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C G Brown

Publications and source records attributed to C G Brown.

At least 37 records · Page 2Linked to original sources
PubMed1998

Theileria annulata: the expression of two novel macroschizont antigens on the surface of infected mononuclear cells differs during in vitro attenuation of a virulent cell line.

The first part of this study of the biological mechanisms underlying attenuation of virulent Theileria annulata macroschizont-infected cell lines screened four pairs of T. annulata (Hisar) in vivo- and in vitro-derived macroschizont-infected cell lines (lines) and identified a single in vivo-derived line, which induced lethal tropical theileriosis. The other seven lines were relatively avirulent. Analysis of the clinical, hematological, and parasitological responses of cattle immunized with different passages of the virulent line after in vitro culture showed that it was partly attenuated by passage (p) 50 and avirulent by p130. Clones representing the three glucose phosphate isomerase (GPI) isotypes, which constituted the newly isolated virulent culture, were obtained from p3 by limiting dilution; p50 and p130 consisted of one isotype. The second part of the study raised monoclonal antibodies (MAbs) against macroschizont-infected cells, as reagents for detecting antigenic differences between virulent and avirulent parasites, and identified two MAbs that recognized the surface of infected cells as well as macroschizonts. MAb EU1 recognized an antigen expressed by all the lines tested, whether in vitro- or in vivo-derived, whether uncloned or cloned, and irrespective of extent of subpassage in culture. MAb EU106 recognized an antigen whose expression by the virulent line and its clones disappeared on passage in culture. This antigen was not expressed at all by the avirulent in vitro-derived line prepared with cells from the same calf. Both antigens were expressed by lines infected with other stocks of T. annulata, including two lines known to induce lethal disease. The different profiles of expression of the two novel antigens, recognized by MAbs EU1 and EU106, by the line undergoing attenuation suggest (1) that the two antigens interact differently with the bovine immune system; and (2) that there are two, very different, potential roles for these antibodies in the development of vaccines against T. annulata infections.

Animals↗
PubMed1998

Identification of a Theileria annulata antigen expressed in multiple stages of the parasite life cycle.

In order to identify sporozoite surface molecules which may be involved in invasion and could act as potential vaccine candidates, a number of Mabs were raised in mice against T. annulata sporozoites. These were assayed for their ability to block sporozoite invasion of bovine peripheral blood mononuclear (PBM) cells in vitro. One of these, Mab 4B11, was found to neutralize sporozoite invasion to a high degree and to recognize a group of sporozoite antigens on Western blots. A T. annulata lambdagt11 genomic expression library was screened with Mab 4B11 and a positive clone containing a 900-bp insert (KP8) analysed further. Data from Southern and Northern blotting indicated that the gene containing the KP8 sequence, termed sporozoite and macroschizont gene 2 (spm2), was expressed both in T. annulata sporozoites and in later parasite life-cycle stages, macroschizont-infected leucocytes and piroplasms. The KP8 sequence was expressed in E. coli as a fusion protein with glutathione-S-transferase (GST) using the vector pGEX1lambdaT. Bovine antiserum raised against GST-KP8 recognised a single high molecular weight molecule on Western blots corresponding to one of the antigens recognised by Mab 4B11, expressed in sporozoites, macroschizont-infected leucocytes, and piroplasms. While our evidence suggests that the spm2 molecule alone is not responsible for sporozoite neutralization, it is a multistage antigen likely to function both in T. annulata sporozoites and in subsequent parasite life-cycle stages.

Amino Acid Sequence↗
PubMed1998

In vitro cultivation of an African strain of Babesia bigemina, its characterisation and infectivity in cattle.

An African (Kenyan) strain of Babesia bigemina, Muguga (B(2-1)), was inoculated into a calf from a stabilate and blood from the calf was used to establish the parasite in vitro. The strain has been cultured continuously for 20 months, initially in bovine erythrocytes with 60% adult bovine serum, later, with 50%. Cultures were incubated at 37 degrees C in RPMI 1640 medium with a gas mixture of 1% O2, 5% CO2, 94% N2. Adaptation in vitro was demonstrated when serum from a calf which had recovered from infection with B(2-1) bound to proteins of Mr 46 kDa, 49 kDa, 52 kDa, 61 kDa and 72 kDa on Western blots of B(2-1) antigens from cattle blood but did not recognise the 49 kDa or 52 kDa antigens from in-vitro-derived parasites. These proteins were considered specific for B(2-1), as they were not recognised by the same serum on profiles of a Mexican isolate of B. bigemina or an African isolate of B. bovis (Kwanyange). After 9 months of in vitro culture, a stabilate of the cultured parasite was injected into two splenectomised calves and one intact calf. The calves experienced a drop in packed cell volume and low parasitaemias but recovered spontaneously. Two of these animals, one splenectomised and one intact, were challenged with virulent B(2-1) and experienced only mild babesiosis, in contrast to a previously uninfected calf also challenged with B(2-1), which had to be euthanised after 5 days with severe babesiosis.

Africa↗
PubMed1998

Phenotypic and genotypic alterations associated with the attenuation of a Theileria annulata vaccine cell line from Turkey.

Attenuated vaccines, produced by prolonged in vitro culture of the macroschizont stage of the life-cycle, are the main method of controlling Theileria annulata infections. Little is known about the mechanism(s) of attenuation. Here we present data from a Turkish cell line demonstrating that attenuation is associated with reduced ability to differentiate into microschizonts and a reduction in matrix metalloproteinase activity. We also show that attenuation results in a change in the structure of the parasite population. Using the technique of differential mRNA display, we demonstrate that gene expression profiles differ between non-attenuated and attenuated macroschizont infected leucocytes. One differentially expressed gene is of parasite origin. These data are discussed in the context of a multifactorial model for virulence.

Animals↗
PubMed1998

Metastasis of Theileria annulata macroschizont-infected cells in scid mice is mediated by matrix metalloproteinases.

Theileria annulata (Ta)-infected leucocytes are able to disseminate in scid mice. The dose of virulent parasites of the Ta-Ode line required to achieve quantifiable dissemination was found to be 2 x 10(6) cells given i.p. Dissemination was higher on day 11 post-inoculation than on day 18. The attenuated Ta-Ode cells were found to disseminate very poorly compared to their virulent progenitors, which correlates with a marked reduction in matrix metalloproteinase (MMP) expression. A daily i.p. injection of mice with BB94, a synthetic inhibitor of MMPs, almost completely ablated dissemination compared to controls. This provides strong evidence that metastasis of Theileria annulata macroschizont-infected host cells is mediated by host MMPs induced by the parasite. This has important implications for explaining a number of pathological features of tropical theileriosis in cattle.

Animals↗
PubMed1998

Theileria lestoquardi--maturation and quantification in Hyalomma anatolicum anatolicum ticks.

The maturation and quantification of Theileria lestoquardi (T. hirci) parasites in unfed and partially fed adult Hyalomma anatolicum anatolicum ticks was studied using (1) methyl green pyronin (MGP) staining of salivary glands, (2) in vitro infection of peripheral blood mononuclear cells (PBM) with parasites harvested from infected ticks and (3) a semi-quantitative polymerase chain reaction (PCR). With MGP staining the greatest infection rate was seen in unfed ticks. Feeding resulted in a gradual reduction in the number of infected acini with a concomitant increase in the maturity of the parasites. In vitro infection of sheep PBM with titrated group-up tick supernate (GUTS) demonstrated that infectivity peaked between 2 and 4 days of tick feeding whereas GUTS prepared from unfed ticks was not infective. The polymerase chain reaction (PCR) was both sensitive and specific, detecting T. lestoquardi DNA in unfed and partially fed ticks, with a maximum sensitivity of 0.022 infected acinus/tick in 2-day fed ticks, though it gave no indication of the infectivity of the parasite.

Animals↗
PubMed1998

Bovine babesiosis in dual purpose calves in the state of Yucatan, Mexico.

Thirty-eight dual purpose calves in the 3 agricultural zones of the state of Yucatan, Mexico were monitored for antibodies to Babesia bovis and B. bigemina from close to birth up to 12 months of age. The results showed that infection with both parasites is widespread throughout the 3 zones. Many of the animals had produced antibody to both parasites by 6 months of age indicating high transmission rates resulting in enzootic stability for both parasites. Any changes to agricultural practices in the state that could affect disease stability through either reduction in numbers of tick vectors or the introduction of susceptible adults will have to be accompanied with measures such as vaccination to limit losses due to clinical babesiosis.

Age Distribution↗
PubMed1998

Nitric oxide causes the macroschizonts of Theileria annulata to disappear and host cells to become apoptotic.

The proliferation of Theileria annulata macroschizont-infected cell lines in vitro was significantly inhibited by nitric oxide (NO) generated by S-nitroso-N-acetyl-DL-penicillamine (SNAP). Incubation with SNAP caused the macroschizonts to disappear and host cells to become apoptotic. SNAP-derived NO also significantly inhibited the incorporation of tritiated thymidine by cultures of cells in which the schizonts had been induced to differentiate into merozoites by maintenance at 41 degrees C instead of 37 degrees C, the temperature used for culturing macroschizont-infected cells. These results point to NO as the mediator of macrophage anti-T. annulata activity and provide new evidence that the protective immune mechanisms which allow cattle to recover from primary infection and resist challenge may be attributed principally to the products of activated macrophages. These findings indicate that effective inactivated vaccines against T. annulata should include antigens able to stimulate the type of CD4+ T cell response which elicits macrophage activation and NO synthesis.

Animals↗
PubMed1997

Revaccination with the same Theileria annulata infected cell line may not be feasible for boosting immunity against tropical theileriosis.

A model for studying re-immunisation using skin grafting was developed as the allogeneic responses produced by T. annulata cell lines were similar to those produced by skin grafting. Appearance of schizonts and piroplasms post-immunisation was either delayed or prevented by already existing allogeneic responses. Isolation of parasite infected cell lines from lymph node biopsies and peripheral blood after cell line immunisation was also delayed or prevented by already existing allogeneic responses. Ability to isolate cell lines after immunisation correlated with protection i.e. if no parasite infected cell line of donor origin was isolated after immunisation, there was no protection. Allogeneic responses delayed or prevented the appearance of MHC I restricted parasite specific cytotoxic T lymphocytes post-immunisation. If the parasite transfer was prevented after immunisation; animals were fully susceptible to challenge. These experiments showed that allogeneic responses, generated in animals after immunisation with T. annulata schizont cell culture vaccine, can block parasite transfer and further development or enhancement of immunity against the parasite at the time of second immunisation with the same cell line. The observations are of immediate importance in endemic areas where T. annulata infected cell culture vaccines are being used. They are even more relevant in countries where animals are regularly moved between theileriosis free and endemic areas. It may not be advisable to re-immunise animals with the same cell line as that used for first vaccination.

Animals↗
PubMed1997

Protective immune responses to Theileria annulata of relevance to vaccine development.

A series of projects on Theileria annulata funded by the European Union (STD1/STD2/STD3) have provided convincing evidence that macrophage and natural killer (NK) cell-dependent immune mechanisms may directly control the proliferation of different stages of T. annulata in cattle. The evidence for this conclusion and the implications for vaccine development are discussed in the following paper.

Animals↗
PubMed1997

Use of the polymerase chain reaction for identification and quantification of Theileria parva protozoa in Rhipicephalus appendiculatus ticks.

The polymerase chain reaction (PCR) was adapted for detection of Theileria parva sporoblasts in Rhipicephalus appendiculatus ticks by comparison with staining of histological preparations of ticks with methyl green and pyronin (MGP). Two 32mer primers (IL174 and IL179) were used to amplify Theileria parva (Muguga isolate) DNA from the TPR 1 region of the genome by the PCR. Detection of T. parva was carried out with dissected salivary glands and whole ticks preserved in ethanol. Adult ticks which fed as nymphs on a T. parva infected calf were used in three experiments. Firstly, 70 whole ticks divided into 7 batches representing the rising and falling parasitaemia of the calf were used to show that detection of infection by the PCR was significantly correlated with MGP staining. Secondly, 120 dissected ticks were used from 4 different batches representative of the overall infection profile within the ticks to show a high correlation between PCR quantification within tick salivary glands and MGP count data of the paired gland. Thirdly, 120 ticks were used in batches selected for high and low infections. Bloodmeal contaminants from partially fed adult ticks, present in 60 out of the 120 ticks used, did not inhibit the PCR amplification of T. parva DNA. This experiment also showed a great increase in infection detection in partially fed batches of ticks compared to the untreated batches.

Animals↗
PubMed1997

Bovine cells infected in vivo with Theileria annulata express CD11b, the C3bi complement receptor.

Bovine cells from cattle infected with Theileria annulata were phenotyped with monoclonal antibodies recognizing bovine leukocyte antigens. Macroschizont-infected, transformed cell lines prepared from peripheral blood mononuclear cells of cattle, infected with sporozoites, were assessed by flow cytometry; parasitized cells in tissues from infected cattle were examined by immunocytochemical techniques. Co-expression of markers for different cell lineages by the cell lines precluded a definite conclusion as to their phenotypic origins. For, while the pattern of leukocyte antigens expressed by these in vivo-derived schizont-infected cells, which included CD11b, was indicative of a myeloid origin, the possibility that they were NK cells could not be excluded. The monoclonal antibody (MAb) IL-A15, which recognizes CD11b, reacted with a high proportion of parasitized cells in sections of tissues from infected cattle at all stages of acute disease. Mononuclear cells infected with parasites at all stages of differentiation, from macroschizont to microschizont, expressed CD11b. Such parasitized cells occurred throughout the lymphoid tissues, being found in the thymus, spleen and lymph nodes, particularly the prescapular node draining the site of infection, the hepatic, mesenteric and precrural nodes, as well as in the reticulo-endothelial tissue of the liver, kidney, lung, abomasum, adrenal and pituitary glands. These observations provided the first evidence for a myeloid origin for the parasitized T. annulata cells found in infected bovine tissues and blood and suggested a mechanism whereby schizonts could transfer from cell to cell during mechanical infection with schizont-infected cells.

Animals↗
PubMed1997

Allograft responses can interfere with the development of immunity against Theileria annulata following vaccination with parasite infected cell lines.

Theileria annulata macroschizont-infected cell lines are successfully used as vaccines in several countries. The inoculated animals produce a strong allogeneic response against the MHC antigens of the immunizing cell line followed by an anti-parasite response. Immunity against the parasite wanes in the absence of challenge and re-immunization is sometimes recommended. However, it is not known if allogeneic responses generated by the first immunization with a T. annulata infected cell line will interfere with the boosting of immunity against the parasite at the time of re-immunization with the same cell line. Animals were primed against MHC antigens by skin grafting, followed by immunization with a T. annulata infected cell line prepared from the skin donor. A strong anti-MHC response was produced. This interfered with parasite transfer and the development of an anti-parasite immune response; the effect was more marked when a low vaccine cell dose was used. There was a negative correlation between the ease of isolating infected cells from the animals after cell line immunization, and the subsequent response to challenge. Where no cell lines could be isolated, the animals were fully susceptible to sporozoite challenge. These observations are of immediate importance in endemic areas where cell lines of T. annulata schizonts are being used as vaccines to control the disease.

Animals↗
PubMed1997

Analysis of the ventricular fibrillation ECG signal amplitude and frequency parameters as predictors of countershock success in humans.

OBJECTIVE: The purpose of this study was to assess from the ventricular fibrillation ECG signal whether certain amplitude parameters, or frequency parameters derived using fast Fourier transform analysis, are predictive of countershock success (defined as a stable supraventricular rhythm following countershock). DESIGN: Retrospective, descriptive study. SETTING: Emergency medical service at a university hospital. PATIENTS: Twenty-six patients with out-of-hospital cardiac arrest, whose initial ECG rhythm was identified as ventricular fibrillation. METHODS AND RESULTS: In all patients, advanced cardiac life support was performed in the out-of-hospital setting and a semiautomatic defibrillator was used for countershock therapy and simultaneous on-line ECG recording. For each patient, ECG data were stored in modules in digitized form over a period of 20 min and analyzed retrospectively. Using fast Fourier transform analysis of the ventricular fibrillation ECG signal in the frequency range of 0.3 to 30 Hz (mean +/- SD), median frequency, dominant frequency, edge frequency, and amplitude were as follows: 5.17 +/- 1.05 Hz, 4.56 +/- 0.99 Hz, 10.74 +/- 3.46 Hz, and 1.33 +/- 0.44 mV before successful countershock (n = 20); and 4.21 +/- 1.17 Hz (p = 0.0034), 3.31 +/- 1.57 Hz (p = 0.0004), 9.46 +/- 2.93 Hz (p = 0.5390), and 1.15 +/- 0.69 mV (p = 0.0134) before unsuccessful countershock (n = 134). Using software filters to completely eliminate interference due to manual cardiopulmonary resuscitation from the ventricular fibrillation power spectrum, only amplitude remained statistically different (p < or = 0.03) in predicting countershock success. CONCLUSIONS: We conclude that in patients, median frequency, dominant frequency, and amplitude are predictive of countershock success in humans.

Electric Countershock↗
PubMed1997

Median fibrillation frequency in cardiac surgery: influence of temperature and guide to countershock therapy.

OBJECTIVE: This study was designed (1) to investigate the effects of normothermic and hypothermic perfusion on the median frequency of the fibrillating myocardium, and (2) to elucidate whether frequency-guided countershock therapy improves countershock success during the reperfusion phase of cardiac surgery. DESIGN: Prospective, randomized study. SETTING: University hospital cardiac surgery room. PATIENTS: Thirty patients (first part of the study) and 38 patients (second part of the study) scheduled for elective coronary artery bypass surgery. METHODS AND RESULTS: During cardiopulmonary bypass, ventricular fibrillation (VF) was induced at a core body temperature of 34.1+/-0.2 degrees C (normothermia) (n=15) or at a core body temperature of 29.8+/-0.2 degrees C (hypothermia) (n=15). Using fast Fourier transformation of the ECG signal, median fibrillation frequency was recorded continuously for a period of 120 s. At the end of surgery, countershock was performed as soon as VF was recognized on the ECG monitor (X Hz group; n=19) or countershock was not performed until median fibrillation frequency had increased to the threshold of at least 5 Hz (5 Hz group; n=19). Median fibrillation frequency in the normothermic fibrillation group was statistically higher than in the hypothermic group. In the X Hz and 5 Hz countershock group, median fibrillation frequency before the first countershock attempt was 3.6+/-0.2 Hz and 5.4+/-0.1 Hz (p<0.0001), respectively. In the X Hz group, six countershocks resulted in supraventricular rhythm, 10 in VF, two in electromechanical dissociation, and one in asystole. In the 5 Hz group, 16 countershocks resulted in supraventricular rhythm, two in VF, and one in asystole (p=0.008). CONCLUSIONS: During normothermia, median fibrillation frequency is significantly higher than during hypothermic perfusion conditions. During the reperfusion phase of cardiac surgery, countershock success rate is significantly higher when a threshold of at least 5 Hz had been reached before the first countershock attempt.

Coronary Artery Bypass↗
PubMed1996

Epidemiology of tropical theileriosis (Theileria annulata infection of cattle) in an endemic region of Tunisia: characterisation of endemicity states.

A serological survey on tropical theileriosis was conducted on a sample of 54 farms in a region within the semi-arid bioclimatic zone of Tunisia. Screening of cattle sera at a dilution of 1/160 using the indirect immunofluorescent antibody test with the schizont antigen of Theileria annulata, revealed the presence of animals with positive sera in 92.15% of the sampled farms. The exposure of calves to infection in the first season was shown to be significantly lower than in older cattle. Three endemic situations were identified based on the serological profiles of herds and the incidence and age distribution of disease cases. Endemic stability was observed in farms showing a sero-prevalence of 100% in cattle of four theileriosis seasons or more and by the incidence of the highest disease levels in cattle at their second and third theileriosis season. High endemic instability was identified on the basis of low sero-prevalence rates and the occurrence of the highest disease incidence in cattle at fourth theileriosis season or more.

Age Distribution↗
PubMed1996

Conservation of neutralizing determinants between the sporozoite surface antigens of Theileria annulata and Theileria parva.

The sporozoite surface antigens SPAG-1 of Theileria annulata and p67 of Theileria parva are postulated to contain determinants necessary for host cell invasion and/or recognition and are both being considered as candidates for inclusion in subunit vaccines. Preliminary data suggest that these are related molecules. In this paper we describe the investigation of the relationship between these sporozoite antigens further by analysis of the immunological cross-reactivity using Mabs and sera raised against each antigen. The cross-reactions were examined by carrying out Western blots, IFA tests, and in vitro sporozoite neutralization assays. In addition, sequence comparison data which clearly establish that these surface antigens are encoded by related genes are presented. The regions of SPAG-1 identified as containing cross-reactive epitopes recognized by p67 antiserum correlated to regions of high predicted homology between p67 and SPAG-1, which are located at their respective N- and C-termini. Furthermore, p67 and SPAG-1 were found to contain cross-reactive determinants responsible for neutralization of sporozoite infectivity in vitro, and at least some of these were located in the C-termini of both molecules. The relevance of these findings to the possible roles for these molecules in host cell invasion is discussed.

Amino Acid Sequence↗