In vitro isolation of Cowdria ruminantium from plasma of infected ruminants

A new and simple technique for isolation of C. ruminantium in bovine and ovine vascular endothelial cells (aorta, pulmonary artery) is described. Unlike previous studies, no efforts were made to retard cell growth by irradiation or chemicals. Instead, heparin-derived plasma samples obtained from only those animals exhibiting prolonged or extremely high temperatures were used. In this way, C. ruminantium was isolated from 27 of 37 samples (73%) and from 22 of 26 animals (85%). A total of six Zimbabwean stocks of C. ruminantium were isolated in cell culture.     

Characterization of Ehrlichia ruminantium replication and release kinetics in endothelial cell cultures

Ehrlichia ruminantium is the causative agent of Heartwater, a fatal tick-borne disease affecting ruminants in African countries and West Indies and can be used as an inactivated vaccine for wild and domestic animals. In order to improve E. ruminantium production yields we characterize E. ruminantium growth kinetics in terms of duplication time, maximum production yield, and peak of infectivity. After a 24 h period for E. ruminantium attachment/internalization and a lag phase of 12 h, the exponential growth occurred within 36-108 h post-infection (hpi) with a net increase of up to 2.2 orders of magnitude. Maximum E. ruminantium infectivity was observed at 120 hpi and was defined as the best time of harvesting (TOH) for propagation of E. ruminantium cultures. This study showed that considering the quality constraint of the final product (E. ruminantium vaccine), the E. ruminantium suspension should be harvested at 113 hpi. Overall, the characterization of E. ruminantium progression through the average infection cycle, not only can contribute to the maximization of E. ruminantium production yield, with important consequences for the large scale production and utilization of an inactivated Heartwater vaccine, but also to elucidate growth mechanisms of some of the other ehrlichial species, with emerging impact in human and animal health.     

In vitro infection by Ehrlichia ruminantium of baby hamster kidney (BHK), Chinese hamster ovary (CHO-K1) and Madin Darby bovine kidney (MDBK) cells

The Welgevonden stock of Ehrlichia ruminantium, aetiological agent of heartwater, was propagated in baby hamster kidney (BHK) cells, Chinese hamster ovary (CHO-K1) cells and Madin Darby bovine kidney (MDBK) cells. The cultures required supplementation of the medium with cycloheximide for reliable growth of E. ruminantium. Growth of the Welgevonden stock in BHK and CHO-K1 cells could lead to the development of suspension cultures suitable for the mass production of E. ruminantium for an inactivated elementary body vaccine.     

Possible death of a buffalo calf (Syncercus caffer) due to suspected heartwater (Ehrlichia ruminantium)

Rickettsial organisms resembling Ehrlichia ruminantium (the causative organism of heartwater) were demonstrated in brain smears and formalin-fixed brain sections derived from a buffalo calf that died on a private game reserve in northern KwaZulu-Natal. The possibility that the tick-free environment of a quarantine boma may have affected the calf's immunity, is discussed. These findings suggest that monitoring heartwater in wild ruminants and making brain smears as a routine during post mortem evaluations of wild ruminants, should be encouraged.     

In vitro response of purified ovine peripheral blood lymphocytes to phytohemagglutinin-M.

Peripheral blood was collected from three normal yearling castrated male lambs. Lymphocytes were isolated by Ficoll-Hypaque gradient centrifugation. Lymphocyte response to phytohemagglutinin was evaluated in an isotopic uptake stimulation test under varying culture conditions in order to standardize the assay. Results were analyzed as log10 counts per minute and stimulation indices. Culture conditions consisting of 2 microL (20 micrograms) phytohemagglutinin-M/culture, 1 X 10(6) cells/mL and a 72 hour incubation period were selected for use in further studies.     

Identification of Ehrlichia ruminantium (Gardel strain) IFN-gamma inducing proteins after vaccination with a killed vaccine

IFN-γ is considered as a key factor in protection against heartwater of ruminants, caused by the obligate intracellular bacterium Ehrlichia ruminantium. In this study, a better definition of the molecular masses of IFN-γ inducing proteins of the Gardel strain of E. ruminantium was obtained by the use of continuous flow electrophoresis (CFE) and sensitized polyclonal lymphocytes. Out of 15 E. ruminantium CFE fractions tested within the 14–39 kDa region, eight were commonly reacted to by all goats. Interestingly, half of these fractions fall within the 23–29 kDa region, shown previously to contain polymorphic B-cell epitopes. Thus, the results suggest that this region also contains T-cell epitopes potentially involved in protection. Also, several proteins were found to be more immunogenic than the serologically immunodominant MAP1 protein. Finally, high activity within the 15–19 kDa region was observed, which confirms previous work done with CD4+ T-cell lines obtained from cattle immunized with a South African strain of E. ruminantium. The proteins falling within the molecular weight ranges defined in this study may have potential as vaccine antigens.     

Transmission of Anaplasma phagocytophila (human granulocytic ehrlichiosis agent) in horses using experimentally infected ticks (Ixodes scapularis)

Most human granulocytic ehrlichiosis (HGE) studies carried out in horses use needle inoculation of infected leucocytes or cell cultures. This route of inoculation does not accurately reflect natural infection of the tick-borne agent. To investigate whether tick transmission influences the course of granulocytic ehrlichiosis in the horse model, experimental transmission through infected laboratory-reared Ixodes scapularis ticks was attempted into two healthy horses. One additional horse served as negative control and was exposed to uninfected ticks. Eleven days after exposure to nymphal or adult ticks infected with Anaplasma phagocytophila (HGE agent) the two horses developed severe clinical and laboratory signs consistent with granulocytic ehrlichiosis. Bacteraemia was determined at various time points in the two horses by observation of morulae within neutrophils and by detection of A. phagocytophila genomic DNA by PCR of peripheral blood leucocytes. Further, both horses seroconverted. In contrast the control horse stayed uninfected. The results demonstrate that A. phagocytophila can be experimentally transmitted by infected nymphal and adult ticks and that the agent is able to produce a severe disease, similar to naturally occurring cases. Therefore, tick transmission is highly reproducible and can be successfully used in the equine animal model in order to study HGE.     

The isolation of ovine lymphocytes and granulocytes from whole blood using hydroxyethylcellulose.

Sheep erythrocytes do not aggregate spontaneously but will sediment when mixed with a solution of hydroxyethylcellulose. The resulting leucocyte-rich supernatant can be used in the preparation of purified lymphocyte and granulocyte suspensions.     

In vitro release of interferon-gamma by trichophytin-stimulated whole blood cell cultures from ringworm-vaccinated and control calves experimentally inoculated with Trichophyton verrucosum

Of a group of seven calves, four were vaccinated against bovine ringworm with the vaccine Ringvac bovis LTF-130 (Alpharma, Oslo, Norway), while three calves were left unvaccinated. All calves were inoculated epicutaneously with a virulent strain of Trichophyton verrucosum . Clinical signs were recorded. In response to stimulation with trichophytin, in vitro interferon-γ (IFN-γ) production in whole blood cell cultures was assessed in samples obtained pre-and post-vaccination and pre- and post-inoculation. A commercial enzyme immunoas-say kit was used to measure IFN-γ levels (Bovigam, CSL, Victoria, Australia). Control calves developed typical ringworm lesions, whereas vaccinated calves did not. Following vaccination, release of IFN-γ in whole blood cell cultures indicated the presence of circulating trichophytin-specific lymphocytes. After inoculation with T. verrucosum , IFN-γ production was demonstrated in samples from both vaccinated and control calves. This study showed that vaccination with Ringvac bovis LTF-130 elicits a protective immune response suggesting involve-ment of the cellular branch of the immune system. Experimental infection of naïve nonvaccinated calves with T. verrucosum , also indicated stimulation of a cell-mediated immune response essential for resolution of lesions.     

Continuous in vitro propagation of Cowdria ruminantium (Welgevonden stock) in a canine macrophage-monocyte cell line

The Welgevonden stock of Cowdria ruminantium, aetiologic agent of heartwater, was continuously propagated in DH82 cells, a continuous canine macrophage-monocyte cell line. Cultures of DH82 cells were readily infected provided that the culture medium was supplemented with cycloheximide. Cultures were split at regular 3-day intervals and infection rates ranged between 60% and 95%. Cultures were continuously propagated through more than 125 passages over a period of more than one year.     

In vitro isolation of equine piroplasms derived from Cape Mountain zebra (Equus zebra zebra) in South Africa

Twenty blood samples of zebras (Equus zebra zebra) from the Karoo National Park and the Bontebok National Park in South Africa, all seropositive for Theileria equi, were subjected to in vitro culture to identify carrier animals and to isolate the parasites. Sixteen animals had a detectable parasitaemia in Giemsa-stained blood smears examined before culture initiation, the remaining four animals were identified as T. equi carriers by in vitro culture. Cultures were initiated either in an oxygen-reduced gas mixture or in a 5% CO2-in-air atmosphere. Out of the 20 blood samples, 12 cultures of T. equi and two cultures of T. equi mixed with Babesia caballi were established. None of the four animals seropositive for B. caballi could be identified as carrier animals, whereas two seronegative samples became culture-positive for B. caballi.     

Determination and quantification of the in vitro activity of Aloe marlothii (A. Berger) subsp. marlothii and Elephantorrhiza elephantina (Burch.) skeels acetone extracts against Ehrlichia ruminantium

An Ehrlichia ruminantium culture system was utilized for the anti-rickettsial evaluation of two ethnoveterinary plants, Elephantorrhiza elephantina and Aloe marlothii. Well-established E. ruminantium cultures were incubated with the plant leaf acetone extracts and compared to oxytetracycline and untreated controls. Effectivity was established by comparing the percentage parasitised cells and the calculation of both EC50 and extrapolated EC90 in microg/ml. The plant extracts were also screened for antibacterial activity using bioautography. Elephantorrhiza elephantina and A. marlothii demonstrated anti-ehrlichial activity with an EC50 of 111.4 and 64.5 microg/ml and EC90 of 228.9 and 129.9 microg/ml, respectively. The corresponding EC50 and EC90 for oxytetracycline was 0.29 and 0.08 microg/ml. Both plants appeared to produce their inhibitory activity by a similar mechanism, unrelated to that of the tetracyclines. Both the plant acetone extracts demonstrated antibacterial activity against Escherichia coli and Staphylococcus aureus (ATCC strains).     

Direct isolation of the agent of enzootic abortion of ewes (Chlamydia psittaci) in cell cultures

Isolation of Chlamydia psittaci in McCoy cell coverslip cultures from clinical material from field cases of enzootic abortion of ewes proved more sensitive than diagnosis by examination of smears stained by Ziehl-Neelsen. Enzootic abortion could be diagnosed in the absence of fetal membranes by culture of fetal lung or liver tissue.     

In vitro activation of bovine macrophage and peripheral blood mononuclear cells by Nocardia rubra cell wall skeleton (N-CWS)

Nocardia rubra cell wall skeleton (N-CWS) was used for immunotherapy to bovine leukemia virus (BLV)-positive cattle with enlarged subcutaneous lymphatic nodules. Electron microscopical observations showed the infiltration of macrophage and T cells around N-CWS treated lesions. Antitumor effect induced by N-CWS was examined in vitro. The maximum cytolytic activity of macrophage was observed, when the cells were incubated with 10 micrograms/ml of N-CWS. Chemiluminescence response of peripheral blood mononuclear cells (PBMC) using N-CWS as stimulant was observed with a high level of activity for a long period, 5 hr. Mitogenic effect of N-CWS to PBMC was observed in the presence of macrophages but not without macrophages. Furthermore, interleukin 2 activity was recognized in supernatant of PBMC cultured with N-CWS. Maximum cytotoxic T lymphocyte response was induced when PBMC were cultured with 0.1 micrograms/ml of N-CWS.     

In vitro response of lymphocytes of normal and ovine squamous cell carcinoma-bearing sheep to phytomitogens and tumour extracts

A comparison was made of the blastogenic responses of peripheral lymphocytes (BRPL) of normal sheep with those of sheep bearing ovine squamous cell carcinoma (OSCC). In normal tumour-free sheep, BRPL to phytomitogens were found to vary in different age groups, and BRPL to OSCC extracts were found to become significantly elevated in sheep over one year old. In tumour-bearing mature sheep, BRPL to phytomitogens and to OSCC extracts decreased significantly with increasing maturity of tumours. The results are interpreted to show that increasing tumour volume, due to natural growth or enhancement, associates with increasing suppression of cell-mediated responses, and that such suppression may be a cause and a result of increased tumour volume.