagnosis of fasciolosis in experimentally and naturally infected sheep

The value of cercarial antigen for diagnosis of experimental and natural sheep fasciolosis was studied by enzyme linked immunosorbent assay (ELISA) and enzyme linked immunotransfer blot (EITB). In ELISA, the antibody levels of experimentally infected sheep with Fasciola gigantica appeared at 2 weeks post infection (PI), gradually increased till 7 weeks PI and nearly remained at the same level from 7 to 13 weeks PI (the end of experiment). Also, the sensitivity and specificity of cercarial antigen for diagnosis of naturally sheep fasciolosis were 100 and 90%, respectively.In EITB, in the sheep experimentally infected with F. gigantica, the band of 32.5kDa molecular weight polypeptide appeared at 2 weeks PI and continued till the end of experiment. Also, the cercarial antigen recognized 32.5kDa molecular weight band with all sera from naturally infected sheep with fasciolosis (n = 25). This band did not cross-react when tested with sera from infected sheep with Cysticercus tenuicollus (n = 20). This study suggests that, the 32.5kDa molecular weight polypeptide could be used as sensitive and specific epitope for the serodiagnosis of sheep fasciolosis.     

Serodiagnosis of haemonchosis with a somatic antigen (Hc26) in several breeds of sheep.

Sera from 53 sheep belonging to Castellano, Churro, Manchego, and Merino breeds were analyzed to test the diagnostic value of a 26-kD antigen from adult Haemonchus contortus at prepatency and early and late patency of experimental haemonchosis. Animals that received zero, 1, or 2 infections with the parasite were tested. In addition, sera from 20 experimentally infected and 10 noninfected Texel sheep were used to test the antigen. Sera from 37 infected animals at prepatency as well as at patency in primary and secondary infection were found positive with the 26-kD antigen. However, sera from 10 animals with the lowest worm burdens (second infection) did not recognize the antigen during early patency (day 28 postinfection). IgG1 was the only isotype implicated in antigen recognition because IgG2, IgA, and IgM, in the same sera, showed no reactivity with the peptide. Antigen specificity was confirmed because hyperimmune sera against infective larvae and adult stages of the most common gastrointestinal nematodes found in natural infections in sheep (Trichostrongylus colubriformis and Teladorsagia circumcincta) did not recognize this peptide. The antigen was recognized only by anti-adult H. contortus hyperimmune sera and appeared to be absent in the L3 parasite stage. In addition, the partial N-terminal amino acid sequence of the diagnostic peptide is reported.     

Molecular characterization of Theileria orientalis piroplasm protein encoded by an open reading frame (To ORF2) in a genomic fragment

In the present study, a novel antigenic protein expressed in the piroplasm stage of Theileria orientalis was characterized. A 4,707 bp genomic fragment amplified by PCR contained two open reading frames (ORFs). The deduced amino acid sequence of the first ORF showed significantly high similarlity to the ubiquitin carboxy terminal hydrolases/proteases while the second ORF (To ORF2) showed homology to several surface antigens of plasmodia. To ORF2 was expressed to determine whether the protein product is expressed by the parasite. In western blot analysis, bovine antiserum from a T. orientalis-infected calf recognized the recombinant protein containing a C-terminal part of the ORF expressed by baculovirus system. Western blot analysis with the anti-To ORF2 mouse serum recognized a 48 kDa protein in T. orientalis piroplasm lysates. Indirect immunofluorescence antibody test by confocal scanning laser microscopic analysis showed that antisera against the recombinant protein recognized T. orientalis piroplasm in the infected erythrocyte. The results from this study indicate that To ORF2 protein is expressed at the piroplasm stage and is immunogenic. This novel antigenic To ORF2 protein could be exploited for vaccine development against bovine piroplasmosis.     

Protein analysis of Babesia caballi merozoites by two-dimensional polyacrylamide gel electrophoresis and western blotting

Babesia caballi merozoites were prepared by combining two improved methods of cultivation and purification of merozoites using Percoll-gradiation, and the protein compositions of merozoites were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. The relative molecular masses of the major proteins and protein masses separated by electrophoresis were >94, 80-70, 50-45, 34-30, 30-28 and 18 kDa. By Western blotting, twelve proteins or protein groups were recognized by pooled sera from two horses experimentally infected with B. caballi. Among twelve proteins, five new proteins (54, 30-26, 24, and two 18 kDa) were identified, and the 48 kDa protein was revealed to consist of 2 components in the B. caballi merozoite. One protein (54 kDa) of B. caballi was also recognized by the pooled sera from two horses experimentally infected with B. equi.     

Dynamics of the humoral immune response of calves infected and re-infected with Cooperia punctata

The dynamics of the humoral immune response of calves were analysed after primary infection and re-infection with the intestinal nematode Cooperia punctata. 12 male 5 month-old Holstein-Friesian calves were randomly divided into two groups A and B. At the beginning of the experiment Group A animals were each infected experimentally with a single oral dose of 130,000 infective third stage larvae (L3) of C. punctata. The animals of Group B were kept as non-infected controls. The two calves from Group A with the highest infections died of cooperiosis at 32 and 44 days after infection (DAI), respectively. On DAI 100 the calves were treated with the recommended dose of oxfendazole. On DAI 180 the remaining four calves of Group A and three animals of Group B (B1) were infected with 260,000 L3 of C. punctata, while the other three calves of Group B (B2) served as non-infected controls. Monitoring of the humoral immune response predominantly demonstrated an IgG1 response against both adult and L3 antigen of C. punctata. Moreover, re-infections increased the levels of these immunoglobulins. IgA levels were less increased than IgG1 and no significant increase was observed in IgG2 and IgM levels. Immunoblotting analysis showed that total IgG present in the serum of the primary infected animals mainly reacted against adult proteins of 12-14 and 17-20 kDa and against L3 proteins of 33 and 43 kDa. After re-infection total IgG reacted with the same adult proteins but also with an adult 29 kDa protein.     

Immunoglobulin E recognition of Dirofilaria immitis antigens is more specific than immunoglobulin G.

The chronological development of the serum IgE and IgG response to microfilaria, third and fourth stage larvae, and male and female adult Dirofilaria immitis was examined by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunoelectrotransfer blot (EITB). Dirofilaria immitis-specific IgE and IgG levels peaked 16-18 weeks post-infection after increasing in response to the fourth larval molt. Specific IgG levels plateaued after patency, while IgE continued to decline. The use of ammonium sulfate cut sera showed there was no quenching or blocking of IgE binding by IgG in the ELISA and EITB methods used in this study. IgE-specific EITB showed 30-49 bands for the five respective extracts that were identified by M(r) or relative mobility. Eighty-five to 100 bands were visualized by IgG-specific EITB for the same five extracts. The isotype-specific ELISA and EITB were shown to be closely related by significant correlations (P < 0.0001) between S/N ratios and the number of bands found on blots. The isotype-specific EITB bands non-specifically recognized were greater in size than 21 kDa for IgG and 45 kDa for IgE. Recognition of bands changed over time with some bands being recognized only by prepatent sera. Ten antigen bands of seven M(r) were consistently and specifically recognized by IgE in the five-stage extracts by sera from prepatent and patent infections; only one such M(r) at 13.9 kDa, was described for IgG. A potentially diagnostic 31.9 kDa antigen band was identified on the IgE-specific EITB of D. immitis female extract and was shown to be recognized by IgE in sera from all infected dogs at all time points examined from 2 weeks until 1 year post-inoculation. Overall, IgE reactivity was more specific for D. immitis infections than IgG reactivity.     

Spondylitis in turkeys associated with experimental Pasteurella anatipestifer infection.

Nine previously vaccinated turkeys were inoculated intravenously with Pasteurella anatipestifer, and blood samples were taken periodically to evaluate the potential of chronically infected turkeys to serve as reservoirs of infection for blood-feeding arthropod vectors. Vertebral osteomyelitis (spondylitis), as yet unreported in the literature in association with infection with the organism, was found in the thoracic vertebrae of five out of nine inoculated turkeys, and P. anatipestifer was isolated from the thoracic vertebrae of three of the five. The organism was isolated from the peripheral blood of six turkeys 24 hours postinoculation and from the peripheral blood of one turkey 7 days postinoculation. The organism was also isolated from the heart blood of two birds at necropsy--from one at 21 days and, following an intramuscular injection of dexamethasone, from the other turkey at 38 days postinoculation.