Developed and resolving lesions in porcine proliferative enteropathy: possible pathogenetic mechanisms.

Proliferative enteropathy, caused by Lawsonia intracellularis, offers the opportunity to examine bacterial mechanisms that influence epithelial cell proliferation. Ultrastructural features of developed and resolving lesions included the presence of enlarged intestinal crypts containing undifferentiated immature epithelial cells and an absence of goblet cells. Numerous intracytoplasmic bacteria, identified as L. intracellularis, were consistently present within affected cells. In recovering intestinal tissue, additional features were (1) the common presence of pale, swollen, protruding epithelial cells, (2) shrunken, degenerate epithelial cells, (3) apoptotic bodies in both epithelial cells and macrophages, (4) the reappearance of normal goblet cells, and (5) reduced numbers of L. intracellularis within lesions. Bacteria were released from cells via cytoplasmic and cellular protrusions into the intestinal lumen. It is speculated that the presence of the intracytoplasmic bacterium, L. intracellularis, may disrupt normal processes of cell growth, differentiation or apoptosis in the intestinal epithelium.

Entry of the bacterium ileal symbiont intracellularis into cultured enterocytes and its subsequent release.

Separate suspensions of two strains of ileal symbiont (IS) intracellularis, an obligate intracellular bacterium and the causative agent of porcine proliferative enteropathy, were added to 40 or 80 per cent confluent monolayers of established cultures of rat (IEC-18) or pig enterocytes (IPEC-J2). Peak numbers of intracellular organisms were detected within the enterocytes six days later, but no cytopathic effects were evident. After an initial close association with the cell membrane of the enterocytes, single bacteria were internalised after three hours within membranes-bound vacuoles. The formation of an electron-dense projection between cell membranes and external bacteria was only evident if the bacterial suspensions were centrifuged on to the monolayers. The release of internalised bacteria into the cytoplasm, with the breakdown and loss of membrane-bound vacuoles, was also evident three hours after infection. Internalised bacteria were associated with, but not observed within, coated membrane pits. Mitochondria were closely associated with internalised vacuoles and with released bacteria. Two to six days after infection, multiplication of the bacteria free in the cytoplasm was frequently observed. In infected cells six days after the inoculation of monolayers, groups of bacteria were found within large, balloon-like, cytoplasmic protrusions, and the subsequent release of bacteria from the monolayer provided a means of bacterial exit from the cells. Many events in the in vitro culture model closely resembled events observed at the cellular level in animals infected with IS intracellularis and the model provides a useful basis for investigating the pathogenetic mechanisms of this bacterium.

Sequential pathology of tick-borne fever.

The sequential pathology and clinical responses of 12 sheep infected with the Sourhope strain of Cytoecetes phagocytophila are described. During the experimental period of 3 weeks, the clinical effects were mild, as is usual. There was transient leucopenia, affecting mainly lymphocytes, eosinophils and neutrophils. Parasitaemia was detected from days 5 to 16, approximately. A mild but consistent histopathological response up to day 21 comprised (i) pulmonary alveolitis with minimal shedding of cells, (ii) follicular and especially parafollicular lymphoreticular reactions in lymph nodes and spleen, (iii) mesangial glomerulitis, and (iv) in the cranial nervous system marked choroiditis but minimal reaction in the brain. Two forms of intracytoplasmic inclusions were found, a smaller, granular basophilic structure occurring in clumps and an intravacuolar pleomorphic form. In tissue sections they were mainly associated with lymphoid and macrophagic cells. The significance of these findings is discussed including the problems of differential diagnosis in complex infections.

The effect of high and low dose Theileria parva infection on the reproductive function of Boran/Friesian heifers.

Two separate experiments were carried out to find the effect of infection with low and high doses of Theileria parva sporozoites on the cyclical status of Boran/Friesian cross heifers. In each experiment, ten heifers were infected and another four heifers acted as controls. Routine monitoring of estrous behavior, determination of serum progesterone concentrations and palpation of the ovaries per rectum were used to assess the cyclical status of each animal. Two and eight animals died in the T . parva low (TpL) and high (TpH) dose experiments, respectively. Five of eight recovered animals in the TpL group cycled regularly postinfection, while three others had extended periods of ovarian dysfunction postinfection. Both infected and recovered animals in the TpH-group similarly had extended periods of ovarian dysfunction postinfection. All controls in both experiments cycled regularly throughout the study period. Differences in cyclical status were not related to the febrile response, parasitological or hematological differences. The animals that continued to cycle throughout lost the least amount of weight. Animals that stopped cycling lost between 16 and 29% of their initial body weights postinfection. In conclusion, this study confirmed previous anecdotal evidence that theileriosis has an adverse effect on reproduction. Results of this study suggested that this was as an indirect result of weight loss associated with infection and not directly attributed to pathology of target organs.

Caecal intussusception in two ponies.

One case of caecocaecal intussusception (case 1) and one case of caecocolic intussusception (case 2) in ponies are described. Case 1 showed mild abdominal discomfort for seven days followed by sudden death whereas case 2 showed continuous moderate pain for three weeks. At post mortem examination, case 1 showed intussusception of the base of the caecum into the body whereas in case 2, the entire caecum had invaginated into the right ventral colon. Histopathological examination showed that the lesions in both animals had been present for a long time.

Early lesions of proliferative enteritis in pigs and hamsters.

Gnotobiotic pigs and conventional hamsters were given suspensions of intestinal mucosa from a pig with proliferative hemorrhagic enteropathy and killed 10 or 21 days later. Affected animals had evidence of marked proliferation of immature enterocytes in the intestinal crypts. Numerous Campylobacter-like organisms were in the cytoplasm of enterocytes, and in some instances, bacteria were closely associated with enterocytes. Some intracellular bacteria lying below the microvillous border were within membrane-bound structures. Immunofluorescence and electron immunogold staining with specific antibodies indicated that these organisms were antigenically different from curved bacteria in the crypt lumen of early lesions. This study indicates that the life cycle of the intracellular organisms may involve entry into crypt enterocytes from the intestinal lumen with subsequent intracellular multiplication.

Serological diagnosis of the porcine proliferative enteropathies: implications for aetiology and epidemiology.

Campylobacter mucosalis and C hyointestinalis have been associated with the proliferative enteropathies of pigs. An examination of the antibody response to these organisms and to the intracellular campylobacter-like organism was undertaken. Antibody to the campylobacter-like organism was predominantly IgM, short lived, and could be detected by an immunofluorescence test using bacteria released from lesions as antigen. The majority (75 per cent) of pigs with proliferative enteropathy at necropsy were antibody positive and a small number (4 per cent) of pigs in which lesions were not observed were found to have antibody. Antibody appeared to be correlated with the presence of lesions rather than with exposure to infection and was independent of the presence of antibody to C mucosalis or C hyointestinalis. In natural outbreaks of the disease antibody to the campylobacter-like organism was more prevalent than clinical signs in the affected animals.

Anti-muscle antibodies in Graves' ophthalmopathy.

The nature of the humoral immune response in patients with Graves' ophthalmopathy has been investigated using a solid phase 125I Protein A binding assay. Retro-orbital muscle (R.O.M.), skeletal muscle (Sk.M.), R.O.M.-membranes, thyroid, kidney, liver, harderian gland, acetylcholine receptors (AchR), actin and myosin were used as target antigens. No significant difference in antibody binding profile to R.O.M. and Sk.M. was found indicating that the ophthalmic immunoglobulins (OIgs) were not recognising a R.O.M. specific antigen(s). Comparison between R.O.M. and R.O.M.-membranes, however, revealed that these antigens were detecting very different antibody populations. Using the former, it appeared that the predominant antibody population being measured was anti-myosin whereas the latter appeared to be detecting primarily anti-AchR antibodies. Anti-actin antibodies were also present in some of the sera. Thus a spectrum of anti-R.O.M. antibodies appears to be present in Graves' ophthalmopathy but the cross-reactivity of these with non-R.O.M. skeletal muscle and their similarity to those found in myasthenia gravis prevent them as yet being used to explain the specific immunopathology observed in this disease.

The lack of specificity of ophthalmic immunoglobulins in Graves' disease.

Immunoglobulins (Igs) binding to retro-orbital muscle (ROM) antigens, known as ophthalmic Igs (OIg), were measured using a 100,000 X g sediment of porcine ROM as antigen in a solid phase [125I]protein A binding assay. Serum samples from 50 control subjects bound from 0.60-2.42 times the amount of [125I]protein A as did the normal reference serum samples, defined as the OIg ratio. Serum from 95 patients with hyperthyroid Graves' disease had OIg ratios from 0.64-9.99, with 24 (25%) being positive [OIg ratio greater than 2.05 (mean + 2 SD of the normal group)]. Ten patients with euthyroid Graves' ophthalmopathy had OIg ratios from 1.01-6.33, with 6 (60%) being positive. Among those Graves' disease patients with ophthalmopathy (n = 19) and the euthyroid Graves' ophthalmopathy patients there was a good correlation between the severity of eye signs and the OIg ratio. The OIg-positive serum samples cross-reacted with skeletal muscle and thyroid as well as with ROM antigen. This lack of specificity contradicts previous reports, but does not rule out a role for these antibodies in the etiology of Graves' ophthalmopathy.

Demonstration of a new intracellular antigen in porcine intestinal adenomatosis and hamster proliferative ileitis.

In the proliferate enteropathies of the pig, intracellular Campylobacter-like organisms can be demonstrated in the apical cytoplasm of affected cells. A new antigen (omega) can be visualised at the site of bacterial parasitism using certain rabbit sera in a sandwich immunofluorescence test; the rabbit sera tested were largely derived from rabbits immunised with bacterial antigens including Campylobacter spp., but it appeared that the reaction was not related to the immunisation procedure and was also present in at least one "normal" rabbit serum. Rabbits immunised with bacteria extracted from the tissues sero-converted to omega-antigen yet did not react with Campylobacter spp., cultured from pig intestine. The omega-antigen was also present in the lesions of hamster proliferative ileitis.

Behaviour of Campylobacter sputorum subspecies mucosalis in gnotobiotic pigs.

Gnotobiotic pigs were dosed orally with Campylobacter sputorum subspecies mucosalis, either alone, or combined with rotavirus or non-pathogenic Escherichia coli and Streptococcus bovis to study the behaviour of C s mucosalis in defined conditions, to assess intracellular parasitism of enterocytes by C s mucosalis, and if possible to establish an experimental model of porcine intestinal adenomatosis. C s mucosalis colonised the gut of gnotobiotic pigs, persisting for up to 47 days after infection, but did not induce adenomatosis. Despite evidence of limited penetration of the mucosa up to two days after infection, the majority of C s mucosalis remained in the gut lumen. Rotavirus did not enhance invasion of enterocytes by C s mucosalis. The presence of E coli and S bovis caused an increase in the total numbers of C s mucosalis in the gut, but did not affect their distribution. Thus C s mucosalis was largely non-pathogenic in gnotobiotic pigs.

A preliminary report on hepatic pigmentation in Falkland Islands sheep.

Pigmentation of hepatocytes and Kupffer's cells with lipofuscin and melanin-like pigment is described. The occurrence of pigmented reticulo-endothelial cells in the lungs and lymphoid tissue was also noted. No discernible impairment of function accompanied the pigmentation.

Ultrastructural characteristics of in vitro parasite-lymphocyte behaviour in invasions with Theileria annulata and Theileria parva.

Host-parasite relationships have been studied by electron microscopy using glutaraldehyde-OsO4-fixed pellets of lymphoid cultures infected in vitro by Theileria annulata and T. parva. Intracellular presence of the parasite resulted in a progressive and marked lymphoblastoid transformation. The schizont stage periodically provoked the formation of, and adopted an intimate association with, cytoplasmic annulate lamellae in the interphase cell. Annulate lamellae developed from the outer nuclear membrane of the host cell by a delamination process and were taken into the cytoplasmic matrix of the schizont by phagotrophy through the cytostome. Schizont nuclei themselves were seen to divide at the prometaphase stage of host cell mitosis, the division being characterized by the development of intranuclear spindle microtubules anchored in spindle pole bodies. A hypothesis is propounded that Theileria parasites, consequent on interiorization, provoke the blastoid transformation and the formation of annulate lamellae through the influence of components of their genomic material on host cell deoxyribonucleic acid (DNA) and that the annulate lamellae represent a species of messenger ribonucleic acid (mRNA) and serve as a monitoring device for the schizont, facilitating the accurate timing of the host cell cyclical events.

Presence of serum agglutinins to Campylobacter sputorum subspecies mucosalis in pigs.

Pigs affected with intestinal adenomatosis had agglutinating antibodies to Campylobacter sputorum subspecies mucosalis. The titres obtained varied, depending on the strain used as antigen and appeared to rise with the age of the affected animal. Examination of sera from experimental animals showed that pigs between 30 and 77 days old rarely had antibody, even when they had been exposed to infection. Evidence is also presented indicating that the serological response is specific and that in a limited examination of sera derived from pigs at slaughter almost all animals had high levels of antibody.

Pneumonia in sheep associated with dipping in carbolic dips.

Fourteen outbreaks of pneumonia in sheep following dipping in carbolic dips were investigated at seven laboratories. Deaths commonly occurred between one and three days after dipping and fat young sheep of the Suffolk and Border Leicester breeds were most usually involved in outbreaks. The pneumonia was characterised by hyperaemic, oedematous and cyanotic lungs, and the main histological features were hyperaemia, fluid and cellular exudation into alveolar spaces, and widespread epithelialisation of alveolar walls. The microscopic changes differed from pneumonic pasteurellosis and no significant organisms were recovered from any of the lungs. One of 27 Dorset Horn lambs dipped in a batch of dip which had been incriminated in some episodes developed an epithelialising interstitial pneumonia, while two out of three Suffolk lambs dipped in twice the recommended strength of the same batch of dip became ill with severe pneumonia, apparently following skin absorption.

Experimental infection of piglets with cryptosporidium.

Piglets from five litters were doses orally with cryptosporidium originally derived from diarrhoeic calves. The piglets were either nursed by the sow, artificially reared after sucking colostrum, or weaned on to creep feed. Colostrum-fed, artificially reared piglets obtained from two litters and exposed in the first week of life developed clinical signs of inappetence, vomiting and diarrhoea and shed oocysts in the faeces. Histologically the parasite was observed throughout the small and large intestine attached to epithelial cell surfaces and its presence was associated with extensive mucosal damage, particularly in the posterior small intestine, stunting and fusion of villi, immaturity of villous epithelial cells and oedema with increased cellularity of the lamina propria. Piglets from two other litters, both sucking and colostrum-fed artificially reared, exhibited similar but milder clinical signs. Histological lesions were less severe and cryptosporidium infection less extensive. When weaned piglets were exposed they remained clinically healthy although histologically there was evidence of cryptosporidium attachment in the small intestine and minor mucosal damage. There appears to be a good correlation between the extent of intestinal infection, the degree of mucosal damage and the severity of clinical disease induced by cryptosporidium in piglets.