C.G.D. Brown

Detection of Theileria annulata in cattle and vector ticks by PCR using the Tams1 gene sequences.

A Polymerase Chain Reaction (PCR) and Southern blot hybridization for the detection of Theileria annulata are described. The PCR used primers amplifying a 785 base-pair fragment of the T. annulata gene which encodes the 30 kDa major merozoite surface antigen, Tams1. The sensitivity of the PCR in bovine blood was 1 piroplasm in 1 microl of blood. T. buffeli, T. parva, Babesia bigemina, B. bovis and B. divergens were not detected. The PCR detected down to 1 infected acinus/tick in resting and partially fed adult Hyalomma anatolicum anatolicum ticks and was negative for T. lestoquardi and T. equi, which are transmitted by this tick but are not infective to cattle. The specificity of the PCR was checked using 30 stocks of T. annulata, all of which were detected. Three stocks of T. lestoquardi, 4 of T. equi and 1 each of T. buffeli, T. parva, B. bigemina, B. bovis and B. divergens were used to ascertain there were no cross-reactions. A nested PCR using separate primers for the first reaction and the same primers for the second reaction detected T. annulata to the same sensitivity and specificity in saponin-extracted DNA samples stored for long periods at -20 degrees C.

Innate and Adaptive Immune Responses Co-operate to Protect Cattle against Theileria annulata

For many years it was assumed that Theileria annulata resembled T. parva, parasitizing lymphocytes and causing lymphoproliferative disease, with the two species being controlled by similar protective immune responses. Patricia Preston et al. here review the evidence that has led to a different view of T. annulata. It is now thought that the schizonts of T. annulata inhabit macrophages and B cells, and that tropical theileriosis is not a lymphoproliferative disease. Both innate and adaptive responses contribute to recovery from infection and resistance to challenge and cytokines produced by infected and uninfected cells influence the outcome of infection. Partial protection has been stimulated recently by defined recombinant antigens; efficacy depended upon the delivery system.

Infection of bovine monocyte/macrophage populations with Theileria annulata and Theileria parva

Infection and transformation of cells of the bovine immune system by Theileria annulata and T. parva were compared. Preliminary experiments with mammary gland macrophages indicated that they were permissive to infection by T. annulata but only to a limited extent by T. parva. Further experiments involved several purified subpopulations of bovine cells including bovine monocytes, T cells and MHC class II positive and negative populations. These subpopulations were incubated with T. annulata or T. parva sporozoites in limiting dilution cultures. T. annulata preferentially infected macrophage type cells and also MHC class II positive cells, whereas the frequency of MHC class II negative cells infected by this parasite was negligible. T cells also showed a very low level of infection. In complete contrast, T. parva preferentially infected T cells and did not infect cells phenotypically defined as monocytes at all. These results suggested that class II expression was necessary for T. annulata infection and not necessary for, though not a barrier to T. parva infection. T. annulata infected cell lines all expressed class II molecules to varying degrees. Other available phenotypic markers were only expressed at very low levels or no longer expressed. The immunological significance of the different cell preferences and phenotypes of infected cell lines of T. annulata and T. parva is discussed.

Nitric oxide inhibits establishment of macroschizont-infected cell lines and is produced by macrophages of calves undergoing bovine tropical theileriosis or East Coast fever

Nitric oxide (NO) was produced when bovine peripheral blood mononuclear cells (PBMC) or purified, adherent PBMC (macrophages) were incubated in vitro with bovine recombinant interferon gamma (Bo rIFN‐γ). NO was produced by cells from naive, uninfected calves as well as by cells from cattle either infected with or recovered from infection with Theileria annulata or Theileria parva. PBMC of cattle undergoing tropical theileriosis (T. annulata infection) or East Coast fever (T. parva infection) synthesized NO spontaneously in vitro. NO was also induced when PBMC of immune, but not of naive, cattle were cultured with T. annulata macroschizont‐infected cell lines. Macrophages alone were not stimulated to produce NO by such infected cells. In vitro establishment of macroschizont‐infected cell lines was suppressed either by incubating sporozoites with S‐nitroso‐N‐acetyl‐DL‐penicillamine (SNAP), a NO releasing molecule, prior to invasion of PBMC or by pulsing developing cultures of trophozoite‐infected cells with SNAP. Proliferation of established macroschizont‐infected cell lines was not affected by SNAP. Taken together with the well documented roles of NO in neurotransmission, vasodilatation, cell and tissue damage and immunosuppression, the results presented here indicate that NO may not only protect cattle against T. annulata and T. parva but, if produced in excess, play a prominent role in the pathogenesis of tropical theileriosis and East Coast fever.

Tropical theileriosis in Bos taurus and Bos taurus cross Bos indicus calves: response to infection with graded doses of sporozoites of Theileria annulata

This work extends basic knowledge of tropical theileriosis in taurine and crossbred cattle. Infection of Bos taurus and Bos taurus cross Bos indicus (Sahiwal) calves with graded doses of sporozoites of Theileria annulata (Hissar), an Indian stock of the parasite, showed the following to be dose dependent in both cattle types: the time to appearance and population size of macroschizonts, microschizonts and piroplasms, time and severity of pyrexia, anaemia manifested by erythrocyte counts and haematocrit. All infections were accompanied by a prompt and severe panleucopenia. This effect was dose related in both the taurine and the Sahiwal crossbred calves. Lymphocyte counts returned to preinfection levels in the blood of animals which recovered, but death from theileriosis was characteristically accompanied by a persistent and severe lymphocytopenia. Flow cytometry using monoclonal antibodies to bovine mononuclear cells was used to identify the lymphocyte subsets involved in lymphocytopenia. The outcome of infection was dose dependent in the crossbred calves but not in taurine calves. Although the results obtained did not differ qualitatively between the two cattle types, they provided some preliminary evidence for resistance to tropical theileriosis in Sahiwal crossbred calves.

Cytokines inhibit the development of trophozoite-infected cells of Theileria annulata and Theileria parva but enhance the proliferation of macroschizont-infected cell lines

Summary The following bovine (Bo) and human (Hu) cytokines—Bo rTNF‐a, Bo rIFN‐g, Hu IFN‐a, Hu rIL‐1, Hu rIL‐2—significantly inhibited the in vitro development of trophozoite‐infected cells of three stocks of Theileria annulata and of Theileria parva (Muguga). However, none of these cytokines inhibited the proliferation of established T. annulata or T. parva macroschizont‐infected cell lines. Indeed, Bo rTNF‐a and Hu rIL‐2 consistently enhanced the proliferation of macroschizont‐infected cell lines of both species and the blastogenesis of uninfected lymphocytes in trophozoite‐infected cultures. These results suggest that cytokines could help in resistance to challenge infections by preventing the further development of trophozoite‐infected cells but provide no evidence that any of the above cytokines directly help to resolve primary infections by inhibiting the growth of macroschizont‐infected cells. These findings also suggest that both TNF‐a and IL‐2 could play a role in the pathogenesis of Theileria infections by promoting the proliferation of macroschizont‐infected cells and the associated lymphoid hyperplasia.

Bovine Cells Infected in Vivo with Theileria Annulata Express CD11b, the C3bi Complement Receptor

Forsyth, L.M.G., Jackson, L.A., Wilkie, G., Sanderson, A., Brown, C.G.D. and Preston, P.M., 1997. Bovine cells infected in vivo with Theileria annulata express CD11b, the C3bi complement receptor. Veterinary Research Communications, 21 (4), 249-263Bovine 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.

Synthesis of tumour necrosis factor-alpha and interferons by mononuclear cells from Theileria annulata-infected cattle

Bovine macrophage‐derived tumour necrosis factor‐alpha/ cachectin (TNF‐α) was synthesized when peripheral blood mononuclear cells (PBMC) and purified adherent PBMC from naïve and Theileria annulata‐infected cattle were incubated in vitro with concanavalin A (Con‐A) or bovine recombinant interferon gamma (Bo rIFN‐γ). TNF‐α production was also induced when adherent PBMC were cultured with T. annulata macroschizont‐infected cells. In contrast, non‐adherent PBMC from sublethally infected cattle produced interferon (IFN) when incubated with Hu rIL‐2, Con‐A, phytohaemagglutinin (PHA) or T. annulata macroschizont‐infected cells growing as cell lines in vitro. Whilst PBMC from lethally infected cattle spontaneously produced IFN‐γ during advanced stages of infection, the sera of such animals contained type 1 IFN (alpha/beta). IFN was also produced by T. annulata macroschizont‐infected cell lines maintained in vitro. This work suggests that cytokines serve as crucial links between proliferating Theileira‐infected cells and the characteristic clinical symptoms of tropical theileriosis.

Studies on the transmission and course of infection of a Kenyan strain of Theileria mutans

Theileria mutans (Aitong) isolated from cattle exposed in the Narok District of Kenya and blood-passaged through cattle 8 times, appeared to have lost its original pathogenicity for cattle. It was demonstrated that the parasite was trans-stadially transmissible by the tick Amblyomma variegatum but not by Rhipicephalus appendiculatus . Four tick–bovine passages were made using A. variegatum , and infective parasites were also harvested from A. variegatum nymphs which had been fed for 5 days on rabbits. Blood containing piroplasms, or lymphoid cells infected with schizonts taken from cattle, at the 2nd tick–bovine passage were shown to be infective on inoculation. The course of the T. mutans infections in the cattle was studied. In tick-induced infections macroschizonts occurred transiently, persisting longest in circulating lymphoid cells. Microschizonts were rarely detected. The macroschizonts were morphologically distinct from those of other Theileria species described from East Africa. In tick-induced infections, the piroplasm parasitaemia increased rapidly and there was evidence of restored pathogenicity of the parasite since high piroplasm parasitaemias were associated with a marked anaemia.

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).