key: cord-0755276-efyfvrkl
authors: Saif, L. J.
title: Development of nasal, fecal and serum isotype-specific antibodies in calves challenged with bovine coronavirus or rotavirus
date: 1987-12-31
journal: Veterinary Immunology and Immunopathology
DOI: 10.1016/0165-2427(87)90159-0
sha: 9e6e2f114c42061a63519a9fe684088dbfd9e146
doc_id: 755276
cord_uid: efyfvrkl

Abstract Unsuckled specific pathogen free calves were inoculated at 3–4 weeks of age, either intranasally (IN) or orally (O) with bovine coronavirus or O plus IN (O/IN) or O with bovine rotavirus. Shedding of virus in nasal or fecal samples, and virus-infected nasal epithelial cells were detected using immunofluorescent staining (IF), ELISA or immune electron microscopy (IEM). Isotype-specific antibody titers in sera, nasal and fecal samples were determined by ELISA. Calves inoculated with coronavirus shed virus in feces and virus was detected in nasal epithelial cells. Nasal shedding persisted longer in IN-inoculated calves than in O-inoculated calves and longer than fecal shedding in both IN and O-inoculated calves. Diarrhea occurred in all calves, but there were no signs of respiratory disease. Calves inoculated with rotavirus had similar patterns of diarrhea and fecal shedding, but generally of shorter duration than in coronavirus-inoculated calves. No nasal shedding of rotavirus was detected. Peak IgM antibody responess, in most calves, were detected in fecal and nasal speciments at 7–10 days post-exposure (DPE), preceeding peak IgA responses which occurred at 10–14 DPE. The nasal antibody responses occurred in all virus-inoculated calves even in the absence of nasal shedding of virus in rotavirus-inoculated calves. Calves inoculated with coronavirus had higher titers of IgM and IgA antibodies in fecal and nasal samples than rotavirus-inoculated calves. In most inoculated calves, maximal titers of IgM or IgA antibodies correlated with the cessation of fecal or nasal virus shedding. A similar sequence of appearance of IgM and IgA antibodies occurred in serum, but IgA antibodies persisted for a shorter period than in fecal or nasal samples. Serum IgG1 antibody responses generally preceeded IgG2 responses and were predominant in most calves after 14–21 DPE.

The tropism of bovine coronavirus and rotavirus for intestinal epithelial cells is well-established, as is the viruses' ability to cause villous atrophy and malabsorptive diarrhea in young calves (Mebus et al., 1973; Doughri and Storz, 1977; Mebus and Newman, 1977; Bridger et al., 1978) . Recent reports suggest bovine coronavirus also possesses a tropism for respiratory epithelial cells (Thomas et al., 1982; McNulty et al., 1984; Reynolds et al., 1983; Saif et al., 1986) , a characteristic shared by certain coronaviruses infecting other species (Mclntosh et al., 1974; Kemeny et al., 1975) . Bovine coronaviruses have been isolated from respiratory tissues of calves with respiratory disease in the United Kingdom (Thomas et al., 1982) . However experimental intranasal (IN) challenge of calves with bovine coronaviruses generally produced only mild or no signs of respiratory disease, even though all calves developed upper respiratory tract infections and diarrhea (McNulty eta|., 1984; Reynolds et al., 1985; Saif et al., 1986) . Although several authors have reported respiratory symptoms preceeding or concurrent with rotavirus infections in children (Tallett et al., 1977; Hieber et al., 1978; Lewis et al., 1979) there is no evidence that rotaviruses multiply in human or porcine respiratory tissues either in vivo (Theil et al., 1978; Lewis et al., 1979; Stals et al., 1984) or in vitro (Tallett et al., 1977) . Studies of such localized infections of either respiratory and intestinal tissues (bovine coronavirus) or intestinal tissues alone (rotavirus) should provide comparative information on development of active immune responses at both the local mucosal site of viral replication and a distant mucosal site. Both rotaviruses and coronaviruses cause localized mucosal infections and there is no evidence for hematogenous spread of these viruses to a distant mucosal site (Mebus et al., 1973; Doughri and Storz, 1977; Mebus and Newmann, 1977) . (Saif et al., 1983; Sail et al., 1986) . Calves were fed 1% normal bovine colostrum in Similac (commercial infant formula) 2X/day from birth through 5 days of age, and Similac alone thereafter.

Nasal and rectal swabs or fecal samples were collected daily through 14 days post-exposure (DPE) and placed in cell culture medium (2 swabs in 8 ml) (Saif, 1986) . Nasal epithelial cells were collected from the nasal swab samples, processed and stained for IF using fluorescein-conjugated bovine anticoronavirus or anti-rotavirus sera as described previously (Saif et al., 1986) .

The processed nasal swab fluid was tested for rotavirus or coronavirus using a cell culture immunofluorescence (CCIF) test (Saif et al., 1986; Bohl et al., 1982) . Feces or rectal swab samples were processed as described previously and tested for presence of rotavirus using CCIF and ELISA assays (Bohl et al., 1982; Saif et al., 1986) , or for coronavirus using immune electron microscopy on 10% fecal suspensions (IEM, Saif et al., 1977) .

Antibody assays A plaque reduction virus neutralization (VN) test was used to assay rotavirus and coronavirus antibody titers (80% neutralization end points) in serum, and in processed fecal and nasal samples as described previously for bovine rotavirus . The VN antibody titers to bovine coronavirus were assayed in Madin Darby bovine kidney (MDBK) cells using plaque-purified NCDV bovine coronavirus (L.J. Saif and T. Mohamed, unpublished) . IgG 1, IgG2, IgA and IgM antibodies to each virus were determined in sera, and in processed fecal and nasal samples using isotype-specific ELISA.

Monoclonal antibodies to each bovine isotype (except IgM) were used in ELISA to validate the specificity and sensitivity of the absorbed polyclonal anti-bovine Ig heavy chain-specific sera used (Saif et al., 1983; .

Nasal and fecal sheddin 9 of virus and clinical signs of disease Information on the onset and duration of nasal and fecal shedding of coronavirus and rotavirus, and diarrhea is summarized in Table 1 rotavirus showed similar patterns of fecal shedding and diarrhea, the latter persisting for a shorter period than in coronavirus-inoculated calves. 

For further details see Figure 1 legend. (Figures 1 & 2) . In the latter calves, & 4) .

Serum isotype-specific antibody responses in calves in each of the 4 groups were similar (Figures 5 & 6) . IgM antibodies were detected at peak levels in all calves by 7 DPE after which they declined to low levels. IgA antibodies, first appeared at 14 DPE in all but 2 calves (Groups B and C), reached undetectable levels by 21-35 DPE in all calves.

Low levels of passive IgG 1 antibodies in serum at the time of challenge, due to the initial colostrum feeding, were detected in 6/8 calves. Activelyproduced IgG 1 antibodies were first detected in all calves at 14 DPE, whereas

IgG 2 antibodies were first detected in 2/8 calves at 14 DPE, and the remaining calves (except #181, Figure 6 ) at 21DPE. IgG 1 antibodies predominated in all but 1 calf (in which IgM predominated) by the end of the sampling period. The VN antibody responses closely paralleled IgG 1 responses, but were detected earlier (7 DPE), and were of lower titers than IgG I antibodies except at 7 DPE.

The patterns of diarrhea and fecal shedding of rotavirus and coronavirus observed in this study were similar to those reported in other studies of SPF or gnotobiotic calves (Sail et al., 1983; McNulty et al., 1984; Reynolds et al., 1985: Sail and Smith, 1985; Saif et al., 1986; Van Zaane et al., 1986) .

As noted in previous studies nasal shedding of coronavirus was detected in both 0 and IN-inoculated calves. However, no respiratory shedding of rotavirus was evident in the present study in calves or in prior studies in children (Lewis et al., 1979; Stals et al., 1984) .

The appearance of isotype-specific antibodies in feces and serum following intestinal infection of calves with rotavirus were similar to those reported in our previous studies (Saif and Smith, 1984; Saif et al., 1986) and one other report (Van Zaane et al., 1986) in which younger calves were used.

Predominance of IgA or IgM antibodies in calf feces correlates with the predominance of IgA rotavirus antibody-producing cells and IgM and IgA containing cells in the intestinal mucosa of rotavirus-inoculated or normal caives, respectively (Allen and Porter, 1975; Vonderfecht and Osburn, 1982) .

Patterns of serum and fecal antibody responses in calves were similar to those noted following infection of children (Sonza, 1980; Riepenhoff-Talty et al., 1981; Stals et al., 1984) , pigs (Corthier and Vannier, 1983 ) and mice (Sheridan et al., 1983) with rotavirus. Rotavirus antibody isotype-specific responses detected using ELISA, and VN tests on sera from calves #148 (upper panel) and #151 (lower panel) inoculated with coronavirus.

children (Riepenhoff-Talty et al., 1981) . Failure to detect fecal IgG 1 (except for 1 calf) and IgG 2 antibody responses, agrees with the results of several previous studies of rotavirus infections in calves (Saif and Smith, 1985; Saif et al., 1986; Van Zaane et al., 1986) . Reasons for occurrence of fecal IgG 1 compared with calves given rotavirus which was not shown to replicate in the upper respiratory tract, either in the present study or others (Tallett et al., 1977; Heiber et al., 1978; Lewis et al., 1979; Stals et al., 1984) . The major differences seen were in the higher magnitude of the nasal IgM and IgA antibody responses in the coronavirus-inoculated calves. In a similar study in children with rotavirus diarrhea, rotavirus IgA antibodies were detected in pharyngeal secretions at levels comparable to those in feces, even in the absence of detectable rotavirus shedding from pharyngeal secretions (Stals et al., 1984) . protection (Lehner et al., 1980) , perhaps due to a paucity of IgA plasma cells in tonsils and salivary glands (Crabbe and Heremans, 1967; Cripps and Lascelles, 1976) . A second possible explanation for occurrence of antibody responses of a similar magnitude and onset at a distant mucosal site (respiratory tract) after oral stimulation with rotavirus may be the existence of the common mucosal immune system (Bienenstock and Befus, 1980) . An ir~unologic link between the intestinal and respiratory tracts has been shown previously (reviewed in Bienenstock and Befus, 1980; Husband, 1985) with either intestinally-derived IgA immunocytes, or possibly intestinal IgA antibodies secreted in serum, transported to the respiratory tract. Either mechanism could account for the predominance of IgA antibodies in nasal secretions of rotavirus-infected calves which failed to show nasal virus shedding. However the parallel rise seen in fecal and nasal IgA antibodies, but their delayed detected in serum, suggests the former explanation may be more likely. In addition, in ruminants the bulk of the IgA in respiratory secretions is locally produced and not serum-derived . Although in this study we cannot exclude the possibility that rotavirus replicated in other tissues of the respiratory tract such as trachea or lung, this has not been observed in other species (Tallett et al., 1977; Hieber et al., 1977; Theil et al., 1978; Lewis et al., 1979; Stals et al., 1984) and absence of nasal shedding of virus under such conditions would be unlikely.

IgA antibodies in nasal secretions could play a role in protection against enteric viruses by contributing additional antibodies to saliva thereby neutralizing virus at the portal of entry.

Others have reported that oral inoculation of rabbits, mice or man with respiratory viruses also led to IgA responses in nasal secretions (Peri et al., 1982; Waldman et al., 1983) .

Studies in sheep have shown that the numbers of IgA antibody containing cells in the upper respiratory tract could be enhanced following intratracheal boosting of intraperitoneally primed animals .

Consequently the oral route may provide an effective route of immunization for stimulation of immunity against viruses which infect either the respiratory or intestinal tracts or both.

In an earlier report (Howard et al., 1986) , replication of M~coplasma bovis in the bovine respiratory tract elicited IgM and IgA responses in tracheobronchial washings, similar to responses in nasal secretions in the present study. However, these investigators also observed an IgG1, and IgG 2 antibody response in such samples by 4 weeks postinoculation. No IgG I or IgG 2 nasal antibody responses were detected in virus-infected calves in the present study.

These differences may relate to the invasiveness of M. bovis which causes extensive lung lesions not evident in coronavirus infections of the respiratory tract (McNulty et al., 1984; Reynolds et al., 1985; Howard et al., 1986; Saif et al., 1986) . Such invasive lower respiratory tract infections might evoke IgG responses since IgG plasma cells predominate in the lower respiratory tract (compared with IgA cells in the upper tract, Morgan et al., 1981) .

Alternatively IgG antibodies may be partially serum-derived in response to the inflammation and damage produced by invasive infections of respiratory tissues.

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