Adaptation of an insect cell line (Grace's Antheraea cells) to medium free of insect hemolymph.

Cultures of the insect cell line derived by Grace from Antheraea eucalypti Scott [Austrocaligula eucalypti (Scott)] were successfully adapted to medium supplemented with fetal bovine serum, whole-egg ultrafiltrate, and bovine plasma albumin instead of insect hemolymph. Cells, now in their 37th passage, have a population doubling time of 2.5 days; those of unadapted cultures, 4.2 days.

Pheromone-mediation of host-selection in bont ticks (Amblyomma hebraeum koch).

The bont tick, Amblyomma hebraeum, is the principal vector to southern African ruminants of heartwater (Cowdria ruminantium infection). The role of feeding male ticks, which emit an aggregation-attachment pheromone, in attracting unfed ticks to cattle was investigated. Calves infested with feeding male ticks were more attractive to unfed adult ticks than were uninfested calves. The presence of the pheromone on previously infested cattle apparently allows unfed ticks to discriminate between hosts on which these parasites have fed successfully (suitable hosts) and those on which they have not (potentially unsuitable hosts). The use of acaricides is thus unlikely to reduce bont tick populations in areas where adequate numbers of alternate (wild) hosts are present. Also, cattle so treated may lose their resistance to heartwater through lessened exposure to infected ticks.

Helical mycoplasmas (spiroplasmas) from Ixodes ticks.

A new spiroplasma isolated from Ixodes pacificus collected in Oregon was serologically and morphologically distinct from known spiroplasmas. The new spiroplasma could also be isolated in tick cell cultures. Discovery of a new fastidious mycoplasma in ticks offers opportunities to explore the possible role of these agents in human and animal diseases.

Amino acid and protein depletion in medium of cell cultures infected with Cowdria ruminantium.

Cowdria ruminantium (Rickettsiales) causes heartwater in ruminants of Africa, and some islands off Africa and in the Caribbean Sea. The in vitro culture method for the organism devised in 1985, which provided for the first time a means for production of adequate quantities of live organisms and their products, is erratic and requires improvement. We studied depletion of amino acids (AAs) and major proteins in culture medium taken daily from infected and uninfected ovine and bovine vascular endothelial cell cultures. AAs of these samples were analyzed by Pico Tag reversed phase HPLC precolumn derivatization, and major proteins determined by capillary electrophoresis using a 57 cm x 75 microns fused silica tube at high pH. In both ovine and bovine cell cultures, significant depletion of arginine and glutamine occurred over a 5-day observation period regardless of whether they were infected or uninfected. This indicates that supplementation of nutrient media with these AAs might improve conditions for growth of the organism. Both AAs are essential for survival of cultured cells, and probably for the rickettsia (although the metabolism of C. ruminantium is poorly understood). Concentrations of several AAs increased in infected cultures, implying de novo synthesis and/or proteolysis on the part of the organism. In fact, several protein fractions did decrease in culture medium throughout the course of infection, while increasing or remaining unchanged in uninfected control cultures. Proteolytic activity by C. ruminantium may be essential for nitrogen metabolism by the organism. It is suggested that studies such as these will facilitate the development of a specific medium for optimal in vitro growth of the heartwater organism, and may also lead to an understanding of the metabolic stratagem of C. ruminantium. This knowledge, in turn, could reveal the mechanism for pathogenesis of heartwater, with implications for control.

Growth of bluetongue and epizootic hemorrhagic disease of deer viruses in poikilothermic cell systems.

Continuous cell lines from the ticks Dermacentor variabilis, D. parumapertus, D. nitens, Rhipicephalus sanguineus and R. appendiculatus, the mosquitoes Aedes albopictus and Culex quinquefasciatus and the African toad Xenopus laevis were tested for their ability to replicate bluetongue (BT) and epizootic hemorrhagic disease of deer (EHD) viruses, and for their sensitivity as potential isolation systems. BT serotype 17 grew to peak titers of 10(4.5)-10(7.5) TCID50 ml-1 in all except one of the tick cell lines, EHD 2 virus attained titers similar to that of BT 17 in the mosquito and toads cells, but failed to replicate in tick cells. Only Aedes albopictus and Xenopus laevis cells were as sensitive to infection with low-passage BT 11 and EHD 2 viruses as control cultures of Vero and BHK cells. At 27 degrees C, persistent infection of Xenopus laevis cells occurred, producing low yields of BT 17 and EHD 2. When shifted to 32 degrees C, these cultures expressed virus in exponential increments. No cytopathic effect (CPE) was seen in any of the tick-virus systems, but infected mosquito and toad cells detached from the monolayer within 3-6 days after inoculation with either virus. In the toad cells, this CPE was presaged by the development of plaques within 48 h after infection. Potential applications of poikilotherm systems in orbivirus research are discussed.

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.

Influence of feeding and starvation on the persistence and transmission of Quaranfil virus by Argas (Persicargas) arboreus (Acari: Argasidae).

Viral antigen was detected in the cytoplasm and in associated membranes of salivary gland acinus cells by indirect immunofluorescence and immunoperoxidase staining. Viral ribonucleoproteins (indicated histochemically by presence of pyroninophilic granules) which had accumulated in the cytoplasm of salivary gland type B (granular) acini of unfed Argas (Persicargas) arboreus Kaiser, Hoogstraal & Kohls were no longer visible 24 h after feeding. Virus in tick salivary glands increased from 300 to 500 plaque-forming units during the brief feeding interval (approximately 1 h), but virus was not detectable by 72 h. Overall salivary gland, ovarian, and synganglion tissue levels of Quaranfil virus decreased in the 96 h after feeding, except for synganglion samples in which virus titers increased during 24 h after feeding. Starvation for 105 d resulted in a sevenfold increase in salivary gland viral content compared with those starved 45 d, whereas synganglion tissue titers for Quaranfil virus became undetectable, and ovarian tissue values were similar to those starved for 45 d. Feeding had a greater effect on viral persistence in tissues for ticks starved 60 additional d (comparing 45 with 105 d) in that no Quaranfil virus was detected in any tissue after 48 h (compared with 72 h). Feeding infected ticks (with short extrinsic incubation) on chicks resulted in a peak of host mortality on days 7 and 8, whereas long extrinsic incubation resulted in sporadic mortality over 20 d of monitoring.

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.

Current status of in vitro cultivation of Cowdria ruminantium.

Heartwater caused by Cowdria ruminantium infection is the most important tick-borne disease of ruminants in southern Africa. The in vitro culture system for this rickettsia, developed less than a decade ago, is responsible for the great majority of research accomplishments currently being reported in the areas of epidemiology, diagnosis and control of heartwater. Despite this progress, cultivation of C. ruminantium remains more of an art than a science, inasmuch as significant discrepancies exist, both among and within laboratories, in the ability to successfully and repeatedly produce cultured organisms. The current status of the in vitro system and its contributions are reviewed, and ongoing research in these areas by the Onderstepoort Veterinary Laboratory and collaborating institutions is discussed.

The pathogenicity and immunologic relationship of a virulent and a tissue-culture-adapted Babesia bovis.

Babesia bovis grown in tissue culture was used to inoculate 12, 2-year-old Holstein steers. All 12 developed serological evidence of infection but only six had a febrile response of greater than or equal to 40 degrees C, and only one had a demonstrable B. bovis parasitemia. An average modest drop of 19% was observed in packed cell volume (PCV) during the period of reaction. All 12 steers were subsequently challenged with virulent B. bovis: seven on day 78 post inoculation (p.i.), two on day 106 p.i., and three on day 251 p.i. No demonstrable clinical response was observed in any of the 12 steers previously exposed to the tissue-culture organism, whereas severe signs of babesiosis occurred in seven 2-year-old, non-vaccinated control steers given a comparably virulent B. bovis challenge. All seven controls showed a febrile response, B. bovis parasitemias, with an average drop of 55% in PCV and a 28% mortality.

Infection rates with Cowdria ruminantium of nymphs and adults of the bont tick Amblyomma hebraeum collected in the field in Zimbabwe.

Cowdria ruminantium (heatwater) infection rates of field populations of the bont tick, Amblyomma hebraeum, were determined at two locations in the southern lowveld of Zimbabwe. At Mbizi Quarantine Station, unfed adult males and females, and nymphs were collected at intervals over a 2-year period using traps. At Lemco Ranch, engorged nymphs were collected on three occasions from weaner calves and allowed to moult to adults. The unfed ticks were fed in small pools on heartwater-susceptible sheep, some of which became infected. The infection rates of the ticks were then estimated statistically. Depending on the date of collection and locality, these rates were in the range 0.0-44.9% for males, 20.0-36.1% for females and 0.0-13.4% for nymphs. Most of these rates are considerably higher than those previously believed to occur.

Attenuation of Babesia bovis by in vitro cultivation.

A virulent strain of Babesia bovis was adapted to grow in erythrocyte culture in the presence of equine serum and in lieu of bovine serum. Four splenectomized calves inoculated with the adapted strain, 429, developed hematologic signs of infection and a low grade fever, but remained free of central nervous system (CNS) signs and recovered. All of six control animals inoculated with a virulent strain reacted severely and five showed CNS signs and died. The calves injected with the attenuated strain were solidly immune when challenged with the virulent strain at 44 or 78 days after vaccination.

Establishment and characterization of a diploid cell line from the tick, Dermacentor parumapertus Neumann (Acarina: Ixodidae).

Establishment of a continuous cell line (RML-14) from embryonic tissues of the tick Dermacentor parumapertus Neumann is reported. The culture medium employed consisted of a combination (2:1) of Eagle's and L-15 (Leibovitz) media supplemented with 20% fetal bovine serum, 10% tryptose phosphate broth, and 0.1% bovine plasma albumin. At the 8th passage, 99% of dividing cells had the female chromosome complement, among which more than 70% had a diploid chromosome number of 22. At the 13th passage, cell population showed approximately a 3-fold increase during the first 8 days of culture. As of December 1976, had been subcultured 40 times.

Clinical mange of the black bear, Ursus americanus, associated with Ursicoptes americanus (Acari: Audycoptidae).

Numerous specimens of a mite, Ursicoptes americanus Fain and Johnston, 1970 (Acari: Audycoptidae), were found in skin scrapings from an aged female black bear, Ursus americanus, afflicted with a severe generalized mange. The mite had not been found previously in association with clinical dermatitis. The male, nymph and larva of Ursicoptes americanus, heretofore unknown, are described and the generic diagnosis is amplified. Discovery of the male aids in resolving the questionable taxonomic status of the family Audycoptidae.

Heartwater in sheep and goats: a review.

Heartwater (cowdriosis) is an important, often fatal, tick-borne disease of domestic and wild ruminants in sub-Saharan Africa and some Indian Ocean and Caribbean islands. The causal agent, Cowdria ruminantium (Cowdry 1925), is a rickettsia closely related to members of the genus Ehrlichia, and is probably a part of a complex of genomic species. Imported breeds of sheep and goats (especially Angoras) are highly susceptible, but indigenous populations of endemic areas may be resistant to infection. Very young stock (less than 9 d old) possess a natural resistance that is unrelated to the immune status of the dams. Symptoms of heartwater vary, but usually begin with fever and may involve neurological signs and respiratory distress. Clinical diagnosis is based on symptoms, history of tick-exposure and post-mortem findings, and is confirmed by demonstration of characteristic rickettsial organisms in vascular endothelial cells. Laboratory diagnosis is retrospective and includes fluorescent antibody and enzyme-linked immunosorbent assays. Serological tests are compromised by non-specific reactions with certain Ehrlichia spp. DNA and oligonucleotide probes have been developed, but are thus far unavailable in many countries affected by heartwater. Treatment with tetracyclines is effective if begun in the early stages of infection. Control is based on a knowledge of the disease cycle in nature, and is achieved through judicious tick control, vaccination or both. A virulent, blood-based vaccine is available. Existence of a carrier state in recovered animals, including wild ruminants, complicates control efforts, and eradication is feasible only in circumscribed foci. Problem areas in fundamental and applied research on heartwater, as it affects sheep and goats, are discussed.

Distinctive staining of colonies of Cowdria ruminantium in midguts of Amblyomma hebraeum.

Mallory's phloxine-methylene blue stain was used to differentiate colonies of Cowdria ruminantium in midgut epithelial cells of nymphal Amblyomma hebraeum that had been infected as larvae. Gut tissues were collected from nymphs that had fed on a susceptible sheep and were fixed in formol-saline on the day of repletion. Paraffin sections, 3-4 micron thick, were then stained and this rendered colonies and cell nuclei densely blue against a uniformly pink background of tick tissues. Colonies were easily distinguished from nuclei by their specific morphology. This method of parasite visualization may be adapted to field-collected ticks for rapid detection of C. ruminantium or to assays of susceptibility of tick populations to various strains of the organism.

Sunday Canyon virus, a new ungrouped agent from the tick Argas (A.) cooleyi in Texas.

A new arbovirus was isolated from Texas, U.S.A., populations of the Cliff Swallow parasits Argas (Argas) cooleyi Kohls and Hoogstraal, 1960. The virus, named Sunday Canyon, is serological urelated to any of 185 arbovirus strains or 20 other viral agents with which it was compared. Morphologically it resembles Bunyamwera viruses and, in common with them, is sensitive to lipid solvents and acid pH, and apparently possesses RNA. Although considerably resistant to a temperature of 41.5 degrees C, it rapidly loses infectivity when incubated at 56 degrees C. It is lethal for newborn white mice and infective for the Vero and Antheraea eucalypti cell lines. Sundays Canyon virus is the second tick-associated, Bunyamwera virus-like agent known from North America and the third virus to be reported from A. cooleyi in Texas.

Isolation of spotted fever group and Wolbachia-like agents from field-collected materials by means of plaque formation in mammalian and mosquito cells.

Three isolations from ticks (Dermacentor occidentalis) of a rickettsia of the spotted fever group and 5 isolations from chipmunk (Eutamias rugicaudus) blood of a Wolbachia-like agent were obtained from plaques formed in Singh's Aedes albopictus (mosquito) and Vero (African green monkey kidney) cell cultures. These organisms could not be isolated by injection of the infected ticks or blood into embryonated chicken eggs, guinea pigs, or voles (Microtus pennsylvanicus), but fluid cultures of Grace's Antheraea eucalypti (moth1 and Singh's A. albopictus cells inoculated with the bloods yielded the Wolbachia-like agent.