Gnotobiotic Lambs Research Articles

Detection of Rotavirus G3P[22]Genotype in Gnotobiotic Rabbit Model

Detection of Rotavirus G3P[22]Genotype in Gnotobiotic Rabbit Model Rotaviruses have been recognized as important agents of acute diarrhea in both humans and animals. The use of animal models, including the gnotobiotic rabbit, mouse, piglets, lambs and calves models, has been essential to the understanding of rotavirus infection, pathology, disease, immunity, and testing of vaccine prototypes. The aim of this study was to describe the first detection of rotavirus G3P[22] in rabbits in Brazil and to demonstrate the importance of monitoring gnotobiotic animals used in vaccine clinical tests.

Development of a RT-qPCR method for the quantification of Fibrobacter succinogenes S85 glycoside hydrolase transcripts in the rumen content of gnotobiotic and conventional sheep

An improved RNA isolation method based on the acid guanidinium–phenol–chloroform (AGPC) procedure using saline precipitation but no column purification was evaluated for quantifying microbial gene expression using reverse transcription quantitative PCR (RT-qPCR) in rumen contents. The method provided good RNA integrity and quantity extracts. The transcript levels of eight glycoside hydrolase (GH) genes of the major rumen fibrolytic bacterium Fibrobacter succinogenes were quantified in the complex microbiota of a conventional sheep and in a gnotobiotic lamb harboring a microflora containing F. succinogenes S85 as the sole cellulolytic microorganism. This study validated the improved RNA isolation method, RT-qPCR conditions to quantify GH transcripts using either the F. succinogenes S85 tuf gene or the 16S rRNA-encoding gene ( rrs) as the reference gene, and demonstrated the need to work with good quality RNAs. Transcripts from all the selected genes cel3, endA FS , celF and endB endoglucanase genes, cedA cellodextrinase gene, mlg lichenase gene, and xynC and xynD xylanase genes of F. succinogenes S85 were detected and quantified at varying levels in the rumen content of the two animal models. This study opens new perspectives in studying microbial gene expression in the rumen of both conventional and gnotobiotic sheep.

Establishment and Development of Ruminal Hydrogenotrophs in Methanogen-Free Lambs

The aim of this work was to determine whether reductive acetogenesis can provide an alternative to methanogenesis in the rumen. Gnotobiotic lambs were inoculated with a functional rumen microbiota lacking methanogens and reared to maturity on a fibrous diet. Lambs with a methanogen-free rumen grew well, and the feed intake and ruminal volatile fatty acid concentrations for lambs lacking ruminal methanogens were lower but not markedly dissimilar from those for conventional lambs reared on the same diet. A high population density (10(7) to 10(8) cells g(-1)) of ruminal acetogens slowly developed in methanogen-free lambs. Sulfate- and fumarate-reducing bacteria were present, but their population densities were highly variable. In methanogen-free lambs, the hydrogen capture from fermentation was low (28 to 46%) in comparison with that in lambs containing ruminal methanogens (>90%). Reductive acetogenesis was not a significant part of ruminal fermentation in conventional lambs but contributed 21 to 25% to the fermentation in methanogen-free meroxenic animals. Ruminal H(2) utilization was lower in lambs lacking ruminal methanogens, but when a methanogen-free lamb was inoculated with a methanogen, the ruminal H(2) utilization was similar to that in conventional lambs. H(2) utilization in lambs containing a normal ruminal microflora was age dependent and increased with the animal age. The animal age effect was less marked in lambs lacking ruminal methanogens. Addition of fumarate to rumen contents from methanogen-free lambs increased H(2) utilization. These findings provide the first evidence from animal studies that reductive acetogens can sustain a functional rumen and replace methanogens as a sink for H(2) in the rumen.

Developments in microbiological risk assessment for drinking water.

1. SUMMARYThis paper considers the development of microbiologicalrisk assessment models for pathogenic agents in drinkingwater with particular reference to Cryptosporidium parvum,rotavirus and bovine spongiform encephalopathy (BSE).The available evidence suggests that there is potential forconsiderable variation in exposures to C. parvum oocyststhrough drinking water, during both outbreak and non-outbreak conditions. This spatial/temporal heterogeneityarises both from variation in oocyst densities in the rawwater and fluctuations in the removal efficiencies of drinkingwater treatment. In terms of risk prediction, modelling thevariation in doses ingested by individual drinking waterconsumers is not important if the dose–response curve islinear and the oocysts act independently during infection.Indeed, the total pathogen loading on the population asrepresented by the arithmetic mean exposure is sufficient forrisk prediction for C. parvum, BSE and other agents of lowinfectivity, providing the infecting particles (i.e. oocysts orBSE prions) are known to act independently. However, formore highly infectious agents, such as rotavirus, ignoring1. Summary, 1912. Introduction, 1922.1 Overview of the risk assessment approach, 1923. Pathogen exposures through drinking water, 1923.1 Pathogen exposures under non-outbreak condi-tions, 1933.1.1 Variation in micro-organism counts withinlarge volume samples, 1933.1.2 Effect of treatment on the spatial distribu-tion of micro-organisms, 1933.1.3 Implication for Cryptosporidium exposures todrinking water consumers under non-out-break conditions, 1953.2 A model for Cryptosporidium oocyst concentrationsin drinking water during an outbreak, 1964. Dose–response curves. Estimating the risk to humansfrom ingesting low pathogen doses, 1974.1 Cryptosporidium parvum, 1974.1.1 Experiments with salmonellas in mice suggestthat pathogens act independently and do notco-operate during infection, 1984.1.2 Acquired protective immunity for Cryptospo-ridium parvum, 1994.1.3 Virulence of different strains of Cryptospori-dium parvum, 1994.2 Human rotavirus, 1994.3 Bovine spongiform encephalopathy, 2004.3.1 The human oral ID

Effect of oral rotavirus/iscom vaccines on immune responses in gnotobiotic lambs

A comparison of the effect on the immune responses in gnotobiotic lambs was made between an iscom vaccine prepared from recombinant rotavirus VP6 protein, an inactivated rotavirus/iscom-matrix vaccine and a vaccine comprising inactivated rotavirus alone. All three vaccines induced immunological priming and some degree of protection was observed after a single oral dose. However, different immune responses were induced in response to a virulent infection. The group vaccinated with the rotavirus/iscom-matrix vaccine showed a Th2-like response characterised by rotavirus-specific antibodies and a down-regulation of IFNγ in jejunal Peyer’s patches. Both Th1-like and Th2-like immune responses were induced in the group receiving the VP6 vaccine as seen by significantly increased expressions of IFNγ and IL-6 in the jejunal Peyer’s patch together with an increased percentage of CD8 + T cells in the intestine and rotavirus-specific antibodies at mucosal surfaces. Iscom vaccines given orally have the ability to induce both Th1-like and Th2-like immune responses in a ruminant model.

Expression of the E2 envelope glycoprotein of bovine viral diarrhoea virus (BVDV) elicits virus-type specific neutralising antibodies

A region of genome from the NADL strain of BVDV corresponding to the coding sequence for the E2 glycoprotein has been molecularly cloned using RT-PCR. The viral cDNA sequence was used to construct vaccinia virus recombinants that expressed either the entire E2 coding sequence or fragments of it. These recombinants were used to immunise mice of three H-2 haplotypes to investigate their ability to elicit a neutralising antibody response against BVDV. Sera from mice immunised with the recombinant expressing full length E2 contained high levels of virus neutralising antibodies that in addition to giving neutralisation of the homologous NADL strain were also able to neutralise the Oregon C24V reference strain. These sera failed to give any neutralisation of the Osloss reference strain providing evidence for the division of BVDV isolates into at least two distinct E2 serotypes. These results were confirmed in gnotobiotic lambs. Expression of E2 fragments revealed the presence of at least two distinct neutralising epitopes, one of which was localised within the carboxy terminal 90 amino acids of the protein.

Characterisation of the primary local and systemic immune response in gnotobiotic lambs against rotavirus infection

This study characterised the primary immune response in gnotobiotic lambs after infection with a lamb rotavirus (RV). Lambs were infected and killed over a 7 week period together with controls. RV-ELISA and neutralising antibodies were determined in serum, nasal secretions, and intestinal scrapings. RV-antibody secreting cells (ASC) were enumerated in blood. Lymphocyte proliferations were determined in blood and gut-associated lymphoid tissues and cytokine expression was analysed in jejunal Peyer's patches (JPPs) and mesenteric lymph nodes (MLNs). Infected lambs cleared the virus by 8–9 days after infection without showing any clinical signs. The first indication of a specific immune response to RV was an increased expression of IL-4 mRNA in the JPPs in the infected group compared to the control group 3 days after infection. Rotavirus-specific IgA ASC in blood and IgA antibodies in serum and nasal secretions were detected from 7 days after infection followed at 10 days after infection by RV-specific IgG ASC and antibodies. Rotavirus-specific IgA antibodies were not detected in intestinal scrapings in the first 10 days after infection, but were detected by 52 days after infection. No RV-specific neutralising antibodies were seen in the intestine during the course of the experiment.

Inhibitory effects of Lactobacillus casei upon the adhesion of enterotoxigenic Escherichia coli K99 to the intestinal mucosa in gnotobiotic lambs

Observations were carried out of the interactions between Lactobacillus casei 294/89 and enterotoxigenic Escherichia coli CCM 612 (O101:K99) in vivo. In gnotobiotic lambs, inoculation with enterotoxigenic E. coli (ETEC) resulted in diarrhea with a typical clinical picture and patho-anatomical findings. E. coli adhered to the mucosa of the digestive tract at counts amounting to 10 5 per cm 2. In these lambs, disturbances of intestinal biochemical processes became evident; proteolytic enzyme activity was significantly reduced. Preventive administration of Lactobacillus casei inhibited the negative effects of ETEC in gnotobiotic lambs, minimized the clinical signs to those of a very moderate diarrhea in the first 12 h after inoculation and significantly reduced the patho-anatomical findings. Enterotoxigenic E. coli counts decreased by 99.1 and 76% on days 2 and 4 after inoculation respectively, and amounted to 10 3 per cm 2. The inhibitory effects of L. casei against E. coli were most obvious in the jejunum and ileum. The numbers of adhering E. coli increased from the duodenum with the length of the gut. ETEC counts in the digestive tract of lambs that had been preventively treated with L. casei amounted to 10 7 ml −1. It can be assumed that, in addition to competitive exclusion, the inhibitory effect of L. casei upon ETEC adherence was also mediated by a Lactobacillus-produced substance that inhibited E. coli adhesion to the gut mucosa.

Intestinal cellular immunity after primary rotavirus infection

Infection of neonatal gnotobiotic lambs with a bovine strain of rotavirus was used to characterize the kinetics of the primary cellular intestinal immune response to this agent. At 2-3 days after infection virus was first detected in the faeces and increased numbers of CD45R+ cells were observed in peripheral blood. These cells persisted in significantly increased numbers in the circulation until 7-8 days after infection. At this time, virus was no longer detectable in the faeces. The increase in CD45R+ cells preceded the appearance of virus-neutralizing antibodies in the serum at 1 week after infection. Maximal antibody titres were reached 2 weeks after infection. Virus-primed cells were first observed 1 week after infection in the jejunal and ileal Peyer's patches, mesenteric lymph nodes and peripheral blood, and persisted in the mesenteric lymph nodes and jejunal Peyer's patches for a further 4 weeks. Analysis of lymphocyte surface antigens indicated that different sub-populations of lymphocytes were responding in the various lymphoid tissues; a majority of CD4+ cells was observed in the mesenteric lymph nodes, whereas B cells predominated in the ileal Peyer's patches.

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

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 10 9 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.

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

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 10(9) 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.

Health effects associated with wastewater treatment, disposal, and reuse

and transfer of fecal indicator organisms (coliforms, enterococci, sulphite-reducing clostridia, and coliphages) downstream of a wastewater treatment plant were investigated at Lake Kallavesi, Finland (R?jala and Heinonen-Tanski, 1998). The observation of indicator organisms up to 18 km from the discharge point and good survival in the summer suggested that direct use of lake water could constitute a health risk. The effect of combined sewer overflows (CSOs) on the occur rence of Giardia and Cryptosporidium was investigated (Gibson et al., 1998). The results suggested that CSOs significantly contribute to the load of Giardia and Cryptosporidium in ambient waters and source waters for drinking and recreational uses. An outbreak of waterborne giardiasis in Temagami, Ontario, in 1994 was assessed (Wallis et al., 1998). The outbreak was traced to leakage from the municipal wastewater system into the lake and exacerbated by spring snow-melt runoff. The influence of rainwater on the con centration of helminth eggs in wastewater was investigated (Capizzi and Schwartzbrod, 1998). Concentrations of parasite eggs vary according to water flow rates and sedimentation; heavy rain following rainless days yielded higher concentrations in raw wastewater at the treatment plant, whereas continuous moderate rain resulted in low variation of parasite eggs. The prevalence of respiratory symptoms, skin disorders, status of spirometric lung functions, and amino acid profiles among wastewater workers were reported (Bener et al., 1998). Wastewa ter workers had a higher prevalence of respiratory symptoms than controls, significant for chronic cough (p < 0.02), chronic phlegm (p < 0.03), chronic bronchitis (p < 0.02), asthma (p < 0.02), dyspneoa (p < 0.001), and nasal catarrh (p < 0.001) and acute symptoms of pruritus (p < 0.003), tinea (p < 0.004), dermatitis (p < 0.001), and nose irritation (p < 0.005). Spirometry tests scored lower in wastewater workers, and most of the plasma amino acids concentrations were elevated among wastewater workers. An investigation examining whether inadequate septic system construction may cause contamination of wells with coliform bacteria or nitrates was reported (Tuthill et al., 1998). The authors found that coliform bacteria and nitrate contamination were neg atively correlated with lot size, and coliform bacteria levels were negatively correlated with casing length, which suggested that septic systems may be a source of coliform bacteria and nitrate contamination in wells. CONTAMINATION OF WATER SUPPLIES

Infective Dose Size Studies on Cryptosporidium parvum Using Gnotobiotic Lambs

Natural waterbourne transmission of cryptosporidiosis was studied by a simulation using low-level accxamulative dosing. Gnotobiotic lambs were fed on a diet artificially contaminated to a predetermined level with Cryptosporidium parvum oocysts and the faecal output of cysts was measured. Results indicate a minimum infectious dose as low as one oocysts.

Effect of anaerobic fungi on the ruminal proteolysis in gnotobiotic lambs

The establishment of a fungal population composed of the main species usually found in ruminants in the rumen of gnotobiotic lambs did not significantly alter the in sacco digestibility of meat meal and soybean cake. The proteolytic activity of the rumen fluid against 14C-casein was not affected by the fungi. Therefore, these microorganisms probably do not play an important role in the degradation of proteins in the rumen.

Role of aerobactin in systemic spread of an opportunistic strain of Escherichia coli from the intestinal tract of gnotobiotic lambs

To assess the role of the aerobactin-related system in the virulence of bovine opportunistic Escherichia coli, and to determine the stage(s) of the overall infectious process at which it is acting, germfree lambs were mixedly infected orally with two derivative strains of this bacterium differing in their ability (Iut+) or inability (Iut-) to express a functional aerobactin-mediated iron transport system. The Iut- strain was compared with the Iut+ strain for colonization of the gut, translocation to the mesenteric lymph nodes (MLN), and spread to other organs and to the body fluids of diassociated lambs. The Iut- mutant was found in smaller numbers in the duodenum, suggesting that aerobactin conferred a significant selective advantage for colonization of this intestinal segment. Although the two challenge strains translocated to MLN, the population level in the MLN was always higher for the Iut+ strain. Moreover, experimental infections resulted in recovery of only the Iut+ strain in the organs other than the MLN and in the body fluids. These results indicate a role for aerobactin in promoting systemic spread of the bacteria from the intestine. Direct evidence was obtained that aerobactin secretion occurred in vivo at both intestinal and extraintestinal sites of infection. In contrast to enterobactin, aerobactin was detected in the duodenum, jejunum, ileum, cecum, liver, spleen, kidney, urine, cerebrospinal fluid, and bile. The highest concentration of aerobactin was found in the urine, even when the samples were devoid of infecting bacteria. All of these findings suggest that aerobactin is released in vivo in a diffusible form and that it may be an important step in the production of disease by intestinal opportunistic E. coli.

Comparative pathogenicity of selected bovine viral diarrhea virus isolates in gnotobiotic lambs

SUMMARY The infectivity and pathogenicity of selected bovine viral diarrhea virus (bvdv) isolates were determined in gnotobiotic, colostrum-deprived neonatal lambs. Five-day-old cesarean-derived gnotobiotic lambs were exposed to 1 of 10 bvdv. isolates via aerosol suspension. These isolates were from tissues or secretions of calves or lambs affected with respiratory tract disease, weak neonatal calves, aborted bovine fetuses, or reference Singer or Draper bvdv. The pathogenicity of each isolate, relative to the others, was evaluated in lambs by measurement of the neutralizing antibody response, virus isolation from nasal secretions or tissues, and postmortem lesions. The bvdv isolates varied in their infectivity and pathogenicity. Singer, the cytopathic reference strain, was the most lymphotrophic isolate and stimulated the greatest neutralizing antibody response. Encephalitis was the most consistent lesion observed and was used as the final determinant of relative pathogenicity of the viruses. The most neuropathogenic isolates were the 2 viruses originating from lambs affected with respiratory tract disease, the 2 weak neonatal calf isolates, and 1 isolate from an aborted bovine fetus. The least pathogenic isolates were the 2 reference isolates, Draper and Singer; the 2 mucosal disease isolates; and 1 isolate originating from an aborted bovine fetus.

Biochemical and haematological evidence of endotoxic shock in gnotobiotic lambs with watery mouth disease

Eight gnotobiotic lambs deprived of colostrum were infected by mouth when two hours old with nonenterotoxigenic strains of Escherichia coli. All developed clinical signs of watery mouth disease and seven died within 24 hours. The mean concentrations of several blood constituents were determined in samples taken at intervals until 24 hours after infection in infected lambs and in four control lambs. The biochemical and haematological changes observed in the lambs developing watery mouth disease were those characteristic of endotoxic shock.

Interactions BetweenEimeria ovinoidalisandEscherichia coliin the Gnotobiotic lamb

The effects of Eimeria ovinoidalis on the establishment of Escherichia coli K99 + Ent + Nal r in the digestive tract were observed during the entire cycle of parasite development in nine gnotobiotic lambs. As compared to a saprophytic strain of autochthonous E. coli 35a from lamb colon and caecum which becomes established within 24 h, the development of a pathogenic allochthonous strain exhibited different profiles. Depending on the animal, the strain either did not become established or its development was retarded and then remained at an intermediate level (10 7 /g), or was maximal (10 9 -10 10 /g). In the first two cases, the mechanisms which inhibited or limited the development of E. coli K99 + in healthy animals were not effective when there was coccidia development. Keywords: Ecology; Interactions; Enterotoxigenic; Escherichia coli ; Eimeria ovinoidalis ; Lamb; Gnotobiotic.

The establishment of rumen microbial communities

In young ruminants, the strictly anaerobic bacteria that colonise the rumen are accompanied by a wide range of facultatively-anaerobic organisms, together with some aerobic bacteria. During the development of the rumen, the flora resolves into a spatially-organised set of domains, each populated by characteristic, but to some extent overlapping, consortia. Within and between these consortia, donor/acceptor, predator/prey and competitive relations can be found. The results of experiments on the establishment of functioning microbial communities in gnotobiotic lambs are considered. It seems that any attempt to introduce components of the rumen microflora must take account of the principles of microbial ecology, as they relate to the colonisation of ecosystems. In particular, it is necessary to provide suitable ecological niches for colonisation, which are suitable with respect to sources of energy, carbon, nitrogen minerals and trace nutrients for growth, at ambient pH and Eh values compatible with the tolerance of the desired organisms. The further manipulation of functions that have already been subject to intense natural selective pressure is likely to be particularly difficult. However manipulation to control rumen disorders, utilise exotic substrates or to detoxify toxic compounds produced by plants may be more promising.

Replication of atypical ovine rotavirus in small intestine and cell culture.

Colostrum-deprived lambs experimentally infected with an atypical ovine rotavirus isolated from naturally scouring animals, and a naturally infected colostrum-deprived lamb, were examined by immunofluorescence and immunoperoxidase labelling, and electron microscopy. A hyperimmune serum to the virus was produced in a gnotobiotic lamb and used to demonstrate antigen in the villous epithelial cells of the small intestine from infected animals. Scanning and transmission electron microscopy of tissues from infected animals revealed giant multinucleate syncytia composed of fused epithelial cells. Infected cells contained intracytoplasmic virus particles which resembled group A rotaviruses in morphology and morphogenesis. Small numbers of infected cells in MA104 cultures inoculated with ovine atypical rotavirus could be detected by immunofluorescence but virus growth could not be maintained by passage. Virus particles were seen by thin-section electron microscopy but their morphology and morphogenesis were abnormal; they were each composed of a single electron-dense shell, with no core, and were associated with envelopes of smooth membrane rather than rough endoplasmic reticulum.