Viremia and nasal and rectal shedding of rotavirus in gnotobiotic pigs inoculated with Wa human rotavirus

Respiratory symptoms with rotavirus shedding in nasopharyngeal secretions have been reported in children with and without gastrointestinal symptoms (Zheng et al., 1991, J. Med. Virol. 34:29-37). To investigate if attenuated and virulent human rotavirus (HRV) strains cause upper respiratory tract infections or viremia in gnotobiotic pigs, we inoculated them with attenuated or virulent HRV intranasally, intravenously, or orally or via feeding tube (gavage) and assayed virus shedding. After oral or intranasal inoculation with attenuated HRV, the pigs remained asymptomatic, but 79 to 95% shed virus nasally and 5 to 17% shed virus rectally. After inoculation by gavage, no pigs shed virus nasally or rectally, but all pigs seroconverted with antibodies to HRV. No viremia was detected through postinoculation day 10. Controls inoculated intranasally with nonreplicating rotavirus-like particles or mock inoculated did not shed virus. In contrast, 100% of pigs inoculated with virulent HRV (oral, intranasal, or gavage) developed diarrhea, shed virus nasally and rectally, and had viremia. The infectivity of sera from the viremic virulent HRV-inoculated pigs was confirmed by inoculating gnotobiotic pigs orally with pooled HRV-positive serum. Serum-inoculated pigs developed diarrhea and fecal and nasal virus shedding and seroconverted with serum and intestinal HRV antibodies. Pigs inoculated intravenously with serum or intestinal contents from the viremic virulent HRV-inoculated pigs developed diarrhea, virus shedding, and viremia, similar to the orally inoculated pigs. This study provides new evidence that virulent HRV causes transient viremia and upper respiratory tract infection in addition to gastrointestinal infection in gnotobiotic pigs, confirming previous reports of rotavirus antigenemia (Blutt et al., Lancet 362:1445-1449, 2003). Our data also suggest that intestinal infection might be initiated from the basolateral side of the epithelial cells via viremia. Additionally, virus shedding patterns indicate a different pathogenesis for attenuated versus virulent HRV.     

Diarrheal response of gnotobiotic pigs after fetal infection and neonatal challenge with homologous and heterologous human rotavirus strains.

Pigs exposed in utero to human rotavirus (HRV) strain Wa serotype 1 from 15 to 36 days prior to birth responded immunologically by modifying their clinical response to neonatal oral challenge with a pathogenic dose of homologous Wa or heterologous M serotype 3 HRV. In these cases, diarrhea was prevented in 12 of 14 pigs and greatly reduced in the other two. However, fecal virus shedding was not significantly modified, since it was detected in 12 of 14 pigs. These results suggest the existence of a closer antigenic relationship between these two different HRV serotypes which may only be expressed in an in vivo test system. Exposure of fetal pigs to HRV DS-1 serotype 2 failed to cause infection or to induce any protection when pigs were challenged at birth with HRV Wa. This model for cross-protection studies in gnotobiotic piglets offers good possibilities for the evaluation of potential HRV vaccine candidates, for the in vivo study of antigenic similarities between rotavirus serotypes, and for the understanding of protective immune responses against diarrhea and virus shedding.     

Cytokine responses in gnotobiotic pigs after infection with virulent or attenuated human rotavirus

To understand the role of cytokines during rotavirus infection, we assessed the kinetics of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) (proinflammatory), IL-12 (Th1 inducer), gamma interferon (IFN-gamma) (Th1), IL-4 and IL-10 (Th2), and transforming growth factor beta (Th3) cytokine responses by enzyme-linked immunosorbent assay in serum and intestinal contents of neonatal gnotobiotic pigs and IL-12, IFN-gamma, IL-4, and IL-10 cytokine-secreting cell (CSC) responses of mononuclear cells from ileum, spleen, and blood by ELISPOT. Pigs received the virulent Wa P1A[8]G1 strain of human rotavirus (HRV) (VirHRV), attenuated Wa HRV (AttHRV), or mock (controls). The TNF-alpha levels peaked earlier and remained elevated in serum of the VirHRV group but peaked later in the AttHRV group. In serum, IL-6 was significantly elevated at postinoculation day (PID) 1 in the VirHRV group and at PID 3 in both HRV groups. The IL-12 was detected in serum of all pigs including controls with significantly elevated peaks in both HRV-infected groups, indicating a role for IL-12 in the induction of immune responses to rotavirus infection. Only low and transient IFN-gamma responses occurred in serum and intestinal contents of the AttHRV-infected pigs, compared to significantly higher and prolonged IFN-gamma responses in the VirHRV-infected pigs. This observation coincides with the diarrhea and viremia induced by VirHRV. The number of IFN-gamma-secreting cells was significantly higher in the ileum of the VirHRV group than in that of the controls. The number of IL-4 CSCs was significantly higher in ileum of both HRV groups than in that of the controls. Significantly higher levels of IL-10 in the serum occurred early in the VirHRV group, compared to lower levels in the AttHRV group. However, the number of IL-10 CSCs was significantly higher later in ileum and spleen of the AttHRV than in the VirHRV group, suggesting a delayed initiation of a Th2 response induced by AttHRV. A significantly higher percentage of pigs had IFN-gamma and IL-10 responses in serum after VirHRV infection than after AttHRV infection or in controls. These data indicate a balanced Th1/Th2 response during rotavirus infection, with higher cytokine levels early after infection with VirHRV compared to that with AttHRV. Mapping the kinetics and patterns of cytokine responses after rotavirus infection has important implications for induction of protective immunity by HRV vaccines. Higher protection rates may be associated with more balanced Th1- and Th2-type responses, but induction of higher earlier IFN-gamma (Th1) and proinflammatory cytokines triggered by VirHRV may also play an important role in the higher intestinal immunoglobulin A responses and protection rates induced by VirHRV.     

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.     

Human communication with pigs: The behavioural response of pigs to specific human signals

A knowledge of the influence of behavioural and non-behavioural characteristics of humans on the behavioural responses of pigs to humans is required for the development of approach and handling procedures by stockpersons which achieve and maintain human—animal relationships in which the animals are not fearful of humans.Three experiments were conducted to compare the nature of several possible signals which humans may release when interacting with pigs. The nature of the signals was compared by quantifying the approach behaviour of the pig to the experimenter releasing the appropriate signals in four 3-min tests over an 8-day period. Pigs approached the experimenter significantly more (P < 0.05) when he did not approach, squatted, had bare hands or did not initiate interactions compared with when he did approach, stood erect, had gloved hands or initiated interactions with the pig, respectively.     

Evaluation of immune response to bovine rotavirus following oral and intraperitoneal inoculation in mice

With a view to use mice as an experimental model for studying immune response to bovine rotavirus (BRV), the kinetics of humoral and cellular immune responses to BRV in mice were evaluated by immunizing through intraperitoneal and oral route with UK strain of BRV. Following immunization with BRV, anti-rotavirus antibodies was developed in mice. The mean log antibody titres as measured by ELISA in mice immunized by intraperitoneal route were significantly higher than those immunized by oral route. Significant cellular immune response was observed in BRV-immunized mice on stimulation with BRV antigen, as measured by lymphocyte proliferation assay. The thymidine uptake by splenic and mesenteric lymph-node cells of intraperitoneally immunized mice on stimulation with BRV was 21328 +/- 1225 and 739 +/- 55 CPM, respectively. The splenic cells showed significantly higher stimulation (stimulation index 12.98) as compared to those of mesenteric cells (stimulation index 1.57). Foot pad inoculation test showed maximum virus-specific delayed type hypersensitivity reaction at 24 hr post-challenge following primary immunization and at 18 hr post-challenge following secondary immunization. The results indicate that BRV immunization by intraperitoneal route generates more efficient immune response in mice than by oral route and this route may be used for immune response studies involving BRV infection.     

Prophylactic use of human endotoxin-core hyperimmune gammaglobulin to prevent endotoxaemia in colostrum-deprived, gnotobiotic lambs challenged orally with Escherichia coli

Abstract The efficacy of human IgG polyclonal antibody to endotoxin-core in preventing endotoxaemia and subsequent disease was studied in colostrum-deprived gnotobiotic lambs challenged orally at about 5 h old with 10⁹ cfu Escherichia coli . Human endotoxin-core hyperimmune gammaglobulin was given intravenously to 5 lambs at 1.9 g IgG/kg bodyweight prior to challenge. Human albumin was given intravenously to 3 control lambs. Bacteraemia was observed in all lambs, but the incidence was lower ( P < 0.01) and the onset later ( P < 0.05) in gammaglobulin pre-treated lambs. These lambs showed no signs of disease, whereas clinical endotoxaemia, manifesting as watery mouth disease, was diagnosed in 2 of the 3 control lambs which were killed between 18 and 22 h after challenge. Thus, prophylactic treatment of colostrum-deprived lambs with human IgG enriched in endotoxin-core antibodies was effective in reducing the degree of bacteraemia and preventing endotoxaemia, leukopenia and clinical disease following oral challenge with E. coli .     

Functional maturation of the human antibody response to rotavirus

Infant Abs induced by viruses exhibit poor functional activity compared with those of adults. The human B cell response to rotavirus is dominated by use of the V(H)1-46 gene segment in both adults and infants, but only adult sequences are highly mutated. We investigated in detail the kinetic, structural, and functional advantage conferred by individual naturally occurring somatic mutations in rotavirus-specific human Abs encoded by the immunodominant V(H)1-46 gene segment. Adult Abs achieved enhanced binding through naturally occurring somatic mutations in the H chain CDR2 region that conferred a markedly prolonged off-rate and a desirable increase in antiviral potency. Three-dimensional cryoelectron microscopy studies of Ag-Ab complexes revealed the mechanism of viral inhibition to be the binding of high-affinity Abs at the viral RNA release pore in the double-layer particle. These structure-function studies suggest a molecular basis for the poor quality of Abs made in infancy following virus infection or immunization.     

Association of rat,pig, and fowl biotypes of lactobacilli with the stomach of gnotobiotic mice

We grouped 20 isolates of lactobacilli from the stomach of conventional rats, 21 isolates from pig stomachs, and 19 isolates from the crop of fowls according to their ability to ferment N-acetylglucosamine, dextrin, cellobiose, gum arabic, and xylan. Most of the isolates did not resemble previously describedLactobacillus species. Representative group A isolates were associated with germ-free mice. Only a rat isolate was able to colonize the keratinized squamous epithelium of the stomach of gnotobiotic mice.     

Protective immunity and antibody-secreting cell responses elicited by combined oral attenuated Wa human rotavirus and intranasal Wa 2/6-VLPs with mutant Escherichia coli heat-labile toxin in gnotobiotic pigs

Two combined rotavirus vaccination regimens were evaluated in a gnotobiotic pig model of rotavirus infection and disease and were compared to previously tested rotavirus vaccination regimens. The first (AttHRV/VLP2x) involved oral inoculation with one dose of attenuated (Att) Wa human rotavirus (HRV), followed by two intranasal (i.n.) doses of a rotavirus-like particle (2/6-VLPs) vaccine derived from Wa (VP6) and bovine RF (VP2) rotavirus strains. The 2/6-VLPs were coadministered with a mutant Escherichia coli heat-labile toxin, LT-R192G (mLT) adjuvant. For the second regimen (VLP2x/AttHRV), two i.n. doses of 2/6-VLPs+mLT were given, followed by one oral dose of attenuated Wa HRV. To compare the protective efficacy and immune responses induced by the combined vaccine regimens with individual rotavirus vaccine regimens, we included in the experiments the following vaccine groups: one oral dose of attenuated Wa HRV (AttHRV1x and Mock2x/AttHRV, respectively), three oral doses of attenuated Wa HRV (AttHRV3x), three i.n. doses of 2/6-VLPs plus mLT (VLP3x), three i.n. doses of purified double-layered inactivated Wa HRV plus mLT (InactHRV3x), mLT alone, and mock-inoculated pigs. The isotype, magnitude, and tissue distribution of antibody-secreting cells (ASCs) in the intestinal and systemic lymphoid tissues were evaluated using an enzyme-linked immunospot assay. The AttHRV/VLP2x regimen stimulated the highest mean numbers of intestinal immunoglobulin A (IgA) ASCs prechallenge among all vaccine groups. This regimen induced partial protection against virus shedding (58%) and diarrhea (44%) upon challenge of pigs with virulent Wa HRV. The reverse VLP2x/AttHRV regimen was less efficacious than the AttHRV/VLP2x regimen in inducing IgA ASC responses and protection against diarrhea (25% protection rate) but was more efficacious than VLP3x or InactHRV3x (no protection). In conclusion, the AttHRV/VLP2x vaccination regimen stimulated the strongest B-cell responses in the intestinal mucosal immune system at challenge and conferred a moderately high protection rate against rotavirus disease, indicating that priming of the mucosal inductive site at the portal of natural infection with a replicating vaccine, followed by boosting with a nonreplicating vaccine at a second mucosal inductive site, may be a highly effective approach to stimulate the mucosal immune system and induce protective immunity against various mucosal pathogens.     

The response of young Romney lambs to immunization with Trichostrongylus colubriformis larvae

1988. The response of young Romney lambs to immunization with Trichostrongylus colubriformis larvae. International Journal for Parasitology 18: 1035–1038. Groups of weaned Romney ewe lambs were immunized with two doses of 28,000, 35,000 or 42,000 (2000 kg⁻¹) infective larvae of Trichostrongylus colubriformis at 8, 12 or 16 weeks of age, respectively. Each group and helminthfree control lambs of similar age were challenged with T. colubriformis at the same dose rate as used for immunization. Faecal egg counts (FEC) and haematological observations were made during the experiment, and at slaughter, 42 days after challenge, worm burdens were determined and small intestinal histology was examined.

Lambs in each immunized group were identified as ‘responders’ or ‘non-responders’ on the basis of both FEC and worm burdens. A significant (P<0.001) decrease in the worm burdens recovered as a proportion of the challenge infections in both unimmunized and immunized lambs with increasing age was observed.

Globule leukocyte numbers increased with the age of lambs. In addition, within each age group globule leukocyte numbers reflected individual responsiveness to immunization, significantly (P<0.01) greater numbers being present in ‘responders’ than ‘non-responders’ or unimmunized lambs. No difference in haematological responses were found in relation to the lambs' responsiveness to immunization.     

Some properties of piglet,calf, pig and bovine conglutinin

In sera of newborn piglets which were prevented from sucking maternal colostrum a low titre of conglutinin activity was demonstrated. For comparison of properties of this piglet conglutinin sera derived from adult pigs, cows and calves were used. Conglutinin from precolostral piglet serum behaves in different way as compared with immunoconglutinin from adult pig following reduction with 2-mercaptoethanol and in reaction with EDTA and thus resembled bovine natural conglutinin. In density gradient ultracentrifugation and inhibition reaction with zymosan andn-acetyl-d-glucosamine reacts piglet conglutinin as pig immunoconglutinin. Conglutinin present in precolostral calf serum behaves like a typical natural conglutinin of adult cattle.     

A behavioural comparison of gnotobiotic with normal neonate pigs,indicating stress in the former

The environmental conditions for gnotobiotic pigs differ in many ways from those of normally reared animals. This was reflected in observed differences of behaviour, particularly utterances, whose variations in frequency and type suggest that gnotobiotic pigs are subjected to greater stress. It is argued that this could be a cause of mortality.     

Differential response in growth and mycorrhizal colonisation of soybean to inoculation with two isolates ofGlomus clarum in soils of different P availability

Soybean (Glycine max cv. Mikiwashima) seedlings were inoculated with two tropical isolates of the vesicular-arbuscular mycorrhizal fungusGlomus clarum (Gc); isolateGcA was isolated from soils of low phosphate (P) and isolateGcB from soils of high P availability. In soil with low P,GcA was more efficient in increasing growth, nodulation and nitrogenase activity ofG. max thanGcB. Upon contact with the root surface, pre-infection hyphae ofGcA penetrated the root directly and rapidly colonised the cortical cells, while those ofGcB grew extensively on theroot surface with little host penetration. Mycorrhizal colonisation was higher in roots inoculated withGcA. Dual inoculation with the two isolates did not increase the effect of the single inoculation withGcA. In soils of high P status, both isolates formed pre-infection hyphae with few entry ooints and percentage mycorrhizal root colonisation was consequently low. The variation in efficacy of the isolates emphasizes the significance of evaluating host specificity when selecting efficient VA mycorrhizae strains for field studies.     

Pathogenesis and immune responses in gnotobiotic calves after infection with the genogroup II.4-HS66 strain of human norovirus

We previously characterized the pathogenesis of two host-specific bovine enteric caliciviruses (BEC), the GIII.2 norovirus (NoV) strain CV186-OH and the phylogenetically unassigned NB strain, in gnotobiotic (Gn) calves. In this study we evaluated the Gn calf as an alternative animal model to study the pathogenesis and host immune responses to the human norovirus (HuNoV) strain GII.4-HS66. The HuNoV HS66 strain caused diarrhea (five/five calves) and intestinal lesions (one/two calves tested) in the proximal small intestine (duodenum and jejunum) of Gn calves, with lesions similar to, but less severe than, those described for the Newbury agent 2 (NA-2) and NB BEC. Viral capsid antigen was also detected in the jejunum of the proximal small intestine of one of two calves tested by immunohistochemistry. All inoculated calves shed virus in feces (five/five calves), and one/five had viremia. Antibodies and cytokine (proinflammatory, tumor necrosis factor alpha [TNF-α]; Th1, interleukin-12 [IL-12] and gamma interferon [IFN-γ]; Th2, IL-4; Th2/T-regulatory, IL-10) profiles were determined in serum, feces, and intestinal contents (IC) of the HuNoV-HS66-inoculated calves (n = 5) and controls (n = 4) by enzyme-linked immunosorbent assay in the acute (postinoculation day 3 [PID 3]) and convalescent (PID 28) stages of infection. The HuNoV-HS66-specific antibody and cytokine-secreting cells (CSCs) were quantitated by ELISPOT in mononuclear cells of local and systemic tissues at PID 28. Sixty-seven percent of the HuNoV-HS66-inoculated calves seroconverted, and 100% coproconverted with immunoglobulin A (IgA) and/or IgG antibodies to HuNoV-HS66, at low titers. The highest numbers of antibody-secreting cells (ASC), both IgA and IgG, were detected locally in intestine, but systemic IgA and IgG ASC responses also occurred in the HuNoV-HS66-inoculated calves. In serum, HuNoV-HS66 induced higher peaks of TNF-α and IFN-γ at PIDs 2, 7, and 10; of IL-4 and IL-10 at PID 4; and of IL-12 at PIDs 7 and 10, compared to controls. In feces, cytokines increased earlier (PID 1) than in serum and TNF-α and IL-10 were elevated acutely in the IC of the HS66-inoculated calves. Compared to controls, at PID 28 higher numbers of IFN-γ and TNF-α CSCs were detected in mesenteric lymph nodes (MLN) or spleen and Th2 (IL-4) CSCs were elevated in intestine; IL-10 CSCs were highest in spleen. Our study provides new data confirming HuNoV-HS66 replication and enteropathogenicity in Gn calves and reveals important and comprehensive aspects of the host's local (intestine and MLN) and systemic (spleen and blood) immune responses to HuNoV-HS66.     

Rice response to inoculation with N2-fixing and P-solubilizing microorganisms

Summary Inoculation effect ofA. chroococcum, P. striata andA. awamorii on yield and nutrients uptake in rice was studied under green house conditions. The organisms appreciably increased the yield and uptake of nutrients with or without chemical fertilizers. Phosphorussolubilizing microorganisms and a mixture of the three showed better response than the rest of the treatments among single and mixed culture inoculations respectively. Chemical fertilizers further improved the yield and nutrients uptake. The yield response remained unaffected by replacing superphosphate with rock phosphate and microbial inoculations.