An aerosol challenge model of tuberculosis in Mauritian cynomolgus macaques.

BACKGROUND: New interventions for tuberculosis are urgently needed. Non-human primate (NHP) models provide the most relevant pre-clinical models of human disease and play a critical role in vaccine development. Models utilising Asian cynomolgus macaque populations are well established but the restricted genetic diversity of the Mauritian cynomolgus macaques may be of added value. METHODS: Mauritian cynomolgus macaques were exposed to a range of doses of M. tuberculosis delivered by aerosol, and the outcome was assessed using clinical, imaging and pathology-based measures. RESULTS: All macaques developed characteristic clinical signs and disease features of tuberculosis (TB). Disease burden and the ability to control disease were dependent on exposure dose. Mauritian cynomolgus macaques showed less variation in pulmonary disease burden and total gross pathology scores within exposure dose groups than either Indian rhesus macaques or Chinese cynomolgus macaques. CONCLUSIONS: The genetic homogeneity of Mauritian cynomolgus macaques makes them a potentially useful model of human tuberculosis.

Early lesions following aerosol challenge of rhesus macaques (Macaca mulatta) with Mycobacterium tuberculosis (Erdman strain).

Three rhesus macaques (Macaca mulatta) were challenged with Mycobacterium tuberculosis (Mtb), Erdman strain, as part of studies to investigate lesion development at early time points in tuberculosis (TB) and to assess computed tomography (CT) as a method of monitoring disease progression in vivo. Animals were challenged with either a high, mid or low dose of aerosolized Mtb. The low-dose animal was killed humanely at 24 days post challenge (dpc) and the remaining animals at 25 dpc. Abnormalities in clinical parameters were observed in all animals, but clinical signs relating to respiratory disease were not seen. Pulmonary changes consistent with TB infection were detected by CT at 21 dpc and magnetic resonance imaging (MRI) post mortem. Pulmonary nodule counts obtained from both imaging techniques were directly proportional to the challenge dose and correlated with gross and microscopical lesion counts. On gross and microscopical examination, lesions of similar size and morphology were observed in the lungs of all three animals, with the majority containing necrotic foci. Concomitant gross and microscopical, granulomatous lesions were observed in the tracheobronchial lymph nodes of all animals together with evidence of systemic spread. These findings further contribute to our understanding and knowledge of early lesion formation in the lungs of non-human primates.

Localisation of enteropathogens in paraffin embedded tissue by immunoperoxidase.

An indirect immunoperoxidase technique has been used to identify enteropathogens in formol-sublimate fixed paraffin embedded sections of calf intestine. Infections with bovine rotavirus, bovine coronavirus, Newbury agent SRV -1, and K99+ Escherichia coli have been detected in the intestines from experimentally infected and conventially reared diarrhoeic or normal calves. The ability to visualize enteropathogenic agents in histological sections resulted in the demonstration of virus infected cells at sites not previously shown to be infected using the immunofluorescence technique.

The isolation of reovirus-like agents (rota-viruses) from acute gastroenteritis of piglets.

Isolations of reovirus-like agents (rotaviruses) were made from nine of 23 outbreaks of piglet diarrhoea on different farms and from both weaned and unweaned piglets. The viruses were shown to be morphologically and anti-genically similar to the rotaviruses of children and calves. Gnotobiotig piglets given intranasal inoculations of five different isolates developed acute gastroenteritis, and the virus was re-isolated from the faeces or intestinal contents. The piglet virus was not adapted to replicate in cell culture. We conclude that the pig rotavirus is commonly associated with outbreaks of gastroenteritis and is probably an important aetiological factor in this disease.

A study of the basis of virulence variation of bovine rotaviruses.

Rotaviruses are enteric pathogens of cattle but sub-clinical infections are common. Virulence variation has been identified with bovine rotaviruses and some rotaviruses replicated without clinical signs in non-immune calves. The rotavirus genome is composed of eleven segments of double-stranded RNA and the fourth largest segment codes for a non-glycosylated surface protein, VP4, which has been linked with virulence. In the present study the biological basis of rotavirus virulence variation was studied in vivo and compared with the known properties of the fourth gene. Calves were inoculated orally with a virulent rotavirus or a rotavirus of low virulence which multiplied but failed to cause diarrhoea. They were taken for necropsy at intervals of 2 days after inoculation. Clinical signs, virus in faeces and the percentage of infected small intestinal epithelium were determined. Damage to the small intestine was assessed by measurement of villus heights and crypt-cell production rates. Virulence was associated with a greater level of colonization of the small intestinal epithelium, greater enterocyte damage and preferential infection of the upper small intestine. The fourth gene determines the ability of rotaviruses to spread in vitro and the finding that virulence was associated with greater colonization in vivo raises the possibility that this gene may have an important role in rotavirus virulence.

The pathogenesis of turbinate atrophy in pigs caused by Bordetella bronchiseptica.

The pathogenicity of 3 strains of Bordetella bronchiseptica designated B58, PV6 and B65 was compared by intranasal infection of gnotobiotic piglets. Strain B58 was a phase 1 isolate that produced haemolysin, an adhesin for calf erythrocytes, adenylate cyclase, mouse lethal factor, dermonecrotic factor and cytotoxin. B65 was a variant of B58 that produced no detectable haemolysin, adhesin or adenylate cyclase and 10-fold smaller amounts than B58 of mouse lethal factor, dermonecrotic factor and cytotoxin. Strain PV6 was a phase 1 isolate that produced only haemolysin, adhesin and adenylate cyclase. After nasal infection of gnotobiotic pigs, 10(3.2)-10(6.2) colony forming units ml-1 (cfu ml-1) of strains B58 and PV6 were cultured from nasal washings during the next 25 days. In contrast, only 10(1.0)-10(2.8) cfu ml-1 of strain B65 were recovered during the same period. Only pigs infected with strain B58 had turbinate atrophy when they were slaughtered 25 days after infection and neutralising antibody to cytotoxin was detected only in these pigs. These results suggested that the cytotoxin, which may be the same as the mouse lethal and dermonecrotic factors, was the cause of turbinate atrophy. They also support the view that the adhesin for calf erythrocytes is required for colonisation of the nasal cavity in vivo.

Dysentery in calves caused by an atypical strain of Escherichia coli (S102-9).

Dysentery lasting 4-8 days was produced in five 4-day-old colostrum-fed calves, after inoculation with an atypical strain of Escherichia coli S102-9; peak excretion of S102-9 occurred during the period of dysentery. Two calves were killed when clinical signs were most severe and bacteria were seen attached to the surfaces of enterocytes in the large intestine; microscopic lesions were seen in these areas. The lesions were identical to those previously reported in a natural outbreak of dysentery in calves, from which E. coli S102-9 was isolated, and to those seen in gnotobiotic calves experimentally infected with S102-9. Reinfection of the three surviving calves 16-20 days later with S102-9 and primary infection of two calves aged 24 and 51 days did not cause dysentery. Four of 659 coliforms isolated from field outbreaks of calf diarrhoea resembled the atypical strain S102-9. These four isolates and S102-9 did not produce heat-stable enterotoxin, but all produced a toxin cytopathic for Vero and HeLa cells. Two of the four isolates were inoculated alone into 4-day-old gnotobiotic calves deprived of colostrum; neither calf developed dysentery but microscopic lesions identical to those produced by S102-9 were detected in the large intestines of both animals.

An investigation of temporary workshop clusters reacting with cells of the mononuclear phagocytic system.

Monoclonal antibodies (mAbs) within ten temporary workshop clusters (TC3, TC8, TC9, TC11, TC16, TC17, TC19, TC20, TC21 and TC31) which from preliminary evidence appeared to recognize molecules expressed by cells of the mononuclear phagocytic system were studied by immunohistology and flow cytometry. A number of mAbs were identified that were potentially useful for immunohistological studies of the mononuclear phagocytic system. The results confirmed the specificity of a number of CD11a, CD11b, CD11c and CD44 mAbs. The findings also indicated that for several TCs the mAbs could not be regarded as recognizing the same molecule.

Number and distribution of T lymphocytes in the small intestinal mucosa of calves inoculated with rotavirus.

An understanding of the immune response to rotavirus is needed to develop effective prophylaxis. There is evidence that cell-mediated responses may be involved and to extend these observations, rotavirus antigen and the three major T cell subsets, BoCD4+, BoCD8+, and BoWC1+ gamma/delta lymphocytes were immunostained in tissue sections from calves killed at 2, 4, 6, 8 and 10 days post inoculation and quantified by image analysis. It was established that in control calves, BoCD4+ lymphocytes were predominantly in the lamina propria, while the majority of BoCD8+ and BoWC1+ gamma/delta lymphocytes were in the epithelium. Rotavirus infection was seen throughout the small intestine with the greatest amount of viral antigen detected at 4 days post inoculation in the mid and distal small intestine. Increased numbers of all subsets were detected; small increases in intraepithelial BoCD4+ and BoWC1+ gamma/delta T lymphocytes were observed especially in the distal small intestine, while larger increases in BoCD8+ cells were detected in the epithelium and lamina propria of the proximal, mid and distal small intestine. The timing and location of these increases in T lymphocyte subsets is indicative of a specific immune response involving BoCD8+ and BoWC1+ gamma/delta T lymphocytes.

Variable-interval schedule performance in open and closed economies.

In two experiments, pigeons obtained food according to variable-interval schedules. In the first experiment, equivalent variable-interval schedules with average interreinforcer intervals ranging between 10 and 80 s in different conditions were studied in both open and closed economies. Response rates increased as reinforcement frequency decreased in the closed economy. By contrast, in the open economy response rates decreased for 1 bird and were variable for the other as reinforcement frequency decreased. The second experiment showed that the differences in the functions between responding and reinforcement frequency in the two types of economies were not due to changes in deprivation level. These results suggest that open and closed economies yield different behavioral effects. This conclusion is supported further by a reconsideration of previous findings that appear counter to the conclusion.

Effects of age and diet on small intestinal structure and function in gnotobiotic piglets.

The effects of age and dietary change on the structure and function of piglet small intestine were investigated in gnotobiotic pigs. The small intestinal mucosa was damaged when gnotobiotic pigs were weaned on to a pelleted meal diet; villus height, crypt cell production rate, and activities of brush border enzymes were reduced. Small intestinal damage was associated with reduced weight gain over a three week period; diarrhoea was not observed. The continuous consumption of meal appeared to perpetuate the intestinal damage for three weeks, although evidence of intestinal repair, as indicated by an increased crypt cell production rate, was present three weeks after weaning. Antibodies to soya antigen were detected in serum after weaning and the intestinal damage could have been caused by antibody-mediated immune reactions to soya proteins. Villi of milk fed gnotobiotic pigs shortened significantly with age.

Investigation of a cutaneous delayed hypersensitivity response as a means of detecting Salmonella dublin infection in cattle.

Delayed hypersensitivity reactions developed 48 to 96 h after intradermal injection of killed Salmonella dublin in 25 of 28 cattle which had been inoculated intravenously, and in five of 10 cattle which had been inoculated orally with S dublin 24 to 493 days previously. Control animals showed no delayed hypersensitivity reactions. Persistence of infection in five of the intravenously inoculated and in four of the orally inoculated animals was confirmed by isolation of S dublin from the carcases at necropsy one week after skin testing. Failure to isolate the organism from the carcases of 21 animals which had reacted positively to the intradermal test did not eliminate the possibility of their being carriers of S dublin. Skin testing was concluded to be a reliable means of identifying animals which had been, and possibly still were, infected systemically with S dublin. However recovered animals might be falsely identified as infected. Repeated testing gave misleading results.

Pathology of calves with diarrhoea in southern Britain.

Twenty-one moribund calves with diarrhoea were purchased from 11 farms, their faeces examined for enteropathogens and samples of intestinal tissue removed under anaesthesia. Lesions and presence of enteropathogens on the mucosal surface were scored by histological examination of immunostained paraffin sections. Two or more enteropathogens were detected in 19 calves. Cryptosporidium appeared to be the principal cause of diarrhoea in six calves, rotavirus in four, Salmonella typhimurium in two, bacteria adherent to the surface of the large intestine in two, coronavirus in one and K99+ Escherichia coli in one calf. Diarrhoea in four calves was the consequence of mixed infections in which no one enteropathogen appeared to predominate. In one calf no enteropathogen was detected. Diarrhoea was associated with infections and lesions throughout the small and large intestines. The enteropathogens most frequently associated with lesions in the small intestines were rotavirus, coronavirus and cryptosporidium; in the large intestines they were coronavirus and bacteria apparently adherent to the mucosal surface.

Pathology of natural rotavirus infection in clinically normal calves.

During a longitudinal study of the epidemiology of rotavirus infection in a calf rearing unit, excretion of virus in faeces was detected by enzyme-linked immunosorbent assay in 40 of 48 (83 per cent) unweaned calves aged between three days and five weeks. Fifty per cent of the infected calves had no clinical signs of disease. Enterocytes containing rotavirus antigen and intestinal lesions were found in all of 12 clinically normal calves selected for necropsy between days 1 and 4 of virus excretion. Stunting and fusion of villi, exfoliation, disarrangement and vacuolation of enterocytes and the presence of cuboidal enterocytes were observed in infected calves but not in rotavirus-free control calves. Lesions predominated in the upper small intestine, where rotavirus was most abundant, especially on the first two days of virus excretion. The numbers of enterocytes infected with rotavirus diminished before the lesions resolved.

Immunological responses of fluke-infected and fluke-free cattle to Salmonella dublin and other antigens.

Immune responses to heat-killed Brucella abortus strain 19 and to ovalbumin were compared in 15 fluke-infected and 15 fluke-free Friesian heifers. B abortus was injected 16 weeks and ovalbumin 19 weeks after the oral administration of 1000 metacercariae of Fasciola hepatica. Agglutinating antibody responses to B abortus were similar in both groups. Immediate type hypersensitivity to ovalbumin was apparently suppressed in fluke-infected animals when assessed by active and passive cutaneous anaphylaxis two weeks after sensitisation. However, when assessed by Schultz-Dale responses of intestine, in vitro, 36 weeks after sensitisation there was no difference between the groups. The heifers were subsequently given live Salmonella dublin intravenously. The fluke-infected animals which became carriers of S dublin had the most persistently elevated titres of agglutinating antibodies in their sera and the highest incidence of immediate-type hypersensitivity, as assessed by Schultz-Dale responses of intestine, but the weakest cutaneous delayed hypersensitivity reactions to S dublin. The latter might have been related to lymphopenia which developed after fluke infection. The increased susceptibility of fluke-infected cattle to S dublin cannot be attributed to impaired agglutinin responses but may result from effects on cell-mediated mechanisms.

Responses of fluke-infected and fluke-free cattle to experimental reinfection with Salmonella dublin.

Cattle, aged nine to 18 months, were given sublethal doses of Salmonella dublin either intravenously, orally on two occasions, or by contact for 19 to 22 weeks with a persistent excretor. When challenged by intravenous injection of 10(9) S dublin at 10 to 22 weeks after their first exposure to S dublin 22 out of 36 animals survived. All similarly challenged cattle, which had not been previously exposed to S dublin, died. Infection with Fasciola hepatica did not alter the ability of cattle to survive reinfection with S dublin but the bacteria persisted in the tissues and were excreted in the faeces of fluke-infected animals for longer than in fluke-free animals. The ability to survive reinfection and to eliminate S dublin from the tissues was apparently unrelated to agglutinating antibody titres or delayed hypersensitivity.

Quantitation of small intestinal structure and function in unthrifty piglets.

Litters of piglets were weighed at birth, weaning and five weeks later; pairs of thrifty and unthrifty weaned pigs were identified. Small intestinal structure and function were studied in 28 eight-week-old age-matched littermate pairs and in 16 weight-matched littermates. A comparison of age-matched piglets showed changes in mucosal structure in unthrifty piglets, a slight reduction in absorptive function but no biochemical evidence of small intestinal disease. Morphometric studies of weight-matched piglets revealed no differences between thrifty and unthrifty piglets, suggesting that the differences in the age-matched groups may have been an effect of body-weight. If unthrifty pigs grew slowly as a result of small intestinal damage and malfunction immediately after weaning no evidence of it remained when they were studied five weeks later.

Effects of dietary change and rotavirus infection on small intestinal structure and function in gnotobiotic piglets.

The combined effects of weaning and rotavirus infection on small intestinal structure and function and on growth rate were studied in 28 gnotobiotic piglets. There was little damage by rotavirus to the proximal small intestine, some damage to the mid small intestine and relatively severe damage to the distal small intestine; villi were stunted, crypts lengthened and activities of all brush border enzymes decreased. The damage was short-lived despite the synchronisation of rotavirus infection with simulated weaning. There was no evidence of persistent damage to the small intestine and growth rate was unaffected.

Plasma enteroglucagon and neurotensin levels in gnotobiotic calves infected with enteropathogenic and non-enteropathogenic viruses.

Five gnotobiotic calves were each infected with five viruses. Each calf was inoculated with coronavirus at seven days old, followed by astrovirus, Newbury agent, parainfluenzavirus type 3 and rotavirus at intervals of two weeks. Three of the viruses were enteropathogenic (bovine coronavirus, bovine calici-like virus and bovine rotavirus) and two were not (bovine astrovirus and parainfluenzavirus type 3). Plasma levels of the peptide hormones enteroglucagon and neurotensin and faecal output were measured daily and xylose absorption was studied before and after each infection. A close correlation was found between a rise in plasma enteroglucagon and neurotensin and infection with enteropathogenic viruses. The three enteropathogenic viruses caused increased daily faecal output, and elevated plasma levels of enteroglucagon and neurotensin, while the non-enteropathogens did not. The calici-like virus and rotavirus but not the coronavirus caused xylose malabsorption.

Detrimental effects on villus form during conventional oral rehydration therapy for diarrhoea in calves; alleviation by a nutrient oral rehydration solution containing glutamine.

This paper examines the possibility that treatment of diarrhoea with conventional oral rehydration solutions (ORSs) may be detrimental to villus structure by imposing nutrient deprivation and that such detrimental effects may be reduced or avoided by using a nutrient ORS. A conventional WHO-type ORS (W) was compared with two nutrient solutions (N and G) both containing high glucose concentrations and the latter containing glutamine; their effects on enteric structure were assessed by morphometric analysis of samples obtained from diarrhoeic calves after 96 h treatment. Comparisons were also made with samples from controls and diarrhoeic calves at the stage where oral rehydration would have begun in the treated groups. As in our previous ORS studies, diarrhoea was induced with enterotoxigenic Escherichia coli (09:K30:K99). We measured villus length and width, crypt depth and width and calculated villus surface area in proximal, mid and distal small intestine (PSI, MSI, DSI), using standard morphometric techniques. Proximal and distal spiral colon samples (PC, DC) were examined for crypt depth and width; mitoses per crypt were counted in samples from all regions. Non-diarrhoeic calves showed the expected gradient of villus length through PSI, MSI and DSI, hence data for each region are normalized as a percentage of the control value for that region. PSI showed the greatest loss of villus length and surface area (50%) with diarrhoea. In MSI and DSI the villus loss was greater with solution W and N or G, as were increased mitoses and crypt depth. Crypt depth and mitoses also increased in the colon with solution W. Colonic crypt width increased with diarrhoea and conventional oral rehydration but less so with G; there is reason to believe that such changes have functional significance. Crypt changes in colon, MSI and DSI were least with solution G. The changes developing in diarrhoeic calves prior to treatment were thus less apparent in those treated with a nutritional ORS, particularly if it contained glutamine.