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Biomedical subjects

L J Saif

Publications and source records attributed to L J Saif.

At least 73 records · Page 4Linked to original sources
PubMed1998

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.

Animals↗
PubMed1997

Detection of group B rotaviruses in fecal samples from diarrheic calves and adult cows and characterization of their VP7 genes.

Groups A, B, and C rotaviruses have been identified in cattle. Group B rotaviruses are associated with sporadic cases of diarrhea in calves and adult cows. From diagnostic submissions to our laboratory, 90 fecal samples from cases of calf diarrhea, 81 fecal samples from cases of adult cow diarrhea (winter dysentery), and 20 fecal samples from case control normal adult cows were tested for group B rotaviruses by polyacrylamide gel electrophoresis (PAGE), and reverse transcription (RT)-PCR (targeting 279 bp of the VP7 gene). In addition, 53 fecal samples from diarrheic adult cows were tested for group B rotaviruses by immune electron microscopy (IEM). By RT-PCR, five samples from calves were group B rotavirus positive (5.6%). Fifteen samples from adult cows with diarrhea were group B rotavirus positive (18.5%), and none of the control fecal samples from normal cows were positive for group B rotaviruses. By PAGE, one calf sample (RT-PCR positive) was group B rotavirus positive (short electropherotype), but none of the adult cow samples were positive for group B rotaviruses. By IEM, 5 (9.4%) of the 53 fecal samples from diarrheic adult cows were group B positive (all were also RT-PCR positive). The VP7 genes of three strains (WD653 from an adult cow and the ATI and Mebus calf strains) were sequenced. The VP7 genes from the three bovine strains showed high (over 90%) nucleotide and deduced amino acid homologies, but lower homologies (48 to 61%) were seen between these genes and the genes from rodent (IDIR) and human (ADRV) group B rotaviruses. Although there were some differences of degree, all inoculated gnotobiotic calves (n = 6) showed abnormal feces between 1 and 3 days after inoculation with each of three strains of group B bovine rotaviruses, and group B rotaviruse, were detected in the feces for up to 2 weeks by RT-PCR but for shorter periods by PAGE or IEM.

Animals↗
PubMed1997

Active and passive immune responses to transmissible gastroenteritis virus (TGEV) in swine inoculated with recombinant baculovirus-expressed TGEV spike glycoprotein vaccines.

OBJECTIVE: Baculovirus-expressed transmissible gastroenteritis virus (TGEV) spike (S) glycoprotein vaccines were inoculated parenterally in swine to determine whether such vaccines could induce serum and whey virus-neutralizing (VN) antibodies and protective lactogenic immunity for TGEV-challenge-exposed pigs. ANIMALS AND PROCEDURES: 3 recombinant baculoviruses that expressed full or partial length TGEV Miller strain S glycoproteins were inoculated SC in 17 conventionally raised 11-day-old TGEV-seronegative pigs to determine whether the recombinant S glycoproteins would elicit serum VN antibodies. Eleven TGEV-seronegative pregnant sows were inoculated SC or intramammarily with subunit vaccines (R2-2 or R3-5) or control proteins. Pigs born to 9 of the 11 sows were challenge exposed at 4 to 5 days of age with the virulent Miller strain, and passive immunity was assessed. Serum and whey antibody responses to TGEV were analyzed by VN and ELISA testing. RESULTS: Recombinant S glycoproteins (R2-2 or R3-5) containing the 4 major antigenic sites induced similar VN antibody titers to TGEV in serum and colostrum, but low (some sows) or no VN antibody titer was detected in milk. Subcutaneous inoculation of sows with R2-2 or R3-5 elicited IgG, but not IgA antibodies to TGEV in colostrum. Morbidity was 100%, and mortality ranged from 20 to 80% in TGEV challenge-exposed pigs nursing sows inoculated SC or intramammarily with TGEV S glycoprotein vaccines. CONCLUSIONS AND CLINICAL RELEVANCE: Parenterally administered TGEV S glycoprotein vaccines elicit VN antibodies to TGEV in serum and colostrum that do not fully provide active or passive immunity in swine.

Animals↗
PubMed1996

The gnotobiotic piglet as a model for studies of disease pathogenesis and immunity to human rotaviruses.

Gnotobiotic piglets serve as a useful animal model for studies of human rotavirus infections, including disease pathogenesis and immunity. An advantage of piglets over laboratory animal models is their prolonged susceptibility to human rotavirus-induced disease, permitting cross-protection studies and an analysis of active immunity. Major advances in rotavirus research resulting from gnotobiotic piglet studies include: 1) the adaptation of the first human rotavirus to cell culture after passage and amplification in piglets; 2) delineation of the independent roles of the two rotavirus outer capsid proteins (VP4 and VP7) in induction of neutralizing antibodies and cross-protection; and 3) recognition of a potential role for a nonstructural protein (NSP4) in addition to VP4 and VP7, in rotavirus virulence. Current studies of the pathogenesis of group A human rotavirus infections in gnotobiotic piglets in our laboratory have confirmed that villous atrophy is induced in piglets given virulent but not cell culture attenuated human rotavirus (G1, P1A, Wa strain) and have revealed that factors other than villous atrophy may contribute to the early diarrhea induced. A comprehensive examination of these factors, including a proposed role for NSP4 in viral-induced cytopathology, may reveal new mechanisms for induction of viral diarrhea. Finally, to facilitate and improve rotavirus vaccination strategies, our current emphasis is on the identification of correlates of protective active immunity in the piglet model of human rotavirus-induced diarrhea. Comparison of cell-mediated and antibody immune responses induced by infection with a virulent human rotavirus (to mimic host response to natural infection) with those induced by a live attenuated human rotavirus (to mimic attenuated oral vaccines) in the context of homotypic protection has permitted an analysis of correlates of protective immunity. Results of these studies have indicated that the magnitude of the immune response is greatest in lymphoid tissues adjacent to the local site of viral replication (small intestine). Secondly, there was a direct correlation between the degree of protection induced and the level of the intestinal immune response, with significantly higher local immune responses and complete protection induced only after primary exposure to virulent human rotavirus. These studies thus have established basic parameters related to immune protection in the piglet model of human rotavirus-induced disease, verifying the usefulness of this model to examine new strategies for the design and improvement of human rotavirus vaccines.

Animals↗
PubMed1996

Rotavirus subunit vaccines.

We evaluated rotavirus subunit vaccines for use in humans and animals. Insect cells were co-infected with combinations of individual baculovirus recombinants expressing human, bovine or simian rotavirus VP2, VP4, VP6 or VP7 to produce virus-like particles (VLPs). To determine whether immunization with VLPs could induce active protective immunity, VLPs were administered parenterally to rabbits, and the immune response and protection from rabbit ALA rotavirus challenge were evaluated. Complete or partial protection was attained, showing that parenteral immunization with VLPs induces active protective immunity. We also examined whether heterotypic immune responses could be induced with a limited number of broadly reactive VP7 proteins or with chimeric particles (multiple VP7 types on individual particles). The feasibility of this approach was determined by immunizing mice with VLPs containing a G3 VP7 or G1 VP7 and chimeric G1/G3 VLPs. Broadly reactive neutralizing antibody was induced by the G1 VLPs. VLPs also have been successfully used to boost lactogenic (colostral and milk) immunity in dairy cows. Taken together, these results show that VLPs can be effective immunogens in rabbits, mice and dairy cattle when administered parenterally, a limited number of VLPs may be sufficient to produce a broadly protective vaccine, and G3 VLPs may serve as an effective subunit vaccine for use in bovines.

Animals↗
PubMed1996

The characterization of VP7 (G type) and VP4 (P type) genes of bovine group A rotaviruses from field samples using RT-PCR and RFLP analysis.

Characterization of the VP7 (G type) and VP4 (P type) genes of bovine group A rotaviruses (BRV) from field samples was performed using RT-PCR and RFLP analysis. After RT-PCR amplification of the full length VP7 genes and partial length VP4 genes (nucleotides 1 to 1096), four enzymes, EcoRV, NlaIV, BamHI and HpaII were used for digestion analysis. For VP7, four RFLP profiles were observed after analysis of the digests: they were designated as G6, G6s (subtype, showed about 86% nucleotide and 90% amino acid identity to reference G6 strains), G8 and G10. For VP4, three RFLP profiles were observed: designated as P[1], P[5] and P[11]. The G typing analysis of 86 BRV fecal samples from 5 states, representing at least 11 different herds revealed that 60.5% (52/86) were G6, which included G6s (9/52); 19.8% (17/86) were G10; 7% (6/86) were G8; 10.4% (9/86) were G6 and G10 mixtures including two G6s samples; and 2.3% (2/86) were G6 and G6s mixtures. The P typing analysis of the same 86 fecal samples revealed that 64% (55/86) were P[5]; 28% (24/86) were P[11]; 1.2% (1/86) were P[1] and 6 samples (7%) were mixtures of either P[11] or P[5]. When the same samples were analyzed according to G and P type specificity, all possible combinations of G and P types existed in the field. The G6P[5] type was most prevalent and accounted for 46.7% (41/86) of the samples; 12.8% (11/86) were G10P[11]; 7% (6/86) were G10P[5] and an equal number were G6sP[11]. The G6P[11] (n = 2), G8P[1] (n = 1), G8P[5] (n = 1) and G8P[11] (n = 3) combinations were also observed. The following mixed BRV infections were observed in the field samples; G6sP[5 + 11] (n = 1), G8P[5 + 11] (n = 1), G6 + G10P[5] (n = 1) G6 + G10P[5 + 11] (n = 2), G6 + G6sP[11] (n = 1), G6 + G6sP[1 + 11] (n = 1), G6s + G10P[11] (n = 1) and G6s + G10P[5 + 11] (n = 1). Information on the G and P types and G/P combinations in the field samples should be useful for understanding the epidemiology of BRV and designing vaccination strategies to control BRV in the field.

Animals↗
PubMed1996

Sequence comparison of the VP7 gene encoding the outer capsid glycoprotein among animal and human group C rotaviruses.

The nucleotide sequences of the outer capsid glycoprotein (VP7) genes from the Shintoku bovine and the HF and WH porcine group C rotaviruses were determined and compared with those of the published corresponding genes from the Cowden porcine and Ehime human group C rotaviruses. The VP7 genes of all 5 strains were 1063 nucleotides in length and possess one open reading frame encoding a polypeptide of 332 amino acids. Comparative analysis of the deduced amino acid sequences indicated that 85.2-97.0% identity was observed for the VP7 of the serotypically related strains of group C rotaviruses (Cowden, WH and Ehime) whereas 69.9-74.7% identity was observed among the serotypically distinct strains (Shintoku; Cowden, WH and Ehime; and HF). At least 8 variable regions in the VP7 were recognized among serotypically distinct strains, and these locations were similar to those of the variable regions in the VP7 of group A rotaviruses.

Animals↗
PubMed1996

Sequence conservation and expression of the gene encoding the outer capsid glycoprotein among human group C rotaviruses of global distribution.

Group C rotaviruses have been identified recently from fecal samples of children with diarrhea in the United States. Using reverse transcriptasepolymerase chain reaction and sequence analysis, we sequenced gene 8s encoding VP7 from two U.S. strains (RI-1 and RI-2), and eight other strains isolated from patients on four continents, and compared these with the sequences of four published strains. The gene 8s of the 14 strains were remarkably conserved in size and in predicted primary and secondary structures. When the sequences of the human VP7s were compared with that of the prototype porcine Cowden strain, six regions were found variable in both deduced primary and predicted secondary structures, four of which were predicted to be hydrophilic and might determine serotype specificity. Gene 8 of the human S-1 strain was further characterized by expression in recombinant baculoviruses. The expressed product was immunogenic but failed to elicit neutralizing antibodies. Our sequence analysis indicates that all the human strains characterized to date belong to a single G genotype, which may constitute a single G serotype, pending further antigenic analysis. Whether the human strains and the Cowden strain are the same serotype remains to be determined.

Adult↗
PubMed1996

Mucosal immunity: an overview and studies of enteric and respiratory coronavirus infections in a swine model of enteric disease.

Based on the tenet of a common mucosal immune system, antigenic stimulation at one mucosal site results in the distribution of antigen-specific IgA precursor cells to distant mucosal sites. However, recent studies suggest that functional compartmentalization and limited reciprocity may exist within some components of the common mucosal immune system. Although oral immunization is often very effective in inducing immunity to respiratory pathogens, the converse (respiratory immunization to prevent enteric diseases) may not be as effective. To address this question and to study interactions between the bronchus-associated (BALT) and gut-associated (GALT) lymphoid tissues related to protective immunity, we used as a model two antigenically related porcine coronaviruses which replicate primarily in the intestine (transmissible gastroenteritis virus, TGEV) or respiratory tract (porcine respiratory coronavirus, PRCV). The tissue distribution and magnitude of the antibody secreting cell (ASC) responses (measured by ELISPOT) and cell-mediated immune responses (measured by lymphoproliferative assays, LPA) coincided with the viral tissue tropisms. Immunization via GALT (gut infection with TGEV) elicited high numbers of IgA ASC and high LPA responses in GALT (gut lamina propria, LP or mesenteric lymph nodes, MLN), but lower responses in BALT (bronchial lymph nodes, BLN) and induced complete protection against enteric TGEV challenge. In contrast immunization via BALT (respiratory infection with PRCV) elicited systemic type responses (high numbers of IgG ASC in the BLN), but few ASC and low LPA responses in the gut LP or MLN and induced only partial protection against enteric TGEV challenge. Thus administration of vaccines intranasally may not be optimally effective for inducing intestinal immunity in contrast to the reported efficacy of oral vaccines for inducing respiratory immunity.

Administration, Intranasal↗
PubMed1996

Isotype-specific antibody responses to rotavirus and virus proteins in cows inoculated with subunit vaccines composed of recombinant SA11 rotavirus core-like particles (CLP) or virus-like particles (VLP).

The isotype antibody responses to bovine IND P5, G6 and simian SA11 P2, G3 rotavirus and SA11 rotavirus proteins (VP4, VP6 and VP7) in serum, colostrum and milk were analysed by ELISA in three groups of vaccinated cows and nonvaccinated controls. Pregnant cows were vaccinated intramuscularly and intramammarily with recombinant baculovirus-expressed SA11 rotavirus VLP (triple-layered virus-like particles containing rotavirus VP2, VP4, VP6 and VP7); CLP (double-layered core-like particles containing rotavirus VP2 and VP6); or inactivated SA11 rotavirus, respectively. Rotavirus antigen titers were highest (30-200-fold) in ELISA in the VLP vaccine compared to the inactivated SA11 vaccine. The IgG1, IgG2 and IgM geometric mean antibody titers (GMT) to rotavirus (titers to bovine rotavirus vs SA11 rotavirus did not differ significantly for any isotype or group) and the IgG2 GMT to VP6 in serum at calving in the vaccinated groups were significantly (P < 0.05) higher than in the control group. In colostrum, IgG1 and IgA rotavirus antibody titers were significantly elevated for VLP (IgG1 GMT 832225; IgA GMT 16384), CLP (IgG1 GMT 660561; IgA GMT 10321) and SA11 (IgG1 GMT 131072; IgA GMT 1448) vaccinated cows compared to control cows (IgG1 GMT 11585; IgA GMT 45). The IgG1 and IgA GMT to rotavirus were significantly elevated (6-100-fold) in milk of VLP and CLP vaccinated cows compared to SA11 vaccinated or control cows. The isotype antibody responses to VP6 in serum, colostrum and milk paralleled the responses to rotavirus, but titers were approximately 2-10-fold lower. Only cows vaccinated with VLP had significantly enhanced serum, colostral and milk antibody titers to rotavirus VP4 and VP7. These results demonstrate that rotavirus antibody titers in serum, colostrum and milk are significantly enhanced by use of non-infectious VLP, CLP and inactivated SA11 rotavirus vaccines, but the VLP or CLP vaccines induced the highest antibody responses, corresponding to their higher rotavirus antigen titers measured by ELISA.

Animals↗
PubMed1996

Group A rotavirus veterinary vaccines.

Group A rotaviruses cause diarrhea in young livestock and poultry; consequently, vaccination strategies have focused on induction of active or passive immunity. Gnotobiotic pigs and calves serve as useful models to evaluate induction of active immunity by candidate animal or human rotavirus vaccines. However, live attenuated rotavirus vaccines lacked efficacy when administered orally to calves and pigs in the field, presumably because colostral antibodies inhibited vaccine virus replication. The widespread occurrence of rotavirus antibodies in colostrum led to strategies for maternal rotavirus vaccination to boost lactogenic immunity and transfer passive antibodies to the neonate via colostrum and milk. The variable success of maternal rotavirus vaccines in the field is influenced by vaccine dose, strain, inactivating agent, adjuvant, route of administration, and environmental rotavirus exposure levels. The use of genetically engineered rotavirus-like particle vaccines in cows to boost antibodies in mammary secretions shows promise. Such subunit vaccines possess potential advantages over existing vaccines.

Animals↗
PubMed1996

Pathogenesis of an attenuated and a virulent strain of group A human rotavirus in neonatal gnotobiotic pigs.

Gnotobiotic (Gn) pigs were orally inoculated with Wa strain (G1P1A[P8]) human rotavirus (Wa HRV) serially passaged in Gn pigs (virulent) or cell culture (attenuated) to determine the median virus infectious dose (ID50) and to assess the site of infection and type and progression of morphological lesions and clinical responses induced by these two strains in Gn pigs. The ID50 of virulent Wa HRV was = or < 1 f.f.u. whereas the infectivity of attenuated Wa HRV had to be determined by seroconversion and was approximately 1.3 x 1O(6) f.f.u. Diarrhoea developed at 13 h post-inoculation (p.i.) in pigs inoculated with approximately 1O(5) f.f.u. of virulent Wa HRV and correlated with the presence of viral antigen within villous epithelial cells; villous atrophy developed later at 24 h p.i. and correlated with peak faecal viral titres; recovery from disease correlated with the return of morphologically normal villi. Virus, diarrhoea and villous atrophy were not detected in pigs inoculated with approximately 2 x 10(8)f.f.u. attenuated Wa HRV although HRV-specific serum antibodies were present by 7 days p.i. These findings demonstrate that virulent Wa HRV infection in Gn pigs occurs primarily within intestinal villous epithelial cells with villous atrophy developing as a sequela to infection. However, factors other than villous atrophy appear to contribute to the early stages of HRV-associated disease expression in Gn pigs. The ability of the attenuated virus to elicit virus-neutralizing serum antibodies without disease or pathology indicates promise in the use of such strains for oral immunization.

Animals↗
PubMed1996

Development of mucosal and systemic lymphoproliferative responses and protective immunity to human group A rotaviruses in a gnotobiotic pig model.

Gnotobiotic pigs were orally inoculated with virulent Wa strain (G1P1A[8]) human rotavirus (group 1), attenuated Wa rotavirus (group 2) or diluent (controls) and were challenged with virulent Wa rotavirus 21 days later. On various postinoculation or postchallenge days, virus-specific responses of systemic (blood and spleen) and intestinal (mesenteric lymph node and ileal lamina propria) mononuclear cells (MNC) were assessed by lymphoproliferative assays (LPA). After inoculation, 100% of group 1 pigs and 6% of group 2 pigs shed virus. Diarrhea occurred in 95, 12, and 13% of group 1, group 2, and control pigs, respectively. Only groups 1 and 2 developed virus-specific LPA responses prior to challenge. Group 1 developed significantly greater mean virus-specific LPA responses prior to challenge and showed no significant changes in tissue mean LPA responses postchallenge, and 100% were protected against virulent virus challenge. By comparison, both group 2 and controls had significantly lower LPA responses at challenge and both groups showed significant increases in mean LPA responses postchallenge. Eighty-one percent of group 2 and 100% of control pigs shed challenge virus, and both groups developed diarrhea that was similar in severity postchallenge. The virus-specific LPA responses of blood MNC mirrored those of intestinal MNC, albeit at a reduced level and only at early times postinoculation or postchallenge in all pigs. In a separate study evaluating antibody-secreting-cell responses of these pigs (L. Yuan, L.A. Ward, B.I. Rosen, T.L. To, and L.J. Saif, J. Virol. 70:3075-3083, 1996), we found that the magnitude of a tissue's LPA response positively correlated with the numbers of virus-specific antibody-secreting cells for that tissue, supporting the hypothesis that the LPA assesses T-helper-cell function. The magnitude of LPA responses in systemic and intestinal tissues also strongly correlated with the degree of protective immunity elicited by the inoculum (p = 0.81). We conclude that blood may provide a temporary "window" for monitoring intestinal T cells and that the LPA can be used to assess protective immunity to human rotaviruses.

Animals↗
PubMed1996

Identification of group B rotaviruses with short genome electropherotypes from adult cows with diarrhea.

Two field strains (BB-RVLV and KD) of group B rotaviruses from adult dairy cows with diarrhea displayed short genome electropherotypes. Gnotobiotic calves inoculated with fecal filtrates of each group B rotavirus developed diarrhea, and only group B rotaviruses or antigens were detected in the feces by immunoelectron microscopy and in intestinal epithelial cells by immunofluorescent staining, respectively. The feces or intestinal contents of the cows and inoculated calves were negative for group A and C rotaviruses by enzyme-linked immunosorbent assay, immunoelectron microscopy, or cell culture immunofluorescence assays. Comparison of the genome electropherotypes of the calf-passaged BB-RVLV and KD strains with the original samples and reference bovine group A, B, and C rotaviruses revealed conservative of their short-genome electropherotypes and double-stranded RNA migration patterns characteristics of group B rotaviruses. To our knowledge, our previous study (L.J. Saif, K.V. Brock, D.R. Redman, and E.M. Kohler, Vet. Rec. 128:447-449, 1991) and this report are the first description of bovine group B rotaviruses (in a mixed infection with bovine coronavirus or singly in fecal contents) in adult cows with diarrhea and this is the first report of short-genome electropherotypes among group B rotaviruses.

Animals↗
PubMed1996

Systematic and intestinal antibody-secreting cell responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease.

Neonatal gnotobiotic pigs orally inoculated with virulent (intestinal-suspension) Wa strain human rotavirus (which mimics human natural infection) developed diarrhea, and most pigs which recovered (87% protection rate) were immune to disease upon homologous virulent virus challenge at postinoculation day (PID) 21. Pigs inoculated with cell culture-attenuated Wa rotavirus (which mimics live oral vaccines) developed subclinical infections and seroconverted but were only partially protected against challenge (33% protection rate). Isotype-specific antibody-secreting cells (ASC were enumerated at selected PID in intestinal (duodenal and ileal lamina propria and mesenteric lymph node [MLN]) and systemic (spleen and blood) lymphoid tissues by using enzyme-linked immunospot assays. At challenge (PID 21), the numbers of virus-specific immunoglobulin A (IgA) ASC, but not IgG ASC, in intestines and blood were significantly greater in virulent-Wa rotavirus-inoculated pigs than in attenuated-Wa rotavirus-inoculated pigs and were correlated (correlation coefficients: for duodenum and ileum, 0.9; for MLN, 0.8; for blood, 0.6) with the degree of protection induced. After challenge, the numbers of IgA and IgG virus-specific ASC and serum-neutralizing antibodies increased significantly in the attenuated-Wa rotavirus-inoculated pigs but not in the virulent-Wa rotavirus-inoculated pigs (except in the spleen and except for IgA ASC in the duodenum). The transient appearance of IgA ASC in the blood mirrored the IgA ASC responses in the gut, albeit at a lower level, suggesting that IgA ASC in the blood of humans could serve as an indicator for IgA ASC responses in the intestine after rotavirus infection. To our knowledge, this is the first report to study and identify intestinal IgA ASC as a correlate of protective active immunity in an animal model of human-rotavirus-induced disease.

Animals↗
PubMed1996

Immunohistochemistry of transmissible gastroenteritis virus antigens in fixed paraffin-embedded tissues.

An immunohistochemistry technique was developed using fixed tissues to study the presence and location of transmissible gastroenteritis virus (TGEV) antigens in situ. Experimentally infected gnotobiotic and conventional pigs as well as pigs with natural TGEV infection were examined. The staining technique was based on detection of the major structural protein of TGEV, the nucleocapsid, by using a pool of 3 monoclonal antibodies. Formalin and periodate-lysine-paraformaldehyde (PLP)-fixed intestinal tissues from a gnotobiotic pig inoculated with virulent TGEV were used to determine optimal antibody concentrations and incubation times. The intestinal tissues remained in their respective fixatives for 6 months, and serial sections were removed at sequential times and embedded in paraffin blocks. PLP and 10% neutral buffered formalin were acceptable fixatives and preserved TGEV nucleocapsid antigenicity for up to 6 months. Formalin, in comparison with PLP as a fixative, was better for preserving original tissue morphology and provided better antigen detection. Conventional crossbred pigs were inoculated with virulent TGEV, and animals were euthanized on various postexposure days. Intestinal tissues were positive for TGEV nucleocapsid antigens on postexposure days 2, 4, and 8. The immunohistochemistry technique detected TGEV antigen in stored paraffin-embedded tissues from 14 naturally infected pigs previously confirmed as positive for TGEV using a direct immunofluorescence assay on intestinal mucosal smears, whereas 9 naturally infected pigs confirmed negative for TGEV antigen by the same immunofluorescence assay showed no staining consistent with the presence of TGEV antigen. Immunohistochemistry provides a method to detect TGEV and possibly other closely related coronaviruses such as porcine respiratory coronavirus in situ. A diagnostic test using the same fixed tissues processed for histopathology provides veterinary practitioners an alternative to delivering live pigs or refrigerated fresh intestinal samples containing infectious virus to a diagnostic laboratory. Investigators can utilize this technique to retrospectively screen fixed tissues for TGEV antigen.

Animals↗
PubMed1996

Infection and cross-protection studies of winter dysentery and calf diarrhea bovine coronavirus strains in colostrum-deprived and gnotobiotic calves.

OBJECTIVE: To investigate in vitro antigenic relations, in vivo cross-protection, and isotype antibody responses to a winter dysentery (WD) and calf diarrhea strain of bovine coronavirus (BCV). DESIGN AND ANIMALS: Gnotobiotic and colostrum-deprived calves were inoculated oronasally with a WD (DBA) or a calf diarrhea (DB2) BCV, and were challenge exposed with the heterologous BCV. PROCEDURE: Nasal swab and feces specimens and blood samples were collected. Fecal and nasal specimens were assayed for BCV shedding by antigen-capture ELISA or immune electron microscopy. Bovine coronavirus antigens were detected in nasal epithelial cells by immunofluorescence. Antibody titers to BCV in serum were assayed by virus neutralization (VN), and BCV antibody isotype titers in feces and sera were quantitated by ELISA. RESULTS: All calves developed diarrhea and shed BCV nasally and in feces, then recovered and were protected from BCV-associated diarrhea after challenge exposure with the heterologous BCV. After challenge exposure with either strain, fecal shedding of DBA was detected in 1 of 4 calves and nasal shedding of DB2 was detected in 2 of 4 calves. Immunoglobulin M was the principal coproantibody to BCV early, followed predominantly by IgA. Immunoglobulin G1 coproantibody titers to BCV were low, but increased after challenge exposure. Immunoglobulin G1 antibodies were predominant in serum. After challenge exposure, all serum antibody isotype titers increased except IgG2. The VN antibody responses paralleled serum IgG1 antibody responses. CONCLUSIONS AND CLINICAL RELEVANCE: Immunoglobulin A coproantibodies at challenge exposure were associated with protection against diarrhea. Nasal shedding of BCV after challenge exposure confirmed field data documenting reinfection of the respiratory tract of cattle, suggesting that, in closed herds, respiratory tract infections constitute a source of BCV transmission to cows (WD) or young calves.

Animals↗