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Veterinary immunolugy ELSEVIER Veterinary Immunology and Immunopathology 48 1995 35 54 and immunopatbology Cellular immune responses of pigs after primary inoculation with porcine respiratory coronavirus or transmissible gastroenteritis virus and challenge with transmissible gastroenteritis virus Theresa A Brim a John L VanCott aY2 Joan K Lunneyb Linda J Saif a Food Animal Health Research Program Department of Veterinary Preventive Medicine Ohio Agricultural Research and Development Center The Ohio State Universiry Wooster OH 44691 USA USDA Helminthic Diseases Laboratory Agricultural Research Service Beltsville MD 20705 USA Accepted 29 November 1994 Abstract The contribution of cell mediated immunity to protective immunity against virulent transmissible gastroenteritis virus TGEV infection conferred by primary porcine respiratory coronavirus PRCV or TGEV exposure was assessed in pigs that were challenged with TGEV 24 days after a primary oronasal inoculation with PRCV or TGEV when 11 days old PRCV exposure induced partial protec tion against TGEV challenge in suckling pigs based upon a decreased number of diarrhea cases 42 vs 90 in age matched control pigs limited virus shedding in feces and increases in virus neutralizing serum antibody titers in contrast all 11 day old pigs inoculated with TGEV were completely protected after challenge Weaned pigs were also studied to eliminate any possibility that lactogenic immunity from contact PRCV exposed sows kontributed to protection against TGEV Once weaned none of the PRCV exposed or age matched control pigs had diarrhea after TGEV challenge moreover both groups exhibited less recta1 virus shedding than suckling pigs Vigorous lymphocyte proliferative responses 96 000 counts per minute cpm were detected in mononu clear cells prepared from mesenteric MLN and bronchial BLN lymph nodes of TGEV primed pigs Analyses of these responses indicate that virus specific cell mediated immune responses corre lated with protection against rectal and nasal virus shedding after TGEV challenge Primary inocu lation of 1 l day old pigs with PRCV induced moderate transient virus specific lymphocyte Corresponding author Current address PRISM Productions 5040 Pine Creek Drive Westerville OH 43081 USA Current address Department of Microbiology The Mucosal Immunization Research Group and The Immu nobiology Vaccine Center The University of Alabama at Birmingham Medical Center 845 Nineteenth Street South Birmingham AL 35294 2170 USA 0165 2427 95 09 50 0 1995 Elsevier Science B V All rights reserved SSDlO165 2427 94 05416 9 36 T A Brim et al Veterinary Immunology and Itnmunopathology 48 1995 35 54 proliferation 47 000 cpm in MLN from both suckling and weaned pigs after TGEV challenge Substantial BLN proliferative responses 80 000 cpm correlated with failure to detect TGEV in nasal secretions from these pigs Virus specific lymphocyte proliferation in spleens was delayed in onset and of lower magnitude than that observed in MLN and BLN Virulent TGEV exposure resulted in increased percentages of T cell subsets especially in the lamina propria and MLN mucosa associated lymphoid tissues in proximity to the primary replication site of TGEV in the small intestine Our results confirm that PRCV infection primes anti viral immune responses and thus contributes to partial immunity against virulent TGEV challenge Kqvwords Transmissible gastroenteritis virus Porcine respiratory coronavirus Cellular immunity Lymphocyte proliferation Lymphocyte subsets T lymphocytes 1 Abbreviations AX antibody secreting cells BALT bronchus associated lymphoid tissue BLN bron chial lymph nodes CCIF cell culture immunofluorescence Con A concanavalin A cpm counts per minute FCM flow cytometric analysis GALT gut associated lymphoid tissue IEM immune electron microscopy IL 2R interleukin 2 receptor mAbs monoclonal anti bodies MHC major histocompatibility complex MLN mesenteric lymph nodes PCD postchallenge day PFU plaque forming units PHA phytohemagglutinin PID postino culation day PRCV porcine respiratory coronavirus sIg surface immunoglobulin SLA swine lymphocyte antigen TcR T cell receptor TGEV transmissible gastroenteritis virus 2 Introduction Transmissible gastroenteritis virus TGEV and porcine respiratory coronavirus PRCV two antigenically related porcine coronaviruses with distinct intestinal and res piratory tissue tropisms present a unique model for the study of immunologic interactions among mucosa associated lymphoid tissues TGEV infects and destroys villous enterocytes of the small intestine leading to vomiting severe malabsorptive diarrhea dehydration and mortality approaching 100 in newborn susceptible pigs Saif and Wesley 1992 Because effective vaccines and therapy are unavailable the disease continues to negatively impact the swine industry Infection of pigs with virulent TGEV elicits protective active immunity subsequent to enteric replication of the virus stimulation of inductive sites in gut associated lymphoid tissue GALT and production of virus specific cell mediated immunity anti body secreting cells ASC and intestinal secretory IgA Saif and Wesley 1992 VanCott et al 1993 Brim et al 1994 Protective lactogenic immunity conferred to pigs suckling sows that were immunized via natural exposure or oral inoculation with virulent TGEV is theorized to result from the migration of TGEV specific IgA committed B cells from GALT to the mammary glands where they mature into IgA ASC and produce secretory IgA to TGEV in milk Saif and Wesley 1992 TGEV infects the respiratory tract Kemeny et al 1975 Furuuchi et al 1979 O Toole et al 1989 and stimulates virus specific cellular and ASC immune responses in bronchial lymph nodes BLN VanCott et al 1993 Brim T A Brim et ul Veterinary Immunology and lmmunopafhology 48 1995 35 54 37 et al 1994 however the contribution of respiratory lymphoid tissue to protective immu nity against TGEV is unclear PRCV was first identified in Europe in 1984 Brown and Cartwright 1986 Pensaert et al 1986 and later in the USA Hill et al 1990 Wesley et al 1990 as the cause of mild respiratory disease or subclinical infections without enteritis in swine Conventional sero logic tests are unable to distinguish between PRCV and TGEV although antigenic differ entiation is possible with specific monoclonal antibodies mAbs in a serum blocking ELISA Callebaut et al 1988 Garwes et al 1988 Van Nieuwstadt and Boonstra 1992 Simkins et al 1992 PRCV replicates extensively in the respiratory tract but intestinal PRCV replication is timited to a few villous subepithelial cells Pensaert et al 1986 Cox et al 1990 The mechanisms by which PRCV exposure induces a variable degree of active Van Nieuwstadt et al 1989 Wesley and Woods 1992 Cox et al 1993 and passive immunity Bernard et al 1989 Paton and Brown 1990 De Diego et al 1992 to TGEV infection are unclear A bronchus associated lymphoid tissue BALT mammary immu nologic link has been proposed to account for the appearance of TGEV neutralizing secre tory IgA in milk from PRCV exposed sows Callebaut et al 1990 Our recent studies with PRCV inoculated pigs demonstrated vigorous virus specific cell mediated and ASC primarily IgG immune responses in BLN but little stimulation of mesenteric lymph nodes MLN VanCott et al 1993 Brim et al 1994 Clarification of cellular immune responses to TGEV and PRCV is essential for the control of these infections in pigs Cell mediated immunity may play a direct role in recovery from and protection against reinfection and the terminal differentiation of IgA committed B cells into IgA ASC requires virus specific T cell help Mestecky and McGhee 1987 This article presents data that assesses the contribution of cell mediated immunity to the protec tion conferred by primary TGEV or PRCV exposure of pigs against virulent TGEV infection Changes in lymphocyte proliferative responses and in the distribution of mononuclear cell subsets after virus exposure were evaluated in MLN draining lymph nodes for GALT in BLN draining lymph nodes for BALT and the lower respiratory tract in the intestinal lamina propria an effector site in GALT and in spleens systemic lymphoid organs 3 Materials and methods 3 1 Viruses The virulent Miller strain MX of TGEV Bohl and Kumagai 1965 passaged in gnotobiotic pigs and the PRCV strain ISU 1 Hill et al 1990 passaged in swine testicle cells were titered by plaque assays in swine testicle cells before they were inoculated into pigs The low cell culture passaged Miller strain M6 of TGEV Welch et al 1988 was processed for use as viral antigen in lymphocyte proliferation assays as previously described Brim et al 1994 3 2 Experimental design Eleven day old suckling pigs from crossbred sows that were seronegative for TGEV neutralizing antibodies were inoculated oronasally with virulent TGEV M5C 17 pigs from 38 T A Brim et al Veterinary Itntnunology and Itnmunopathology 48 I 995 35 54 three litters 5 X lo5 plaque forming units PFU per pig or PRCV 16 pigs from two litters 2 X IO PFU per pig One fifth of the total inoculum dose was administered via the intranasal route Twenty three age matched uninoculated pigs from three litters served as controls Each litter was housed in a separate isolation room during the experiments each pig in these eight litters remained with its sow until euthanasia On Postinoculation Day PID 24 35day old pigs from all three groups TGEV PRCV and control were given an oronasal challenge dose of virulent TGEV 1 3 X 10 PFU per pig The rationale for the virus doses given to these pigs was detailed elsewhere VanCott et al 1994 Animals were observed for clinical signs of gastroenteritis and respiratory infection i e vomiting diarrhea nasal discharge coughing and dyspnea Nasal and rectal swab specimens were collected from each pig before virus inoculation and challenge then daily for 8 PID and 8 postchallenge days PCD Blood samples were drawn from selected pigs at the time of inoculation and challenge on various PID and PCD and from all pigs following euthanasia Pigs were euthanized on PCD 0 PID 24 2 4 8 and 12 MLN BLN spleens and segments of the small intestine were harvested for cell preparations that were tested in lymphocyte proliferation assays and flow cytometric analysis FCM Tissues from eight TGEV seronegative unexposed suckling pigs from the same herd were also available for FCM An additional ten pigs from one litter were inoculated with PRCV as above then weaned at 3 weeks of age PID 10 One litter of six age matched weaned pigs served as controls Both litters of weaned pigs were challenged with TGEV when 35 days old PID 24 pigs were euthanized on PCD 0 4 and 12 3 3 Virus neutralization test cell culture immunojhorescence CCIF and immune electron microscopy IEM Neutralizing antibody titers to TGEV and PRCV in serum samples were determined by a plaque reduction test Bohl et al 1972 Virus was detected in rectal and nasal swab samples by a CCIF test with hyperimmune porcine anti TGEV cross reactive with PRCV immunoglobulin conjugated to fluorescein isothiocyanate FITC Welch et al 1988 VanCott et al 1993 Titers were expressed as the number of fluorescing foci per milliliter of swab supernatant fluid In addition rectal swab specimens from PCD 2 and 3 were examined for TGEV particles by IEM Saif et al 1977 3 4 Flow cytometric analysis Mononuclear cells were isolated from MLN BLN spleens and the lamina propria of the small intestines and resuspended in complete culture medium as described previously VanCott et al 1993 1994 Cell viability assessed by trypan blue exclusion was 90 for cells from each tissue Aliquots of mononuclear cells were stored frozen up to 1 year in liquid nitrogen The recovery of frozen cells upon thawing was 5060 for cells from MLN BLN and spleens and 20 25 for ileal and duodenal cells Cells were incubated for 30 min at 4 C with the following mAbs specific for swine leukocyte surface antigens PT85a anti Class I monomorphic MSA3 anti Class II swine lymphocyte antigen SLA DR MSA4 anti CD2 74 12 4 anti CD4 76 2 11 anti CD8 74 22 15 anti SWC3 macrophage granulocyte 23 1 3B2 anti interleukin 2 receptor IL 2R T A Brim ef al Veterinary Immunology and Immunopathology 48 1995 35 54 39 5C9 IA1 1 anti IgM 3Dll 3H5 anti IgA and 3H2 3H7 anti IgG Paul et al 1989 Bailey et al 1992 Lunney 1993 The mAbs were undiluted hybridoma culture supernatants except for the anti Ig mAbs which were ascitic fluid diluted 1 lOO in Earl s balanced salt solution supplemented with 1 bovine serum albumin and 5 fetal bovine serum Following two washes cells were stained for 30 min at 4 C with a FITC conjugated anti mouse Ig reagent goat anti mouse IgG F ab 2 Kirkegaard GIBCO Laboratories Grand Island NY or medium alone background control Mononuclear cells from the lamina propria were also stimulated with 1 pg per well of a second T cell mitogen concanavalin A Con A Sigma Chemicals St Louis MO Doses of viral antigen PHA and Con A were optimized for the cell culture conditions specified Cell cultures were incubated for 96 h MLN BLN spleens or 72 h lamina propria at 37 C in 5 COZ then pulsed with 1 the peak mean virus titer was 2 3 X IO5 focus forming units per ml at PID 1 Fig 1 B Virus was not detected in feces from pigs given PRCV Pigs were challenged with virulent TGEV 1 3 X 10 PFU per pig at 35 days of age to determine the degree of protective immunity afforded by primary PRCV exposure Forty two percent of PRCV inoculated pigs developed diarrhea after TGEV challenge one of 16 pigs and ten of 12 pigs shed virus in feces when the samples were tested by CCIF and IEM A RECTAL VIRUS SHEDDING 10 9 10 2 10 k 100 5 02468 z 5 L 24 26 28 30 32 p 106 2 B NASAL 10 10 t O 10 101 100 0 2 4 6 8 VIRUS SHEDDING 0 2 4 6 8 24 26 28 30 32 0 2 4 6 8 Fig 1 Amount of virus in rectal A and nasal B swab samples measured by cell culture immunofluorescence Pigs were inoculated with TGEV n PRCV 0 or no virus A and samples were collected daily through PID 8 At PID 24 PCD 0 pigs from all three primary inoculum groups TGEV PRCV and no virus were challenged with TGEV samples were collected daily through PCD 8 PID 32 Data are presented as the mean fluorescing focus forming unit per ml of swab supematant fluid of 16 23 samples SEM after primary inoculation and of 6 21 samples iSEM after challenge Data from samples without virus overlap and are indistinguishable from the baseline SEM smaller than the symbols are not shown T A Brim et 01 Veterinary Immunology und Immunopathology 48 1995 35 54 41 respectively However all pigs were completely protected against nasal virus shedding Fig 1 One PRCV exposed pig euthanized at PCD 2 had villous atrophy in the jejunum and ileum but intestinal lesions were not found in any pigs that were reinoculated with TGEV R Moxley personal communication 1992 After receiving a 260 fold higher dose challenge dose of virulent TGEV than the primary dose given to 1 l day old pigs 35day old control pigs had diarrhea without vomiting they exhibited virus shedding in feces for a shorter duration 4 days and with a ten fold lower peak mean virus titer than that observed in the younger pigs Fig 1 A However about half of the 35day old TGEV challenged control pigs shed virus in nasal secretions with a peak mean virus titer that was 50 fold higher than that in the younger pigs after primary TGEV exposure Fig 1 B All primary TGEV inoculated pigs were protected against both diarrhea and virus shedding from the intestinal and respiratory tracts when challenged with virulent TGEV Fig 1 The results for IEM of rectal swab samples and virus neutralizing serum antibodies from these pigs were reported elsewhere VanCott et al 1994 Briefly when rectal swab supernatants from a portion of the challenged pigs were examined at PCD 2 and 3 for virus particles by IEM ten of 12 PRCV inoculated pigs one of five TGEV inoculated pigs and seven of nine control pigs were positive for TGEV All virus inoculated pigs seroconverted and produced neutralizing antibodies to TGEV and both the TGEV and PRCV groups had similar antibody titers at PID 24 before challenge After TGEV challenge serum antibody titers increased nearly lOO fold in the PRCV group compared with less than a two fold increase in the TGEV group The control pigs challenged with TGEV seroconverted by PCD 8 4 2 Flow cytometric analysis of immunostained cells from suckling pigs At the time of tissue harvest single cell suspensions preparations from each tissue were frozen after thawing cell subsets from TGEV challenged pigs were compared with normal age matched unexposed pigs using mAbs immunostaining in FCM analysis Details of the B cell subsets from these pigs were presented separately VanCott et al 1994 The phenotypic characterization of mononuclear cells from unexposed 1 l to 51 day old pigs by FCM Fig 2 revealed that CD2 T cells were least numerous in the ileal lamina propria 1 l 29 and that the small intestine lamina propria yielded the lowest percentages of both CD4 21 and CDS 8 T cells Figs 2 B and 2 C Percentages of CD2 35 58 and CD4 2241 cells in MLN BLN and spleens were similar Figs 2 A 2 D and 2 E Overall no association between the age of the pigs 1 l 5 1 days and variation in percentages of cell subsets was noted After TGEV challenge the percentages of CD2 and CD4 T cells in all five tissues generally increased over those found in age matched unexposed pigs Figs 3 A and 3 B but the changes in control pigs after primary TGEV inoculation were most striking in MLN and the lamina propria duodenum ileum lymphoid tissues adjacent to entero cytes of the small intestine the major replication site of TGEV CDS T cells followed the trends seen with CD2 and CD4 cells after virus exposure Fig 3 C The effect of time after virus exposure on T cell populations was assessed These analyses revealed that MLN CD4 T cells apparently increased after primary TGEV inoculation of 35 day old T A E no such trends were observed in CD4 cells from BLN data not shown In spleen cells from TGEV and PRCV primed pigs the sum of the CD4 and CD8 cells 73 and 64 respectively exceeded the percentage of the CD2 cells 56 and 58 respectively implying that a portion of the T cell population was CD4 CD8 double positive T cells Figs 3 A 3 C The presence of CD2 CD4 CD8 cells in the duodenal and ileal lamina propria T cell populations was suggested by the fact that the sum of the CD4 and CD8 cells was less than the percentage of CD2 cells from unexposed pigs and from pigs in all three TGEV challenged groups Percentages of macrophages ranged from 86 of cells in all tissues and no apparent shift in intensity of SLA Class I or Class II antigen expression in response to viral exposure was evident Fig 4 C data not shown Changes in percentages of cells expressing SLA Class II paralleled changes in B cell percentages Low level expression of IL 2R was not affected by virus exposure Fig 4 D 4 3 Virus speci c lymphocyte proliferative responses of suckling pigs Overall mononuclear cells purified from the mucosa associated lymph nodes MLN and BLN gave vigorous virus specific proliferative responses that were strongly associated with T A Brim et al Veterinary Immunology and Immunopaihology 48 1995 35 54 80 60 40 20 zil 0 80 kD8 c I DUOD ILE MLN BLN SPL m UNEXP El CONT El TGEV 0 PRCV 43 Fig 3 Flow cytometric analysis of mononuclear cells from the duodenal Iamina propria DUOD the heal lamina propria ILE MLN BLN and spleens SPL from pigs challenged with virulent TGEV or from age matched unexposed pigs UNEXP sample size n l 4 Primary virus inoculation of TGEV challenged pigs and sample size were as follows no virus CONTrol n 3 6 TGEV II 1 3 PRCV n 24 Cells were immunostained for the swine cell surface markers CD2 A CD4 B and CD8 C Data are expressed as the mean percentage positive cells after subtraction of background staining with anti Ig FITC f SEM Data not available virus replication in the intestinal and respiratory tracts Before challenge at PID 24 PCD O MLN cells from