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PubMed1983

Infection rates of Theileria annulata in the salivary glands of the tick Hyalomma marginatum rufipes.

Theileria annulata was experimentally transmitted to cattle on two occasions by the two-host tick Hyalomma marginatum rufipes. Transmission was transstadial; engorged nymphs fed on Theileria annulata-infected calves transmitted the disease as adults. Salivary glands of all partially fed and incubated adult ticks were heavily infected with Theileria parasites. Immatures attached rapidly and fed successfully on cattle. However, since the immature stages of this species normally feed on birds, this tick is unlikely to be an important vector in the field.

Animals↗
PubMed1983

Fine structure and invasive behaviour of the early developmental stages of Theileria annulata in vitro.

The interaction, in vitro, between bovine peripheral blood lymphocytes and sporozoites of Theileria annulata (Ankara) was studied by light and electron microscopy. Beginning five minutes following incubation, samples were taken for Giemsa-stained smears and glutaraldehyde-fixed pellets, for light and electron microscopy, respectively. Sporozoites of T. annulata measure an average of 0.9 microns long, 0.8 microns broad and possess a limiting unit membrane, the pellicle; a round-to-ovoid, eccentrically situated, non-chromocentric nucleus; double-membraned, tubular, acristate mitochondria; varying numbers of anisocytic, densely osmiophilic and pleomorphic organelles, the rhoptries which together with the polar ring form the apical complex; and numerous, loosely scattered, electron-dense ribosomal particles. As early as 5 min of incubation, sporozoites had made contact with, and penetrated, lymphocytes. Sporozoites consistently attached to the lymphocyte plasmalemma by their basal end, possibly at specific receptor sites. Apparently only a proportion of lymphocytes (up to 40% and more commonly 10-20%) were susceptible. Two subpopulations of the susceptible lymphocytes were observed; one which appeared to have receptor sites localized on one pole of the plasmalemma and the other subpopulation in which the receptor sites were distributed evenly around the plasmalemmal surface. Within individual susceptible lymphocytes, the number of interiorized sporozoites increased from 1 to 3 at 5-10 min to as many as 15 or more parasites at around 60 min of incubation. Theileria annulata sporozoites were interiorized by the invagination of the host cell plasmalemma which remained intact throughout the process but later fragmented. Within 30 min of interiorization, each sporozoite underwent dedifferentiation by the loss of its rhoptries and transformed into a trophozoite. Around 24 h, the trophozoite, a uninucleate, motile and feeding stage of the parasite, developed into a schizont by an acentric, closed mitosis.

Animals↗
PubMed1984

Chemotherapy of East Coast fever: parvaquone treatment of clinical disease induced by isolates of Theileria parva.

Two experiments were carried out in which parvaquone was used to treat experimentally-induced acute clinical East Coast fever infections. In the first experiment, infections with Theileria parva parva (Kiambu 5) were induced by applying infected Rhipicephalus appendiculatus ticks or by inoculation of triturated infected-tick stabilate. The character of the disease was similar with both methods of infection and following a single treatment with parvaquone at 20 mg kg-1, 5 of 7 cattle in each group recovered. All untreated control cattle died. In the second experiment, 5 stabilate isolates from different locations within East Africa, and representative of the challenge likely to be met in the field, were used. Treatment was administered in 2 X 10 mg kg-1 doses 48 h apart. The isolates used were T. p. parva (Mbita), T. p. parva (Pugu), T. p. parva (Entebbe), T. p. lawrencei (Mara) and T. p. lawrencei/(Manyara); following treatment 3/7, 6/6, 6/7, 5/7 and 6/7 animals recovered, respectively. All untreated control cattle died. There was evidence of a difference in susceptibility of isolates to treatment, and some animals showed prolonged disease episodes. The nature of the response to treatment and the problems in treating a lympho-destructive disease are discussed.

Africa, Eastern↗
PubMed1984

Factors affecting the capacity of Theileria annulata sporozoites to invade bovine peripheral blood lymphocytes.

The number of T. annulata sporozoites invading bovine peripheral blood lymphocytes (PBL) under different conditions (in vitro) was determined. Heat-inactivation of T. annulata sporozoites for 45 min, in a thermostatically controlled, shaking water bath preset and stabilised at 60 degrees C resulted in an almost total lack of invasion of fresh, normal PBL by the sporozoites, indicating that the interiorization process is parasite-effected. The mean number of T. annulata sporozoites interiorization (per 1000 lymphocytes) in cultures set up using sporozoites and PBL, mixed and incubated at 0 degrees C for 1 h in melting ice, was highly significantly reduced (P less than 0.01), indicating the invasion of bovine lymphocytes by T. annulata sporozoites is an active process dependent on active metabolism which is markedly affected by temperature. Pre-treatment of PBL with trypsin significantly reduced the number of invading sporozoites thus incriminating proteins or glycoproteins as constituents of receptors involved in sporozoite-lymphocyte recognition.

Animals↗
PubMed1984

Detection of antibody to Theileria parva schizonts and cell surface membrane antigens of infected lymphoblastoid cells by immunoperoxidase techniques.

Peroxidase-labeled antibody procedures were described for detecting bovine antibodies reactive with intracellular Theileria parva schizonts and cell surface membrane antigens of infected lymphoblastoid cells. Indirect tests were performed where the reacting bovine antibodies were localized with affinity purified rabbit-anti-bovine IgG coupled to horseradish peroxidase. A 4- to 8-fold increase in sensitivity for detecting bovine antibodies was obtained with unlabeled rabbit-anti-bovine IgG which in turn was detected with peroxidase labeled goat-anti-rabbit IgG. The T. parva infected cells used as antigen were attached to poly-l-lysine treated glass slides and all reaction steps were performed on the slides. The intracellular schizonts and cell surface staining reactions were dependent upon the status of the cells; acetone-fixed cells were required for schizont reactions and viable unfixed cells for cell surface membrane reactions. Sera from cattle stimulated in various ways with T. parva were examined by the techniques. Cattle infected by stabilate inoculation or inoculated with infected autologous lymphoblastoid cells developed relatively high levels of antibody to schizonts, but no detectable antibody to cell surface membrane antigens. This would indicate that parasite antigens do not occur on the surface of infected lymphoblasts. Cattle inoculated with infected allogeneic lymphoblasts developed low-levels of antibody to schizonts and readily demonstrable antibody to cell surface antigens. The immunoperoxidase procedures have certain advantages over immunofluorescence in that light microscopy is used; therefore, the reactions do not fade which permits a more detailed examination and provides a relatively permanent record, the preparations can be counterstained, and the reagents may be used for immunoelectron-microscopy. The procedures could provide suitable alternatives to immunofluorescence methods for East Coast fever investigations and other systems having intracellular and/or cell surface membrane antigens.

Animals↗
PubMed1984

Titration in cattle of infectivity and immunogenicity of autologous cell lines infected with Theileria parva.

Cell lines infected with Theileria parva were derived by infection of bovine peripheral blood lymphocytes with sporozoites in vitro. Cattle were inoculated with doses of autologous infected cells ranging from 1 X 10(1) to 1 X 10(8). Infection became established in animals which received 1 X 10(2) or more cells. While 1 X 10(2) cells resulted in sub-patent infection with development of immunity to challenge with sporozoites, larger doses of cells gave rise to patent infections of increasing severity. Thus, doses of 1 X 10(5) and 1 X 10(6) cells sometimes produced lethal infections and with 1 X 10(7) and 1 X 10(8) the outcome was invariably lethal. Based on the previous observation that induction of immunity by allogeneic cells requires transfer of infection into the recipient-host cells, a comparison of the infections produced by autologous and allogeneic cells indicated that the transfer of infection from allogeneic cells occurs at a frequency of maximally 1 X 10(-5). Two pairs of cattle were identified as being mutually non-reactive in the mixed leukocyte reaction (MLR). Doses of 1 X 10(6) and 1 X 10(7) cells of cell lines derived from 1 animal of each pair were inoculated into the autologous host, the non-reactive partner and an animal which was shown to be strongly reactive to the donor in the MLR. In each instance, the reaction in the MLR non-reactive recipient was not significantly different from that of the MLR reactive recipient, but was markedly different from that of the autologous recipient.

Animals↗
PubMed1985

The integrity of host lymphocyte plasmalemma during its invasion by Theileria annulata sporozoites as demonstrated by ruthenium red staining.

The integrity of host lymphocyte plasma membrane during its invasion by Theileria annulata sporozoites was examined in an in vitro system using ruthenium red staining. It was demonstrated that during interiorisation of the sporozoites, the dye was completely excluded from the intracellular milieu, indicating that the continuity of the host-cell plasmalemma was not disrupted throughout the process of penetration, and confirmed a previous observation that the fragmentation of the host lymphocyte membrane only occurs later, after the parasite is intracellular.

Animals↗
PubMed1985

Metabolic energy pathways in Theileria annulata sporozoites and their significance in sporozoite-bovine lymphocyte interactions in vitro.

The existence of metabolic energy pathways has been studied in extracellular T. annulata sporozoites using chemicals known to inhibit specific energy-generating pathways, and their role during invasion of bovine peripheral blood lymphocytes (PBL) by the sporozoites determined in an in vitro system. An inverse relationship was depicted between the dose of various chemicals and the number of T. annulata sporozoites invading PBL: as the concentrations of the inhibitor drugs increased, the number of T. annulata sporozoites within the lymphocytes decreased. An ultracytochemical study demonstrated the presence of the respective pathway marker enzymes, i.e., lactic dehydrogenase (LDH) in the cytosol and within mitochondria, succinic dehydrogenase (SDH) on the mitochondrial membranes and in the contiguous matrix, and cytochrome oxidase (CO) between the inner and outer mitochondrial membranes, in infective T. annulata sporozoites fixed in situ within whole salivary glands of 3-day fed Hyalomma anatolicum anatolicum ticks.

2,4-Dinitrophenol↗
PubMed1985

Comparative autoradiographic study of parasite-host-cell cyclical relationship in lymphoblastoid cell lines infected with Theileria annulata and Theileria parva in vitro.

Cyclical interactions between intracellular schizonts of the Ankara and Hissar strains of Theileria annulata and the Muguga strain of T. parva and the parasitised host lymphoblasts have been studied autoradiographically by following the incorporation of (3H) thymidine into parasite and host cell nuclei, and also by quantitating the number of schizont nuclei per lymphoblast, at various stages and phases of host cell cycle. The synthesis of DNA by Theileria schizonts and the parasitised host lymphoblasts was found to be asynchronous and to occur at different phases of the host cell interphase stage. While the lymphoblast nuclear DNA incorporates (3H) thymidine during the S phase, schizont nuclei were labelled during the G2 phase of the host lymphoblast interphase stage. The replication of schizont nuclei took place before the metaphase stage of host cell cycle, viz, prometaphase, so that the mean schizont nuclear number at host prometaphase and at anaphase--telophase was consistently more or less double the mean nuclear number at interphase.

Animals↗
PubMed1985

The incidence of canine haematozoa in Peninsular Malaysia.

In 3 urban areas in Selangor, Peninsular Malaysia between 1973 and 1981, blood from 4084 dogs was examined for haematozoa. The following frequencies were found: Babesia gibsoni 17.7%; microfilariae of Dirofilaria immitis 9.6%; Hepatozoon canis 1.2%; B. canis 1.1%; Ehrlichia canis 0.2%; Trypanosoma evansi 0.1%. A detailed examination of B. gibsoni infections and microfilariasis due to D. immitis with regards to monthly distribution, breed frequency, sex and age, revealed that pedigree and non-pedigree dogs were equally susceptible to Babesia and microfilariae infections.

Age Factors↗
PubMed1986

Evaluation of purified Theileria annulata sporozoite antigen from the tick Hyalomma anatolicum anatolicum.

Rabbits immunized with purified sporozoites from Hyalomma anatolicum anatolicum produced antibody to Theileria annulata specific antigens as measured by the indirect fluorescent antibody test. Nonspecific fluorescence which compounded the interpretation of the test was observed. Incorporation of Eriochrome black and Evans blue as a counterstain in the diluent buffer for the conjugate significantly reduced the background fluorescence.

Animals↗
PubMed1986

Humoral antibody response of rabbits to experimental infection with Encephalitozoon cuniculi.

Six female rabbits (Oryctolagus cuniculus) were experimentally infected intravenously with approximately 1.5 X 10(7) live spores of Encephalitozoon cuniculi. Head tilt was observed as the single clinical sign in only one of the six animals. Antibody response was registered over 68 days postinfection using the indirect immunofluorescence test (IFT) for IgM and IgG, and the carbon immunoassay (CIA). IgG titers reached a level of 160-2560 after a latent phase of 13-28 days, followed by a 2-4 week relatively steep increase. The IgM seroconversion was faster than that of IgG and occurred at the beginning of the antibody response. Thus, simultaneous detection of both IgM and IgG allowed the infection to be identified as recent. Long, short, and episodic antibody responses could be distinguished: the IgG titer continued to increase on Day 68 in one animal (long response) and began to decrease between Days 45 and 63 in three other animals (short response). In two additional animals the seroconversion was very short, occurring between Days 13 and 41, and 28 and 52, respectively (episodic response). The CIA proved to be specific, reliable, and simple to perform; titers were slightly higher than in the IFT. Parasite pseudocysts were detected scattered throughout the brain on Day 68 in four of the six rabbits. The persistence of antigen in the brain did not correlate with antibody response, which in most cases was shorter.

Animals↗
PubMed1986

Epidemiology of theileriosis in calves in an endemic area of Kenya.

Thirty-one calves born into five Maasai zebu cattle herds over a period of 1 month in the Trans-Mara Division of Kenya, endemic for theileriosis, were recruited for an intensive study of theileriosis. No calves up to 6 months of age died but all developed Theileria infections as judged by slide examination and serology. Parasitosis by T. mutans schizonts in lymph node smears was usually higher than that of T. parva. The T. mutans schizonts usually occurred at an earlier age but persisted at a patent level for a shorter time than those of T. parva. Serological findings using the indirect fluorescent antibody test confirmed the parasitological findings. It was evident that colostral transfer of Theileria antibodies was frequent. Theileria piroplasm parasitaemia had developed in all calves by 111 days of age. The earlier parasitosis by T. mutans reflected the higher infection rates in Amblyomma spp. than in Rhipicephalus appendiculatus. The mean number of R. appendiculatus on the ears of calves during the observations was 9.1 adults and 1.5 nymphs. Clinical episodes of T. mutans and T. parva infection were associated with febrile responses, enlarged lymph nodes, anaemia and other symptoms and about 80% of calves had poor weight gains or weight losses during either clinical infection. It would appear that theileriosis is one of the most important factors in the stunting of calf development in the area.

Anaplasmosis↗
PubMed1986

Induction of immunity against infection with Theileria parva (East Coast fever) in cattle using plasma membranes from parasitized lymphoblasts.

Protection against challenge with Theileria parva was conferred on three of four calves given three or four inocula of plasma membranes prepared from 6 to 12 X 10(8) autologous parasitized lymphoblasts from cultured cell lines. In contrast, calves remained susceptible to infection following immunization with membranes prepared from allogeneic parasitized lymphoblasts. Similarly, calves vaccinated with either gamma-irradiated autologous or allogeneic infected cells also died of East Coast fever after challenge. The results raise the possibility of vaccination against T. parva using subcellular material from infected lymphoblasts.

Animals↗
PubMed1986

Invasion and intracellular development of Theileria annulata sporozoites in lymphoblastoid cell lines already transformed by T. annulata (Hissar), T. annulata (Ankara) and T. parva (Muguga).

Interactions between Theileria annulata sporozoites and lymphoblastoid cell lines already transformed by the Hissar and Ankara strains of T. annulata [T. a. (H) and T.A. (A), respectively] and the Muguga strain of T. parva [T.P. (M)] were studied in vitro. Although sporozoites of the Hissar strain of T. annulata attached to and entered peripheral blood lymphocytes (PBL) and lymphoblastoid cell lines transformed by T. a. (H) and T. a. (A), they neither attached to nor entered the T. p. (M) cell line. Whether the superinfecting T. a. (H) sporozoites developed intracellularly was studied by monitoring daily changes of mean schizont nuclear numbers and by determining electrophoretic mobilities of schizont glucose phosphate isomerase in each cell line using thin-layer starch gel electrophoresis. While the mean schizont nuclear number in freshly-infected PBL underwent a steady increase to the level of those in long standing T. annulata cultures, analysis of variance of similar data in T. a. (H) and T. a. (A) cell lines in which superinfection was demonstrated revealed no significant differences between them and their respective control counterparts, i.e., T. a. (H) and T. a. (A) cultures with no superinfection. Enzyme polymorphism studies showed the formation of uncontaminated species- or strain-specific bands of glucose phosphate isomerase (GPI) isoenzyme activity in the T. p. (M) and in the superinfected T. annulata cell lines.

Analysis of Variance↗
PubMed1986

Antigenic diversity of Theileria annulata macroschizonts.

A series of monoclonal antibodies has been produced which reacts with the intracellular macroschizont of Theileria annulata, and this series has been used to examine the level of antigenic diversity between and within stocks of the parasite in addition to species specificity within the genus. The majority of the monoclonal antibodies (six of eight) reacted with all stocks tested and four were species-specific. Two monoclonal antibodies detected variation between stocks and within stocks. The variation within a stock was manifest as only a proportion of the macroschizonts reacting with the monoclonal antibody. This was demonstrated as being due to the strain containing more than one antigenic type of parasite by cloning the infected lymphocyte cell line and showing that the resulting cloned cell lines were antigenically homogeneous. The variation between stocks allowed them to be divided into three groups on the basis of antigenic profile.

Animals↗
PubMed1987

Field evaluation of parvaquone against naturally occurring East Coast fever.

Parvaquone (Clexon Cooper) was clinically tested for efficacy as a treatment for East Coast Fever (Theileria parva parva infection) in naturally infected cattle. The drug showed a suppressive effect on Theileria schizonts and piroplasms. A recovery rate of 79% was recorded. Best results were obtained when Clexon treatment was initiated in the early stage of the disease, before many red blood cells were invaded and before respiratory distress was evident. A carrier state in animals recovered from East Coast Fever is suspected.

Animals↗
PubMed1987

Cultivation of Besnoitia besnoiti and evaluation of susceptibility of laboratory animals to cultured parasites.

Cultivation of Besnoitia besnoiti on five mammalian cell types showed that Vero (green monkey kidney), L929 (mouse fibroblasts) and BEK (bovine embryo kidney) cells were highly susceptible and were almost destroyed after 5 days of incubation, while MDBK (Madin-Darby bovine kidney) and BL (Theileria annulata-infected bovine lymphoid) cells were less affected. Intraperitoneal infection with culture-derived endozoites was fatal for gerbils (Meriones tristrami) and sand rats (Psammomys obesus). Rabbits showed fever, conjunctivitis and orchitis, but survived the infection. Mice, rats, guinea pigs, hamsters and Microtus guentheri did not show signs of illness, but developed specific antibodies to B. besnoiti. B. besnoiti subcultivated for 2 years in BL cells remained virulent for gerbils.

Animals↗