In vitro isolation of Ehrlichia ruminantium from ovine blood into Ixodes scapularis (IDE8) cell cultures

Four stocks of Ehrlichia ruminantium (Welgevonden, Ball3, Nonile and Blaauwkrans), the causative agent of heartwater in domestic ruminants, were isolated into Ixodes scapularis (IDE8) tick cells using the leukocyte fraction of the blood of infected sheep. Organisms of two of the E. ruminantium stocks (Welgevonden and Blaauwkrans) propagated in IDE8 cells were also successfully used to infect bovine endothelial cells. All stocks were successfully propagated in IDE8 cells using Dulbecco's modified Eagle's medium nutrient mixture Ham F-12 containing 10% foetal bovine serum (FBS). The technique should be included in any attempt to isolate uncharacterized E. ruminantium stocks.     

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.     

Observations on mouse-infective stocks of Cowdria ruminantium: attempts to demonstrate antigenic changes of the Kwanyanga stock in mice

Starting from a stabilate of caprine blood infected with the Kwanyanga stock of Cowdria ruminantium, eight serial passages were made in groups of mice, and eight parallel serial passages in goats. Cross-immunity tests in goats and mice failed to demonstrate any difference between stabilates made after the eighth passage. The Kwanyanga stock was of exceptionally low virulence for Dutch goats.     

Resistance of leopard tortoises and helmeted guineafowl to Cowdria ruminantium infection (heartwater).

Experimental infection trials were conducted to investigate susceptibility of leopard tortoises (Geochelone pardalis) and helmeted guineafowl (Numida meleagris) to infection with Cowdria ruminantium, the causative agent of heartwater, a tickborne disease of domestic and wild ruminants. Ten guineafowl were inoculated intravenously with a virulent dose of C. ruminantium derived from bovine endothelial cell cultures, and four leopard tortoises were exposed to C. ruminantium infection by the feeding of infected Amblyomma hebraeum ticks. Uninfected A. hebraeum ticks (on both tortoises and guineafowl) and Amblyomma marmoreum ticks (on tortoises only) were fed on the animals during weeks 2 and 3 post-exposure in an attempt to detect infection. These ticks were analyzed for C. ruminantium infection by xenodiagnosis and with the C. ruminantium-specific pCS20 polymerase chain reaction (PCR) assay. Attempts to detect infection in ticks fed on either species were negative by both tests. These results suggest that leopard tortoises and helmeted guineafowl are refractory to C. ruminantium infection and, therefore, are unlikely to be capable of introducing heartwater directly into new areas. However, leopard tortoises are efficient hosts of A. marmoreum and A. hebraeum and are likely to be important epidemiologically in the transport and maintenance of these tick vector species.     

Effects of storage and passage of bovine luteal endothelial cells on endothelin-1 and prostaglandin F2alpha production

To establish a storage system for isolated bovine luteal endothelial cells (LECs), we investigated the basal and tumor necrosis factor (TNF) alpha-stimulated production of endothelin-1 (ET-1) and prostaglandin (PG) F2alpha in unfrozen and frozen-thawed LECs until passage 10. LECs were obtained from developing corpora lutea (CL; days 5-7 of the estrous cycle) using enzymatic digestion and magnetic beads coated with lectin BS-1. The LECs were frozen at -80 C or further cultured and/or passaged until passage 10 in DMEM/Ham's F-12 supplemented with 10% calf serum. The hormonal productions of unfrozen and frozen/thawed LECs were compared through passages 2-10. When both the unfrozen and frozen/thawed cells reached confluence, the culture medium was replaced with fresh medium containing 0.1% bovine serum albumin (BSA), and the cells were incubated with TNFalpha (50 ng/ml) for 12 h. The basal productions of ET-1 and PGF2alpha by the unfrozen and frozen/thawed LECs were similar at passage 2. The basal production of PGF2alpha by LECs was not altered by passage and storage at -80 C, whereas the basal production of ET-1 decreased from passage 2 and 3 to passage 4 in the unfrozen LECs and from passage 2 to passage 3 in the frozen/thawed LECs. However, production of ET-1 by the unfrozen and frozen/thawed LECs was similar between passages 4-10 and passages 3-10, respectively. Exposure of LECs to TNFalpha increased (P<0.05) ET-1 and PGF2alpha production by the unfrozen and frozen-thawed LECs in all passages examined. Thus, LECs obtained from developing CLs and stored until passage 10 can be used for study of the physiology of LECs in vitro.     

Cowdria ruminantium DNA is unstable in a SuperCos1 library.

A Cowdria ruminantium genomic library was constructed in a cosmid vector to serve as a source of easily accessible and pure C. ruminantium DNA for molecular genetic studies. The cosmid library contained 846 clones which were arrayed into microtitre plates. Restriction enzyme digestion patterns indicated that these clones had an average insert size of 35 kb. Probing of the arrays did not detect any bovine clones and only one of the known C. ruminantium genes, pCS20, was detected. Due to the high AT content and the fact that C. ruminantium genes are active in the Escherichia coli host, the C. ruminantium clones were unstable in the SuperCos1 vector and most clones did not grow reproducibly. The library was contaminated with E. coli clones and these clones were maintained with greater fidelity than the C. ruminantium clones, resulting in a skewed representation over time. We have isolated seven C. ruminantium clones which we were able to serially culture reproducibly; two of these clones overlap. These clones constitute the first large regions of C. ruminantium DNA to be cloned and represent almost 10% of the C. ruminantium genome.     

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.     

Development and persistence of Cowdria ruminantium specific antibodies following experimental infection of cattle, as detected by the indirect fluorescent antibody test.

Different breeds of cattle were experimentally infected with Palm River, a Zimbabwean isolate, or Ball-3, a South African isolate of Cowdria ruminantium, derived from tissue culture or tick or blood stabilates. C. ruminantium specific antibody responses were detected by an indirect fluorescent antibody test (IFAT) using C. ruminantium-infected bovine aortic endothelial (BAE) cell cultures as antigen. The first detection of antibodies to C. ruminantium generally coincided with the peak of the febrile reaction and the antibodies remained detectable for a period of 8-30 weeks in the Palm River infected group and 18-30 weeks in the Ball-3 infected group. Peak reciprocal antibody titres in both groups ranged from 64 to 2048 between 3 and 6 weeks post-infection. No apparent serological differences were observed among the various C. ruminantium isolates when tested in homologous and heterologous IFATs. Post-infection sera to Anaplasma marginale, Theileria parva parva, Babesia bigemina and Rickettsia conorii did not exhibit reactivity with the C. ruminantium antigen. These results indicate the possible use of C. ruminantium-infected cultures as antigen in IFATs to detect similar C. ruminantium-specific antibody responses in the field in clinically sick, recovered and carrier animals.     

Virulence of two strains ofcowdria ruminantium in mice and their use to predict drug activity against heartwater

A study was made of the infectivity of two mouse-adapted strains of Cowdria ruminantium in mice. The Kwanyanga strain was most virulent in Balb/C mice which died nine days after infection with homogenate of liver from infected mice. CD-1 mice were least susceptible of six strains tested. The du Plessis strain of C. ruminantium was equally virulent in all six mouse strains. The du Plessis strain in CD-1 mice was used as the basis of a drug screen to detect activity against heartwater (C. ruminantium infection) and was highly predictive when active compounds were tested in sheep infected with the Ball 3 strain of C. ruminantium.     

Virulence stability in Flavobacterium psychrophilum after storage and preservation according to different procedures

Experimental infections and lethal dose 50% (LD 50) evaluation were conducted in rainbow trout fingerlings, using a virulent strain of Flavobacterium psychrophilum processed and stored or maintained in different ways; lyophilisation, freezing at -80 degrees C, maintenance in enriched Anacker and Ordal (EAO) medium at 4 degrees C, revival and subsequent in vivo passages in fish. Experiments were performed 1, 8 and 23 months after storing the bacteria. Out of a total of 12 cultures revived for experimentation, one failed to grow and another was found to express modified properties including decreased virulence in spite of in vivo passages. In all other cases, whatever the conditions of preservation, virulence was fairly well maintained after 1 and 8 months of storage. In the last test, after 23 months, the bacteria maintained in the EAO medium at 4 degrees C were found significantly attenuated. Conversely, lyophilised and frozen bacteria only expressed a slight increase in LD0. It was concluded that virulent strains of F. psychrophilum were likely to retain their properties without special provisions within limited periods of time, and that both lyophilisation and freezing at -80 degrees C were reliable methods for long-term preservation of virulence.     

Cowdria ruminantium: stability and preservation of the organism

Blood collected in either sodium heparin or disodium edetate vacutainers from febrile goats infected with 4 isolates of Cowdria ruminantium and cryopreserved with 10% dimethyl sulphoxide at -70 degrees C and -196 degrees C was an effective stabilate to initiate heartwater infections in goats. A homogenized pool of whole Amblyomma variegatum ticks in Snyder's buffer, maintained at -196 degrees C, was used to infect a goat with C. ruminantium. Liver and spleen collected from Swiss mice infected with the Kwanyanga isolate of C. ruminantium were homogenized in Snyder's buffer, maintained at -196 degrees C and were used to initiate infections in mice. Fresh blood collected from febrile goats and maintained at 4 degrees C for as long as 72 h was infectious to mice. Neutrophils separated from blood of C. ruminantium infected goats and maintained in modified RPMI medium at 37 degrees C for 68 h were infectious for a goat. Similarly neutrophils from a 2nd infected goat maintained for 96 h at 37 degrees C were infectious for mice.     

The detection of antibodies to Cowdria ruminantium in serum and C. ruminantium antigen in Amblyomma hebraeum by an enzyme-linked immunosorbent assay

A sensitive and reliable enzyme-linked immunosorbent assay for the detection of antibodies to Cowdria ruminantium in serum and C. ruminantium antigen in Amblyomma hebraeum nymphae is described. For the screening of antibodies, C. ruminantium from A. hebraeum nymphae, partially purified by wheat-germ lectin affinity chromatography, was used as antigen. To screen nymph populations, sera from either Ball 3 strain-infected sheep or Kumm-strain infected mice were used. By using appropriate controls the assays were rendered specific with respect to C. ruminantium.     

Cowdria ruminantium infection in ticks in the Kruger National Park.

Adult Amblyomma hebraeum ticks, the principle vector of heartwater (cowdriosis) of domestic ruminants in southern Africa, were collected in pheromone traps placed in Kruger National Park, an exclusively wildlife sanctuary in South Africa. These ticks transmitted Cowdria ruminantium, the rickettsial agent causing heartwater, to a susceptible goat, resulting in acute, fatal disease. C ruminantium was isolated in bovine endothelial cell culture from the plasma of this animal during the febrile stage of the disease and transmitted to susceptible goats, causing fatal heartwater. The prevalence of C ruminantium infection in 292 ticks was determined by polymerase chain reaction (PCR) analysis to be 1.7 per cent (95 per cent confidence interval 0.71 to 4.0 per cent). A DNA probe analysis, which is less sensitive than PCR, detected infection in three of the five PCR-positive ticks. The remaining infections were below the detection limit of the DNA probe, which is approximately 70,000 organisms. This is the first evidence that a vector-wildlife cycle of transmission of C ruminantium can be maintained independently of domestic ruminants.     

Comparison of diluents for maintaining the viability of Cowdria ruminantium

Thirteen solutions were compared to determine the optimal diluent for preservation of the viability of the Kwanyanga isolate of Cowdria ruminantium. They included the 2 diluents commonly used with C ruminantium and diluents proved effective with other rickettsiae. The capability of each diluent to maintain the viability of C ruminantium over a 3-hour period at room temperature was assessed by comparing the survival distributions of groups of outbred albino mice after they were inoculated IV with infected liver homogenates. The results indicated that the Snyder I diluent was significantly better at maintaining the viability of C ruminantium than were the other diluents studied.     

Histologic and immunochemical study of the pathogenesis of heartwater (Cowdria ruminantium infection) in goats and mice

Eleven adult goats and 32 adult outbred mice were inoculated IV with Cowdria ruminantium-infected blood (Kwanyanga isolate), monitored clinically, then serially euthanatized. Predominant clinical signs of disease in goats were depression, head tremors, seizures, and dyspnea. In mice, dyspnea and depression were the only clinical signs of disease noticed. Tissues were examined histologically and immunohistochemically for C ruminantium colonies or antigen. In goats, C ruminantium was detected only in endothelial cells of the brain, even though gross and microscopic lesions were confined to the thorax. In mice, C ruminantium was detected only in endothelial cells of the heart and lungs.     

Adherence of Bordetella avium to turkey tracheal mucosa: effects of culture conditions

Adherence of Bordetella avium to the tracheal mucosa of turkeys was evaluated, using bacteria grown under different culture conditions. Several solid and liquid media were used at incubation times of 12, 24, 36, or 48 hours with incubation temperatures of 18, 26.5, or 35 C. Adherence of B avium was greatest when the bacteria were grown on solid media at 35 C. Use of Bordet-Gengou or brain-heart infusion agar was associated with significantly greater (P less than 0.05) adherence compared with adherence of bacteria grown on other media. Adherence was greatest, using cultures in the stationary phase of growth; however, with some media, adherence diminished when incubation was extended beyond 36 hours. Adherence of B avium was reduced but not completely prevented when cultures were incubated at 18 C.     

Culture of feline corneal epithelial cells and infection with feline herpesvirus-1 as an investigative tool

OBJECTIVE: To isolate and characterize pure cultures of feline corneal epithelial cells and to assess the extent and nature of feline herpesvirus (FHV)-1 infection in these cells. SAMPLE POPULATION: Healthy eyes from 23 recently euthanatized cats. PROCEDURE: Stroma and epithelium of the rostral portion of the cornea were surgically isolated, and epithelial cells were detached from the stroma by enzymatic incubation. Epithelial cells were cultured in hormone-supplemented media. Cells were passaged, and cytokeratin expression was assessed. Cells were then infected with FHV-1, and cytopathic effects were determined. RESULTS: Cell cultures were readily established from samples obtained from each eye and could be maintained through 6 passages. Cultured cells expressed cytokeratins 3 and 12 but not other cytokeratins. Infection with FHV-1 was rapid and caused widespread cytopathic effects. CONCLUSIONS AND CLINICAL RELEVANCE: Feline corneal cells cultured in vitro during multiple passages maintain consistent morphologic characteristics and intermediate filament expression. They are susceptible to infection with FHV-1 and may provide a useful in vitro model for investigation of ocular drugs.     

Problems with the interpretation of epidemiological data in heartwater: a study on 23 farms

In an epidemiological study undertaken on 23 farms where heartwater occurs endemically, it was found that on an overall average, antibodies to Cowdria ruminantium were detected in 64.3% of the cattle, 6 adult Amblyomma hebraeum ticks were counted per animal and 7.0% of ticks were infected with the heartwater agent. It was found that the seropositivity of the animals was determined largely by the tick loads to which they were subjected and that the influence of the tick C. ruminantium infection rate was less evident. There was no parallel between the prevalence of heartwater on the farms and the immune status of the animals. In general, higher tick counts were recorded in herds where strategic tick control is practised than on farms with a total tick control programme. The method of tick control did not, however, appear to influence the immune status of the cattle, the tick infection rate, or the prevalence of heartwater.     

The development of Cowdria ruminantium in neutrophils

The sequential development of C. ruminantium (Kwanyanga and Kümm isolates) was followed in caprine leukocyte cultures by light microscopy, direct immunofluorescent microscopy (DFA), indirect immunoflourescent microscopy (IFA) and transmission electron microscopy (TEM). During the febrile response, one to several small cocci, large ring forms or rods were observed in neutrophils in blood smears and cytopreparations of neutrophil fractions using Diff Quik stain, Giemsa stain, DFA and TEM. One to several C. ruminantium colonies were seen in up to 35% of neutrophils maintained in vitro for 18 h to 5 days. The organisms were located in neutrophil phagosomes by TEM and were enveloped by two trilamellar unit membranes. Initially, C. ruminantium was tightly enclosed within phagosomes. At 20 h of incubation, organisms were frequently observed undergoing binary fission within enlarged phagosomal vacuoles. At later time periods, neutrophils harboured fully formed colonies (morula) containing numerous organisms. An occasional C. ruminantium-infected macrophage (Kümm isolate), and an occasional infected eosinophil (Kümm and Kwanyanga isolate) were found.     

Detection of Cowdria ruminantium antigen and antibody during the course of heartwater disease in sheep by means of an enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay was used to detect Cowdria ruminantium antibodies during the course of heartwater disease. IgM antibodies reached a maximum on the 4th day after infection and disappeared on the 7th day. IgG antibodies first appeared on the 8th day and continued to increase during the remainder of the observation period of 28 days. The presence of C. ruminantium in the blood fractions of diseased animals was demonstrated by an enzyme-linked immunosorbent assay. The earliest day of C. ruminantium antigen detection was in plasma and serum on the 4th day after inoculation. Of all the blood fractions investigated, the red blood cells showed the highest concentration, and this reached a maximum on the 12th day after infection.