Molecular ABO phenotyping in cynomolgus macaques using real-time quantitative PCR.

Macaques are commonly used in biomedical research as animal models of human disease. The ABO phenotype of donors and recipients plays an important role in the success of transplantation and stem cell research of both human and macaque tissue. Traditional serological methods for ABO phenotyping can be time consuming, provide ambiguous results and/or require tissue that is unavailable or unsuitable. We developed a novel method to detect the A, B, and AB phenotypes of macaques using real-time quantitative polymerase chain reaction. This method enables the simple and rapid screening of these phenotypes in macaques without the need for fresh blood or saliva. This study reports the distribution of the A, B, and AB phenotypes of captive cynomolgus macaques that, while regionally variable, closely resembles that of rhesus macaques. Blood group B, as in rhesus macaques, predominates in cynomolgus macaques and its frequency distribution leads to a probability of major incompatibility of 41%. No silencing mutations have been identified in exon 6 or 7 in macaques that could be responsible for the O phenotype, that, although rare, have been reported. The excess homozygosity of rhesus and cynomolgus macaque genotypes in this study, that assumes the absence of the O allele, suggests the possibility of some mechanism preventing the expression of the A and B transferases.

Epidemiology of microsporidiosis: sources and modes of transmission.

Microsporidia are single-celled, obligate intracellular parasites that were recently reclassified from protozoa to fungi. Microsporidia are considered a cause of emerging and opportunistic infections in humans, and species infecting humans also infect a wide range of animals, raising the concern for zoonotic transmission. Persistent or self-limiting diarrhea are the most common symptoms associated with microsporidiosis in immune-deficient or immune-competent individuals, respectively. Microsporidian spores appear to be relatively resistant under environmental conditions, and species of microsporidia infecting humans and animals have been identified in water sources, raising concern about water-borne transmission. Sensitive and specific immunomagnetic bead separation and PCR-based methods are being developed and applied for detecting microsporidia in infected hosts and water sources for generating more reliable prevalence data. The most effective drugs for treating microsporidiosis in humans currently include albendazole, which is effective against the Encephalitozoon species but not against Enterocytozoon bieneusi, and fumagillin, which has broader anti-microsporidia activity but is toxic in mammals, suggesting a need to identify better drugs. Strategies to capture and disinfect microsporidia in water are being developed and include filtration, coagulation, chlorination, gamma-irradiation, and ozonation.

Defining T-cell-mediated immune responses in rotavirus-infected juvenile rhesus macaques.

The appearance of virus-specific CD4(+) and/or CD8(+) T lymphocytes in peripheral blood of captive juvenile rhesus macaques (Macaca mulatta) was observed following rotavirus infection. These cell-mediated immune responses were measured following experimental or natural infection after rotavirus was isolated from stool specimens of asymptomatic animals. The virus isolated was a new strain of simian rotavirus that we named TUCH (for Tulane University and Cincinnati Children's Hospital). Restimulation of peripheral T lymphocytes by inactivated double- or triple-layered TUCH rotavirus particles containing either VP6 or VP4 and VP7 on their respective surfaces resulted in increased quantities of interleukin-6 (IL-6) and IL-12 in cell culture supernatants. Recall responses to rotavirus by CD4(+) and CD8(+) T lymphocytes were associated with accumulation of intracellular IL-6 and gamma interferon. Antigen presentation of TUCH rotavirus to lymphocytes was mediated via differentiated cultures of monocyte-derived dendritic (HLA-DR(+)) cells. This is the first report demonstrating cell-mediated immune responses to rotavirus in nonhuman primates. Further exploration of rhesus macaques in vaccine trials with human rotavirus vaccine candidates is the major objective of future studies.

Quantitative evaluation of Enterocytozoon bieneusi infection in simian immunodeficiency virus-infected rhesus monkeys.

The association of the microsporidia Enterocytozoon bieneusi with chronic diarrhea and wasting in individuals with acquired immunodeficiency syndrome (AIDS) has been demonstrated. The disease caused by E. bieneusi has been linked to decreased levels of circulating CD4+ T lymphocytes. In this study, we investigated the relationship between the extent of excretion of E. bieneusi in feces of simian immunodeficiency virus (SIV)-infected juvenile macaques and the CD4+ T lymphocyte counts in the peripheral blood. Twelve juvenile rhesus monkeys (Macaca mulatta) were intravenously inoculated with the pathogenic molecular clone SIVmac239. Numbers of CD4+ T lymphocytes were assessed by three-color flow cytometry. The presence of E. bieneusi DNA in feces was assessed by nested PCR. In addition, selected samples of feces were examined by competitive quantitative PCR to assess the level of E. bieneusi infection. Low (n = 5) to undetectable (n = 7) quantities of E. bieneusi were present in feces of the twelve animals in prior to inoculation with SIV. After SIV inoculation the number of animals shedding E. bieneusi increased (n = 10) as did the quantity of E. bieneusi shedding in the feces. Of the twelve juvenile animals, five animals died within 8 months post-SIV inoculation with symptoms of AIDS. Four of the five deceased animals showed shedding of E. bieneusi DNA in feces (> or =100 spores/g) for at least three consecutive months. Increased number of E. bieneusi in feces was accompanied by decreased counts of circulating CD4+ T lymphocytes and increased SIV plasma viral load.

Variability among Cryptosporidium parvum genotype 1 and 2 immunodominant surface glycoproteins.

Published genomic differences between Cryptosporidium parvum genotype 1 (human-derived) and genotype 2 (animal and human-derived) isolates suggest that these may belong to two distinct species. This is of significant interest since genotype 1 isolates are associated with sporadic cases of human cryptosporidiosis in 30-40 % of cases in contrast to 60-70 % of cases caused by genotype 2. The lower genetic sequence similarity between genotype 1 and 2 surface glycoproteins (gp40/15) suggests that antigenic differences should also occur, a feature that was investigated in this study. Using immune and convalescent serum samples from gnotobiotic piglets previously inoculated with genotype 1 and 2 isolates, we demonstrated that C. parvum gp15 was immunodominant for both genotype 1 and 2 isolates. Lower genetic sequence similarity between genotype 1 and 2 Cpgp40/15 did correspond to gp15 protein differences as detected by Western blot. Moreover, we confirmed that gp15 contains epitopes that are also immunodominant. Deglycosylation of C. parvum proteins resulted in decreased ability of gp15, gp23 and gp900 to react with homologous polyclonal antibodies, suggesting that these proteins also express carbohydrate epitopes. Taken together, our data suggest that there is a high phenotypic variability between C. parvum genotype 1 and 2 isolates at the level of gp15. We contemplate that gp15 surface glycoprotein plays an important role in the biology of C. parvum as a potent inducer of immune response and a possible virulence factor.

Development of a reverse transcription-nested polymerase chain reaction assay for differential diagnosis of transmissible gastroenteritis virus and porcine respiratory coronavirus from feces and nasal swabs of infected pigs.

Transmissible gastroenteritis virus (TGEV), a coronavirus, replicates in intestinal enterocytes and causes diarrhea in young pigs. Porcine respiratory coronavirus (PRCV), a spike (S) gene natural deletion mutant of TGEV, has a respiratory tissue tropism and causes mild or subclinical respiratory infections. Conventional antigen-based diagnostic tests fail to differentiate TGEV and PRCV, and a blocking ELISA test to serologically differentiate TGEV/PRCV-infected pigs is conducted on convalescent serum retrospectively after disease outbreaks. A reverse transcription (RT)-nested polymerase chain reaction (PCR) with primers targeted to the S gene deletion region to differentiate TGEV/PRCV was developed. The specificity of the RT-nested PCR was confirmed with reference and recent field strains of TGEV/PRCV, and its sensitivity was analyzed by testing nasal and fecal samples collected from pigs at various days postinoculation (DPI) with TGEV or PRCV. Specific PCR products for TGEV/PRCV were detected only with the homologous reference or field coronaviruses and for 10-14 DPI of pigs with TGEV (feces) or PRCV (nasal samples). The RT-nested PCR assay was more sensitive than antigen-based assays on the basis of duration of virus detection in experimentally infected pigs and was directly applicable to nasal as well as fecal specimens from the field.

Tacrolimus-FK506 induced immunosuppression in gnotobiotic piglets.

Tacrolimus (FK506), an inhibitor of calcineurin, is an immunosuppressive agent used in clinical trials of transplant patients. Although FK506 targets Ca(2+)-mediated T-cell signaling, phenotype(s) of the specific target cells and the corresponding cytokine pathways are not well known. In this study, the impact of FK506 on number and characteristic of T-cells in selected lymphoid tissues of gnotobiotic (GB) piglets was determined. FK506-treated GB piglets were compared with untreated GB and conventional piglets. The T-helper, cytotoxic, natural killer, double-positive, and activated T-cell populations were analyzed in suspensions of mononuclear cells isolated from thymus, mesenteric lymph nodes and peripheral blood. In vitro secretion of interleukin-8 and interferon-gamma in concanavalin A-stimulated lymphoid cell-cultures was measured by ELISA. Daily intramuscular treatment of GB piglets with 1mg/kg of FK506 from birth for 4 weeks resulted in lowered (P<0.05) in vitro secretion of interferon-gamma and interleukin-8. Moreover, depletions of MNC in systemic and mucosa-associated lymphoid tissues were observed in piglets treated with FK506. The depletions of mononuclear cells and low levels of interferon-gamma and interleukin-8 in piglets treated with FK506 were accompanied by lower proportion of CD3+, CD2+CD4+ and CD2+CD8+ T-cell phenotypes in peripheral blood but not in thymus and mesenteric lymph nodes. These results indicate that FK506-treatment causes immunosuppression in GB piglet, and this effect could be exploited further to study opportunistic pathogens in pig model.

Active immunity and T-cell populations in pigs intraperitoneally inoculated with baculovirus-expressed transmissible gastroenteritis virus structural proteins.

The intraperitoneal inoculation of pigs with baculovirus-expressed transmissible gastroenteritis virus (TGEV) structural proteins (S, N, M) in conjunction with thermolabile Escherichia coli mutant toxin (LT-R192G) in incomplete Freund's adjuvant (IFA) was tested in an attempt to elicit active immunity to TGEV in gut-associated lymphoid tissues (GALT). Four groups of 63 (1-5-week-old) suckling, TGEV-seronegative pigs were used to assess the efficacy of the recombinant protein vaccine (group 3) in comparison with sham (group 1), commercial vaccine (group 2), and virulent TGEV Miller-strain-inoculated pigs (group 4). The TGEV-specific mucosal and systemic immune responses were measured after in vivo and in vitro stimulation with TGEV-antigens. The major T-cell subset distribution was analyzed in vivo and in vitro after stimulation of mononuclear cells with TGEV (from mesenteric lymph nodes of group 3 inoculated with TGEV-recombinant proteins). Induction of active immunity was assessed by challenge of pigs with virulent TGEV at 27 days of age. Baculovirus-expressed TGEV proteins coadministered with LT-R192G in IFA induced mesenteric lymph node immune responses associated with IgA-antibodies to TGEV and partial protection against TGEV-challenge. The high titers of serum IgG- and virus-neutralizing-antibodies to TGEV in group 3 pigs most likely reflected the dose of TGEV S-protein administered. At the day of TGEV-challenge, the in vitro stimulation of mononuclear cells from the mesenteric lymph nodes of group 3 pigs with inactivated TGEV resulted in an increase in double positive (CD4+CD8+), natural killer (CD2+CD4-CD8+dim) and cytotoxic (CD2+CD4-CD8+bright) T-cell phenotypes, accompanied by increased expression of interleukin-2 receptor and a decrease of the null (CD2-CD4-CD8-/SW6+) cell phenotype.

Evaluation of the baculovirus-expressed S glycoprotein of transmissible gastroenteritis virus (TGEV) as antigen in a competition ELISA to differentiate porcine respiratory coronavirus from TGEV antibodies in pigs.

The spike (S) glycoprotein of the Miller strain of transmissible gastroenteritis virus (TGEV) was recently cloned and expressed in baculovirus. The recombinant S protein was used as the coating antigen in a competition (blocking) enzyme-linked immunosorbent assay (ELISA) in combination with monoclonal antibodies to the S protein epitope A (conserved on TGEV and porcine respiratory coronavirus [PRCV]) or epitope D (present on TGEV only) to differentiate PRCV- from TGEV-induced antibodies. One set (set A) of 125 serum samples were collected at different times after inoculation of caesarean-derived, colostrum-deprived (n = 52) and conventional young pigs (n = 73) with 1 of the 2 porcine coronaviruses or uninoculated negative controls (TGEV/PRCV/negative = 75/30/20). A second set (set B) of 63 serum samples originated from adult sows inoculated with PRCV and the recombinant TGEV S protein or with mock-protein control and then exposed to virulent TGEV after challenge of their litters. Sera from set A were used to assess the accuracy indicators (sensitivity, specificity, accuracy) of the fixed-cell blocking ELISA, which uses swine testicular cells infected with the M6 strain of TGEV as the antigen source (ELISA 1) and the newly developed ELISA based on the recombinant S protein as antigen (ELISA 2). The sera from set B (adults) were tested for comparison. The plaque reduction virus neutralization test was used as a confirmatory test for the presence of antibodies to TGEV/PRCV in the test sera. The accuracy indicators for both ELISAs suggest that differential diagnosis can be of practical use at least 3 weeks after inoculation by testing the dual (acute/convalescent) samples from each individual in conjunction with another confirmatory (virus neutralization) antibody assay to provide valid and complete differentiation information. Moreover, whereas ELISA 1 had 10-20% false positive results to epitope D for PRCV-infected pigs (set A samples), no false-positive results to epitope D occurred using ELISA 2, indicating its greater specificity. The progression of seroresponses to the TGEV S protein epitopes A or D, as measured by the 2 ELISAs, was similar for both sets (A and B) of samples. Differentiation between TGEV and PRCV antibodies (based on seroresponses to epitope D) was consistently measured after the third week of inoculation.

Immune response of sows vaccinated with attenuated transmissible gastroenteritis virus (TGEV) and recombinant TGEV spike protein vaccines and protection of their suckling pigs against virulent TGEV challenge exposure.

OBJECTIVE: To compare recombinant transmissible gastroenteritis virus (TGEV) spike protein, (SP) R2-2, with attenuated live virus (ALV) vaccine in sows during late pregnancy. ANIMALS: 13 TGEV-seronegative sows and their pigs. PROCEDURE: At prepartum weeks (PPW) 6 and 4, sows of groups 1 and 2 received ALV via the oral/intranasal (O/IN) route. At PPW 2, group-1 sows received ALV IM and group-2 sows received SPR2-2 IM. Group-3 sows received SPR2-2 IM at PPW 4 and ALV O/IN at PPW 2. Sows of group 4 (negative controls) were inoculated O/IN with mock-infected ST cell fluids at PPW 6 and 4 and IM with Sf9 cell lysates at PPW2 (n = 2), or IM with Sf9 cell lysates at PPW4 and O/IN with mock-infected ST cell fluids at PPW2 (2). Serum, colostrum, and milk samples were tested for antibody to TGEV, and a lymphoproliferative (LP) assay was done on blood mononuclear cells. Suckling pigs were challenge exposed with virulent TGEV. RESULTS: Sows of groups 1 and 2 had higher IgG and significantly higher antibody titers in colostrum; their pigs had significantly higher serum antibody titer. At challenge exposure of their pigs, LP responses of group-2 sows were significantly higher than those of sows in the other 3 groups. Mean pig mortality ranged from 43 (group 2) to 92% (group 4). Significant negative correlations were observed among litter mortality and sow LP response, colostral titer, and pig serum titer at time of challenge exposure. CONCLUSIONS: In sows vaccinated twice with attenuated live TGEV, the recombinant SPR2-2 administered IM may be comparable to ALV administered IM as a booster. Vaccination failed to provide complete protection to suckling pigs after challenge exposure.

Contribution of passive immunity to porcine respiratory coronavirus to protection against transmissible gastroenteritis virus challenge exposure in suckling pigs.

OBJECTIVE: To determine the ability of porcine respiratory coronavirus (PRCV) infections to induce passive immunity in suckling pigs to transmissible gastroenteritis virus (TGEV) challenge exposure. DESIGN AND ANIMALS: 4 TGEV seronegative sows and their litters (group A) served as controls, whereas 2 other groups (B and C) of sows (also TGEV seronegative) were oronasally inoculated with live PRCV during 1 or 2 subsequent pregnancies, respectively. PROCEDURE: Effectiveness of passive immunity provided to pigs via colostrum and milk was assessed after TGEV challenge exposure, and TGEV antibody responses in colostrum and milk were analyzed. RESULTS: Mortality in the 3 groups of young pigs correlated with severity of clinical signs of TGEV infection and was highest in control litters (86% in group-A pigs) and lowest in litters of sows inoculated with PRCV in 2 subsequent pregnancies (14% in group-C pigs). Virus-neutralization and IgA and IgG TGEV antibody titers of milk collected from sows at challenge exposure had significant positive correlation with litter survival. Significantly higher numbers of TGEV-specific IgA and IgG antibody-secreting cells were found in group-A pigs than in group-C pigs, suggesting that high titer of maternal antibodies (induced in group-C sows multiply exposed to PRCV) may interfere with active antibody responses. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that, in PRCV-infected pig herds, multiple exposures of pregnant sows are associated with higher IgA and IgG antibody titers to TGEV in milk, and these titers contribute to protection against TGEV infection.

Isolation of rotavirus strains from naturally infected piglets.

Twenty rotavirus strains were isolated in 1991-92 from 60 faecal samples collected from diarrhoeic piglets in the Czech and Slovak Republics. Three isolates were adapted to the growth in the cell line MA-104 and produced cytopathic effect. Rotavirus was demonstrated by immunofluorescence test, electron microscopy, polyacrylamide gel electrophoresis, immunoperoxidase test and ELISA.

Use of DNA fingerprinting for accurate typing of Actinobacillus pleuropneumoniae.

DNA fingerprinting was tested as a method for detailed differentiation of 23 strains of the causal agent of porcine pleuropneumonia--Actinobacillus pleuropneumoniae. The set consisted of twelve reference strains, each representing one serotype, and eleven field strains belonging to serotype 9 which occurs most frequently in the Czech Republic. Nine of the reference strains could be differentiated from each other and from serotypes 1, 9 and 11, but a very close relatedness was demonstrated among the latter three by repeated examination. Four DNA types were identified among the twelve serotype 9 strains. The findings are discussed within the context of the production and efficiency of industrial and autogenous vaccines.

[Present findings on rotaviruses with emphasis on rotaviruses in swine]. / Soucasné poznatky o rotavirech se zamerením na rotaviry prasat.

A summary of thirty pages, concerning rotaviruses, i.e. RNA-viruses, the most frequent causal agents of infectious diarrhoea in children and young farm animals. Most of the chapters are generally focused on rotavirus morphology, structure and biology. Chapter No. 6 is especially detailed, trying to offer a complete summary of available diagnostic methods. The final chapters concern in greater detail the problems of rotaviruses in pigs, such as the immunity, prevention and treatment.