An in-vitro model for studying the interaction of Escherichia coli O157:H7 and other enteropathogens with bovine primary cell cultures.

Sections of kidney, trachea, ileum, colon, rectum and rumen were removed at post mortem from a neonatal calf and, with the exception of the rumen, primary cell lines were established for each of the cell types. The adherence of enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7, enteropathogenic E. coli (EPEC) serotype O111, E. coli K12 (a laboratory adapted non-pathogenic strain) and Salmonella enterica serotype Typhimurium was assayed on each cell type. For all adherence assays on all cell lines, EHEC O157:H7 adhered to a significantly greater extent than the other bacteria. S. Typhimurium and EPEC O111 adhered to a similar extent to one another, whereas E. coli K12 was significantly less adherent by 100-fold. In all cell types, >10% of adherent S. Typhimurium bacteria invaded, whereas c. 0.01-0.1% of adherent EHEC O157:H7 and EPEC O111 bacteria invaded, although they are regarded as non-invasive. EHEC O157 generated actin re-arrangements in all cell types as demonstrated by fluorescent actin staining (FAS) under densely packed bacterial micro-colonies. EPEC O111 readily generated the localised adherent phenotype on bovine cells but generated only densely packed micro-colonies on HEp-2 cells. The intensity of actin re-arrangements induced in bovine cells by EPEC O111 was less than that induced by EHEC O157:H7. The intimate attachment on all cell types by both EHEC O157:H7 and EPEC O111 was clearly demonstrated by scanning electron microscopy.

Field evaluation of an indirect ELISA for detection of brucellosis in lowland Bolivia.

Bovine brucellosis exists endemically at an estimated prevalence of 10% in the developing dairy industry of Santa Cruz in tropical Bolivia. This paper describes field testing of an FAO/IAEA indirect ELISA for brucellosis, as a possible replacement confirmatory test for the complement fixation test (CFT). The ELISA and CFT were compared on sera from 3 cattle populations: a non-vaccinated negative population, an S19-vaccinated negative population, and a brucellosis-positive population of unknown vaccination status. The CFT and ELISA showed excellent specificities of 100% and 98% respectively against the negative non-vaccinated group. The CFT maintained a specificity of 98% against the S19-vaccinated negative group, but ELISA specificity fell to 83% using a cut-off of 20% of positive control, and 94% using a cut-off of 40% of positive control. Against sera from the positive population, the ELISA gave many more positive reactions than the CFT, probably a combination of both higher sensitivity and lower specificity. It is concluded that as Santa Cruz is entering a phase of brucellosis control rather than eradication, the extra sensitivity of the ELISA is not valuable enough to risk a higher level of false positive reactions, especially as S19 vaccination is being increasingly used.

Trichinella spiralis infection in pigs in eastern Bolivia.

Trichinellosis in pigs in Bolivia was first documented in 1993 following a small abattoir survey in a rural community in the Bolivian Altiplano. The present study investigated the presence of antibodies to Trichinella spiralis in pigs in the 2 largest departments in terms of pig production in Bolivia. Three geographically separate abattoir surveys were conducted to cover the major production areas in the Departments of Santa Cruz and Chuquisaca. Sera were tested using an enzyme-linked immunosorbent assay. Of the 1,327 sera analysed from the 3 areas, 13.4% overall tested positive. Results from the 3 individual surveys varied from 10.2% seropositivity to 17.1 per cent. However, within each of the 3 sample areas, highly significant variation in seropositivity was encountered, with those areas with the most extensive production systems having the highest percentage of positive sera. Such variation is probably due to differences in nutrition with foraging and household waste being important components of pig diets in extensive production systems. The results of this study were similar to those obtained from the previous survey in the Altiplano and indicate that trichinellosis is present throughout Bolivia and is a potentially important public health problem.

Characterization of Theileria parva which infects waterbuck (Kobus defassa).

Theileria-free waterbuck (Kobus defassa) born in captivity were successfully infected with Theileria parva sporozoites derived from ticks infected by feeding on African buffalo (Syncerus caffer). All waterbuck underwent mild infections with the development of sporadic schizont and piroplasm parasitosis when inoculated with sporozoite doses lethal to cattle. A carrier state of T. parva was demonstrated by feeding clean R. appendiculatus nymphs on two of these infected waterbuck. Tick batches from these waterbuck on 2 of 5 occasions transmitted lethal Theileria infections to cattle. In a separate experiment, waterbuck cells were infected and transformed in vitro by T. parva sporozoites derived from buffalo but not by cattle-derived T. parva (Muguga) sporozoites. Waterbuck cells infected in vitro with T. parva isolated from buffalo were inoculated into autologous waterbuck but no infections developed. Theileria parva isolates generated in this study from various sources were characterized using anti-T. parva schizont monoclonal antibodies (MAbs), and it was found that buffalo-derived and waterbuck-passaged isolates had different profiles. Species-specific synthetic oligonucleotide probes, restriction fragment length polymorphism (RFLP) analysis with cloned T. parva DNA probes, and DNA sequence analysis of the p67 sporozoite antigen gene confirmed that the waterbuck-passaged parasite was T. parva. The Tpr repetitive probe hybridization patterns from the waterbuck-passaged parasites were different from the other samples tested. The ribosomal genotype of the waterbuck-passaged T. parva was similar to that of cattle-derived T. parva Muguga. Analyses with both probes and MAbs suggested that a minor parasite population present within the T. parva 7014 buffalo-derived stock had been selected during waterbuck passage.(ABSTRACT TRUNCATED AT 250 WORDS)

Transformation of Theileria parva derived from African buffalo (Syncerus caffer) by tick passage in cattle and its use in infection and treatment immunization.

A sporozoite stabilate (St. 199) of Theileria parva was obtained by feeding nymphal Rhipicephalus appendiculatus on an African buffalo (Syncerus caffer) and was used to immunize cattle by the infection and treatment method. Nymphal ticks were applied to one of the steers 90 days later and it was shown that the resultant adult tick had become infected. Using tick/cattle passage, two passage lines of T. parva were established. By the fifth tick/cattle passage, the parasite stocks had changed their behaviour to that of T. parva derived from cattle as the parasite produced relatively high schizont parasitosis and piroplasm parasitaemia in cattle, and had become highly infective to ticks. At various passage levels the parasite populations were characterized by behaviour and by monoclonal antibodies against T. parva schizonts using infected cell culture isolates from cattle during acute infections. The monoclonal antibody profile showed little evidence of antigen change of the parasite during passage through cattle, which was confirmed in a two-way cross-immunity experiment using sporozoite stabilate derived from ticks obtained from the buffalo and fourth passage in cattle. The implication of these results, particularly in relationship to immunization of cattle against T. parva derived from buffalo, is discussed.

Low-dose oral administration of human interferon alpha can control the development of Theileria parva infection in cattle.

Two natural human interferon alpha preparations, (nHuIFN-alpha [Cantell]) and (nHuIFN-alpha [ISI]), were used for the oral treatment of cattle experimentally infected with Theileria parva parva. In the first experiment, 8 Friesian bulls were inoculated with a 1 in 10 dilution of a sporozoite stabilate of T.p. parva (Marikebuni) stock. Four of the cattle were treated daily with 1 international unit/kg body weight (i.u./kg bwt) of nHuIFN-alpha (Cantell) from day -2 to day 8 p.i. None of the 4 calves given IFN developed clinical theileriosis, but 3 of the 4 control calves died of theileriosis while the fourth had a mild infection. Three of 4 treated calves and the 1 surviving control calf developed a detectable antibody response to T.p. parva schizont antigen but, on challenged with a 10-fold higher dose of stabilate, the surviving control animal and only 1 of the 4 treated calves proved to be immune. In a second experiment, 4 groups of 4 calves were inoculated with the same stabilate dilution. Three treatment groups were given either 1 i.u. nHuIFN-alpha (Cantell), 1 i.u. nHuIFN-alpha (ISI), or 10 i.u. nHuIFN-alpha (ISI)/kg bwt from day -2 to day 8 p.i. once daily and the fourth group were controls. Clinical theileriosis occurred in 2 controls, 2 calves given 10 i.u. nHuINF-alpha (ISI), 1 calf given 1 i.u. nHuIFN-alpha (ISI) and no calves given 1 i.u. nHuIFN-alpha (Cantell)/kg bwt. Of these, 2, 1, 0 and 0 cattle died in the respective groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Theilerial parasites isolated from carrier cattle after immunization with Theileria parva by the infection and treatment method.

Groups of cattle were immunized with 10(-2) dilutions of sporozoite stabilates of Theileria parva lawrencei derived from African buffaloes either alone or in combination with Theileria parva parva derived from cattle and concomitant treatment with either long or short-acting formulations of oxytetracyline. At 90 or 120 days after infection, uninfected Rhipicephalus appendiculatus nymphal ticks were applied to individual immunized cattle and the resultant adults ticks were applied to individual susceptible cattle. Theilerial infection developed from ticks fed on 6 out of 11 animals investigated for evidence of a carrier state. Two additional animals were shown by cell-culture isolation to have persistent theilerial infections. Nine cattle infected with the parasites from carrier animals were treated with paravaquone and 7 recovered. These recovered cattle were then challenged with the original immunizing stabilates at 10 degrees dilution together with the original immunized and carrier cattle. Six out of 7 cattle which had recovered from carrier-derived infection succumbed to this challenge and died but none of the original immunized cattle showed theilerial reactions. When a carrier-derived sporozoite stabilate was used to challenge cattle immune to the original immunizing parasite, they proved to be immune. Cattle immune to the carrier-derived parasites were all immune to challenge with the original parasite. A monoclonal antibody profile against T. parva schizonts isolated by cell culture from samples of the experimental animals did not appear to be sensitive enough to determine the antigenic differences between the carrier-derived parasite and the original immunizing parasite. Indications are that the carrier state is not likely to produce new antigenic strains which would be dangerous to immunized cattle.

Isolation of bovine herpesvirus-3 from African buffaloes (Syncerus caffer).

Eleven virus isolations were made from the blood of 45 free living healthy African buffaloes by long term cocultivation of their leucocytes with bovine thymus or spleen cells. The isolates were indistinguishable from each other or from herpesviruses isolated from a severely ill buffalo calf and from a dead buffalo. These viruses possessed the characteristics of the bovine herpesvirus-3 (BHV-3) group and were indistinguishable by serology and restriction endonuclease analysis from the BHV-3 type strains Movar 33/63 and DN599. There was a 93.6 per cent prevalence of indirect immunofluorescent antibody to BHV-3 in the sera of 94 buffaloes in the sample population. No clinical signs or viraemia were detected in five cattle inoculated with 10(8.7) log10 TCID50 of the isolate from the sick buffalo calf. Two of three cattle hyperimmunised with this virus resisted challenge with malignant catarrhal fever herpesvirus, which proved fatal for the other immunised animal and for three control cattle.

Characterization of buffalo-derived theilerial parasites with monoclonal antibodies and DNA probes.

The characteristics of intra-lymphocytic Theileria isolated from African buffalo and from cattle that were infected with buffalo-derived parasites were evaluated using anti-schizont monoclonal antibodies (mAbs) and DNA probes. Antigenic differences were revealed by the reactivities of 27 mAbs with the buffalo-derived parasites isolated from different animals. Antigenic diversity was also seen with Theileria-infected lymphoblastoid cell isolates taken from the lymph nodes and lambda gt11, showed specific hybridization to parasite DNA in Southern blots of restriction enzyme-digested, lymphoblastoid cells infected with buffalo-derived theilerial parasites. Genotypic differences between the buffalo-derived parasites were revealed by the restriction fragment length polymorphisms seen with hybridization of those probes to DNA from cloned and uncloned Theileria-infected cell lines. The evaluation of theilerial parasites derived from buffalo and from cattle which underwent typical T. p. lawrencei reactions, after being infected with buffalo-derived theilerial parasites, did not show any specific phenotypic or genotypic characteristics of these parasites that would distinguish them from T. p. parva and T. p. bovis parasites. The validity of these subspecies distinctions is discussed.

Cytotoxic T-cells elicited in cattle challenged with Theileria parva (Muguga): evidence for restriction by class I MHC determinants and parasite strain specificity.

The MHC restriction and parasite strain specificity of cytotoxic cells elicited in a group of Theileria parva (Muguga)-immunized cattle following homologous challenge, were investigated. The cytotoxic cells were specific for parasitized target cells and in 9 of the 10 animals examined, they were clearly genetically restricted. Cytotoxicity could be inhibited by monoclonal antibodies (MoAb) to class I MHC molecules but not by MoAb to class II molecules, indicating that a large component of the response was restricted by class I MHC determinants. Low levels of inhibition of cytotoxicity were also obtained with a MoAb to the T-cell subset marker BoT8, suggesting that at least part of the response was mediated by BoT8+ lymphocytes. When cytotoxic cells from individual cattle were assayed on panels of parasitized target cells, there was a close correlation between susceptibility of the target cells to lysis and sharing of BoLA-A locus-encoded specificities with the effectors. This observation, taken together with the knowledge that within several of the sets of BoLA-A-matched targets the relevant BoLA-A specificities were on different MHC haplotypes, indicated that the responses were restricted predominantly by BoLA-A products. In individual cattle there was a striking bias in the restriction of the response to one or other BoLA-A specificity. Among the six specificities represented, responses restricted by w6, w8 and KN18 consistently predominated over responses restricted by w7, w10 and w11. In the three cattle tested for parasite strain specificity, two showed complete specificity and one partial specificity for cells infected with the parasite stock used for immunization, T. parva (Muguga).

Isolation of Theileria parva lawrencei-infected lymphoid cell lines from free-ranging African buffaloes (Syncerus caffer).

Lymphoid cells collected from the peripheral blood of 45 free-ranging buffaloes sampled in the Masai Mara Game Reserve in Kenya were cultured in vitro in an attempt to establish cell lines of intralymphocytic theilerial schizonts. Theileria parva lawrencei-infected lymphoblastoid cell lines were established with samples taken from 12 buffaloes, and 11 of these were maintained continuously in vitro. Sixteen of the buffalo samples were contaminated with either trypanosomes or viruses. The successful in vitro isolation of Theileria species from 27 per cent of the buffaloes sampled demonstrates the applicability of this technique for field isolation of T parva lawrencei.

Isolation of Theileria parasites from African buffalo (Syncerus caffer) and characterization with anti-schizont monoclonal antibodies.

Antigenic differences between intra-lymphocytic theilerial parasites isolated from the blood of 18 African buffalo and grown in vitro were assessed with anti-schizont monoclonal antibodies (mAbs). There was marked antigenic diversity both between isolates from different buffalo and between isolates taken at different times from the same buffalo. Many of the isolates from both wild and captive buffalo appeared to consist of mixed parasite populations. Some isolates were found by limiting dilution cloning and mAb testing to contain at least 3 or 4 distinct populations of Theileria. Once cloned, Theileria-infected lymphoblastoid cell lines retained their mAb profiles during prolonged in vitro cultivation and, when recloned, the subclones had the same mAb profile as their parent clone. The implications of these results for further studies on buffalo-derived theilerial parasites are discussed.

Experimental induction of Theileria parva lawrencei carrier state in an African buffalo (Syncerus caffer).

An African buffalo (Syncerus caffer), born in captivity and demonstrated to be Theileria-free, and 2 susceptible cattle were inoculated with a Theileria parva lawrencei sporozoite stabilate. The buffalo had a very mild disease reaction, while the 2 cattle died of acute theileriosis. It was possible to isolate T. p. lawrencei from the buffalo up to 888 days after infection by the application of non-infected Rhipicephalus appendiculatus nymphs and up to 657 days after infection by the establishment of lymphoblastoid cell lines infected with T. p. lawrencei schizonts from peripheral mononuclear blood cells. The infection rate and levels of Theileria in the resultant adult ticks varied from 11 to 70% with 0.3-11 acini infected/tick. Stabilates prepared from these tick batches caused fatal T. p. lawrencei infections in cattle.

Infection of African buffalo (Syncerus caffer) and cattle with Theileria parva lawrencei after serial passage in cattle.

The infectivity of a Theileria parva lawrencei stabilate, from a stock derived from an African buffalo (Syncerus caffer) in the Serengeti National Park, Tanzania, was investigated. In the first experiment a buffalo and three cattle were inoculated with a stabilate from a stock passaged three times in cattle. All cattle developed fatal theilerial infections. Isolations from the buffalo by tick feeding and cell culture isolation showed that it was infected with T p lawrencei at the time of inoculation, but the second isolation made 19 days after inoculation behaved like T p parva in cattle, developing a high parasitosis, while the third isolation made three months later behaved like T p lawrencei with low parasitosis. It was concluded that two biological types of T parva could exist in a buffalo at one time, but it was not shown that the buffalo had become a carrier of T p lawrencei adapted to cattle. In the second experiment two buffaloes and three cattle were inoculated with T p lawrencei (Serengeti) stabilate which had been passaged six times through cattle and ticks. The two buffaloes had mild theilerial infections and developed serological titres in the indirect fluorescent antibody test, but the cattle had fatal infections. Tick and cell culture isolations of T parva were possible during the clinical reactions of the buffaloes, but no carrier state was demonstrated. Theileria-infected cell lines were established from the buffaloes and the cattle and were examined using monoclonal antibodies against T parva schizonts.(ABSTRACT TRUNCATED AT 250 WORDS)

A cell surface antigen associated with Theileria parva lawrencei-infected bovine lymphoid cells.

Attempts to produce monoclonal antibodies specific for a parasite-encoded or induced antigen on the surface of Theileria parva lawrencei-transformed bovine lymphocytes resulted in the identification of the monoclonal antibody J7. This antibody recognizes a membrane glycoprotein on some T.p. lawrencei-infected bovine lymphoid cells but not on any uninfected bovine lymphoid cells. Inoculation of allogeneic recipient cattle with T.p. lawrencei-transformed cells bearing the J7 determinant resulted in transfer of the parasite but not of the J7 epitope to the recipients' own cells. The presence of the J7 determinant could not be correlated with the parasite phenotype nor with the surface phenotype of the infected cells. It is therefore not known whether the J7 epitope is of parasite origin or whether it is a bovine differentiation antigen modified by the presence of the parasite.

Passive endocytosis of sporozoites of Theileria parva in macrophages at 1-2 degrees C.

Phagocytosis by macrophages is generally considered to involve two steps which can be experimentally dissociated: attachment, which is not energy-dependent, and interiorization, which does not occur below 15 degrees C. We have previously reported entry of the sporozoites of Theileria parva into bovine lymphocytes at 2 degrees C by a process of passive endocytosis which appears to depend entirely upon progressive circumferential binding of ligands on the parasite to receptors on the host cell membrane. We report here similar entry into macrophages in vitro at 1-2 degrees C. It is suggested that in vivo small (1 micron) spherical parasites bearing appropriate ligands can invade phagocytic or non-phagocytic host cells by a process of endocytosis that requires little or no expenditure of energy.

Bovine alloreactive cytotoxic cells generated in vitro: target specificity in relation to BoLA phenotype.

Cytotoxic cells of bovine origin were generated in primary MLC using stimulator cells of BoLA w8/w11 phenotype. Bovine lymphoblasts transformed by the protozoan parasite Theileria parva parva acted as target cells in studies of the specificity of cytotoxicity. When responder cells in MLC did not share w8 or w11 with stimulator cells, cytotoxicity was evident with all targets bearing w8 or w11, or both, and was almost entirely restricted to these products of the BoLA-A locus. When responder and stimulator cells shared both w8 and w11, cytotoxicity was also generated. Whether this was specific for the products of other putative Class I loci in cattle, or for the products of a Class II region, remains to be determined. These results suggest that the determinants recognized by appropriately generated bovine alloreactive cytotoxic cells are identical with, or closely related to, determinants characterized by BoLA w8 and w11 defining alloantisera.