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Biomedical subjects

C G Brown

Publications and source records attributed to C G Brown.

At least 19 recordsLinked to original sources
PubMed1999

Tissue damage in cattle infected with Theileria annulata accompanied by metastasis of cytokine-producing, schizont-infected mononuclear phagocytes.

The distribution of schizont-infected cells in six calves undergoing acute, lethal sporozoite-induced infections with Theileria annulata was examined, the calves being killed in the early, middle or late stages of disease. A combination of histological and immunocytochemical techniques showed that schizont-infected cells became disseminated rapidly through the lymphoid tissues from the prescapular lymph node draining the site of inoculation to distant lymph nodes (e.g., precrural, mesenteric and mediastinal) and to the spleen and thymus. The parasitized cells also spread rapidly into non-lymphoid organs, being found in the liver, kidney, lung, abomasum, adrenal glands and pituitary gland by day 7, in the brain by day 12 and in the heart by day 14 after infection. As infection progressed, the schizonts differentiated into merozoites. By the late stages of disease, the cells containing merozoites greatly out-numbered schizont-infected cells. The parasitized mononuclear cells were labelled by antibodies to bovine interferon-alpha1 and tumour necrosis factor-alpha and, during the later stages of the disease, contained erythrocytes parasitized by piroplasms. The results suggested that the parasitized mononuclear cells themselves played a role in the development of clinical disease and in tissue damage. These findings provide new evidence that tropical theileriosis can no longer be viewed as a lymphoproliferative disease resulting from the uncontrolled multiplication and metastasis of lymphoid cells infected with T. annulata schizonts, but is caused by a parasite that lives in, and is disseminated by, cytokine-secreting, proliferating mononuclear phagocytes.

Animals↗
PubMed1998

Stage-specific activity in vitro on the Theileria infection process of serum from calves treated prophylactically with buparvaquone.

An in vitro method for testing activity of buparvaquone in serum on the infection and development of Theileria in its bovine host mononuclear cells is described and results compared with the effect exhibited in vivo. Serum samples were collected over a time course from calves in a clinical trial of 5 mg kg(-1) buparvaquone prophylaxis on Theileria annulata or T. parva experimental infection. To evaluate drug levels and persistence in each animal for a period of 14 days and its effect on the early infection stages, the sera were tested on established macroschizont infected cell lines and against the in vitro infection and development process of the sporozoite and trophozoite stages of the two Theileria species. Results from the in vitro assays show that buparvaquone in serum can completely prevent the establishment of Theileria infection during the first 48 h after administration at 5 mg kg(-1). After seven days, levels are sufficient to delay the establishment of infection. The drug is more effective in the prevention of the de novo development of the parasite in cells than against established macroschizont infected cell culture. At low concentrations, it is more effective against T. parva than against T. annulata. Drug effect peaks during the first 24 h but residual effect persists for 14 days, particularly against T. parva infection. These novel findings demonstrate how high doses of buparvaquone could over-protect calves if used in the 'infection-and-treatment' method of immunisation when drug is administered prophylactically at the same time as infection with live sporozoites. It is suggested that in certain high Theileria risk situations there may be potential for the immunoprophylactic use of buparvaquone without simultaneous infection. The in vitro assay itself has been shown to be of value as a model for Theileria establishment in cattle.

Animals↗
PubMed1998

Chemoprophylaxis of Theileria annulata and Theileria parva infections of calves with buparvaquone.

A clinical trial testing the prophylactic effect of a 5 mg kg-1 dose of buparvaquone on either Theileria annulata or Theileria parva experimental infections of calves demonstrated its efficacy for periods of at least seven days. The drug given 1 h or seven days before 50% lethal T. annulata sporozoite infection protected all eight calves, but prophylaxis was insufficient after 14 days to protect two out of four calves from severe reaction. When immunity was challenged by a lethal second parasite dose a month after the first, all these calves were immune. In the T. parva trial, calves given drug 1 h or seven days before a 25% lethal infection underwent minimal reaction, but some were over-protected and were susceptible to a similar challenge sporozoite dose. Although drug levels remaining 14 days after prophylaxis protected these calves from the mild challenge, some parameters measured were within the range of the 'no drug' control group. These results indicated the effectiveness of a single 5 mg kg-1 dose of buparvaquone for more than seven days but also the potential risk of its use in the infection and treatment method of immunisation. It is suggested that there may be circumstances where simple field prophylactic treatment with buparvaquone may be beneficial.

Animals↗
PubMed1998

Theileria lestoquardi and T. annulata in cattle, sheep, and goats. In vitro and in vivo studies.

Theileria annulata, causing bovine tropical theileriosis, and T. lestoquardi (syn T. hirci), the agent of malignant ovine theileriosis, are both transmitted by ticks of the genus Hyalomma. Their distribution is thus very similar and, should these parasites infect more than one ruminant species, the difficulty in interpreting epidemiological studies is magnified considerably. A pilot series of experiments was thus conducted in which cattle, sheep and goats were infected with sporozoites of a single stock of each of T. annulata and T. lestoquardi from a laboratory colony of H.a.anatolicum. Reciprocal cross-immunity and serological studies and in vitro culture isolations in mononuclear cells of each ruminant species illustrated both the similarity of these organisms and their differences. The importance of these findings in discriminating parasites in epidemiological studies and the control of these diseases with cell culture vaccines is emphasized.

Animals↗
PubMed1998

Theileria annulata: carrier state and immunity.

Recovery from primary infection of Theileria annulata results in the development of a persistent carrier state in the vertebrate host. The carrier state is of great importance in the maintenance of the life cycle by alternate tick/cattle challenge and both contributes to and may be necessary for maintenance of immunity. Therefore, an accurate determination of carrier animals could be useful in determining immune status and may allow the necessary control measures to be implemented. Detailed information on the carrier state of animals following immunization with attenuated cell lines is lacking. In this study, relationship between immune response, persistence of the parasite, and the antibody response has been investigated. Calves were infected with T. annulata sporozoites, low passage (non-attenuated) or high passage (attenuated, vaccine) cell lines and later challenged with a lethal dose of heterologous sporozoites. The presence and persistence of the parasite were monitored by PCR using primers derived from genes coding for ssrRNA and a 30 kDa major merozoite surface protein, by Giemsa stained blood smears to detect the presence of piroplasms and also by attempting to establish infected mononuclear cell cultures from venous blood. Antibody responses were measured by indirect ELISA using a merozoite recombinant antigen and IFAT using piroplasm and macroschizont antigens. Results showed that there was an evident relationship between the persistence of carrier status, antibody response in ELISA and immune response to challenge.

Animals↗
PubMed1998

Different vaccine strategies used to protect against Theileria annulata.

SPAG-1, a sporozoite surface antigen of T. annulata, has previously been shown to elicit partial protection when used, as an hepatitis B core antigen fusion, to immunize cattle. The objective of this study was to try and improve the protective capacity of this antigen by enlisting different vaccine strategies. Cattle were immunized with SPAG-1, as a fusion protein with a His6 tag, either incorporated into ISCOMs, with or without the merozoite antigens TAMS 1-1 and 1-2, or with RWL as adjuvant three times at monthly intervals. Another group of cattle were immunized with p67, the T. parva sporozoite antigen, in RWL to assess whether any cross-protection could be induced. The animals were then challenged with an estimated LD50 of T. annulata sporozoites, and their ability to resist the infection was investigated. Serum responses and T-cell proliferative responses were analyzed throughout the trial. Post-challenge analyses included lymph node biopsies and blood smears to check for the presence of parasites, routine hematological parameters, and observation for clinical manifestations of the disease. The results of this trial will be discussed.

Animals↗
PubMed1998

Immunochemical characterization of in vitro culture-derived antigens of Babesia bovis and Babesia bigemina.

Cross-reactivity between Babesia bovis and B. bigemina becomes a problem in discrimination of the two infections in endemic areas where the two species usually occur in association. With the aim of identifying candidate proteins for use as specific diagnostic tools, culture-derived components of three geographically different stocks of B. bovis (Lismore, Kwanyanga and Mexico) and one of B. bigemina (Mexico) were analyzed by immunoprecipitation using acrylamide gel electrophoresis. The approach taken was based on the analysis of 35S-methionine-labelled parasite antigens released into culture supernatant. A variety of serum samples were tested, including a panel of calf sera experimentally produced against the different stocks of Babesia, serum samples from cattle naturally infected in the field in Brazil, and a panel of anti-B. bovis monoclonal antibodies, previously characterized by the indirect fluorescent antibody test, ELISA and Western immuno-blotting. Approximately 28 and 23 bands (with molecular weights ranging from 200 to 14 kDa) were detected in total protein profiles of B. bovis and B. bigemina culture supernatants, respectively, whereas no bands were seen in the uninfected red blood cell culture supernatant (negative control). The immunoprecipitation analysis showed antigenic diversity amongst the stocks of B. bovis and resulted in identification of at least five B. bovis specific antigens common to the three stocks (molecular weights of 80, 72, 58, 38 and 24 kDa) and four B. bigemina specific antigens (molecular weights of 240, 112, 50 and 29 kDa).

Animals↗
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↗