Collection of free-living nymphs and adults of Amblyomma hebraeum (Acari: Ixodidae) with pheromone/carbon dioxide traps at 5 different ecological sites in heartwater endemic regions of South Africa.

The capture of free-living adults and nymphs of Amblyomma hebraeum, the main vector of heartwater in domestic and wild ruminants in South Africa, by means of attraction-aggregation-attachment-pheromone/carbon dioxide traps at five endemic localities in South Africa is described. Although the traps were used successfully at each of the localities, no determination of their efficiency in relation to the actual abundance of ticks at a particular site was made. This study confirmed that the traps could be used in a variety of ecological areas to locate populations of free-living adult A. hebraeum.

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.

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.

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.

Spiroplasma ixodetis sp. nov., a new species from Ixodes pacificus ticks collected in Oregon.

Eight strains of mollicutes were isolated from pooled suspensions prepared from western black-legged ticks (Ixodes pacificus) collected in Oregon. Morphologic examination by electron and dark-field microscopic techniques showed that each strain consisted of a mixture of motile, tightly coiled helical cells, small coccoid cells with diameters ranging from 300 to 500 nm, and pleomorphic, straight or branched filamentous forms. All cellular forms were surrounded by a single cytoplasmic membrane, and there was no evidence of a cell wall. The organisms were filterable and fastidious in their growth requirements. The optimum temperature for growth was 30 degrees C, but multiplication occurred at temperatures ranging from 23 to 32 degrees C. The strains catabolized glucose but did not hydrolyze arginine or urea. The genome size of strain Y32T (T = type strain) was 2,220 kbp, and the DNA base composition (guanine-plus-cytosine content) of this organism was 25 +/- 1 mol%. The eight isolates were serologically related to each other but were not related to 37 other type or representative strains belonging to the genus Spiroplasma. Strain Y32 (= ATCC 33835) is the type strain of Spiroplasma ixodetis sp. nov.

Pheromonal composition of two species of African Amblyomma ticks: similarities, differences and possible species specific components.

Two species of bont ticks, Amblyomma hebraeum and Amblyomma variegatum, common to Africa were studied to compare types and quantities of compounds known or believed to serve as components of the attraction-aggregation-attachment pheromone (AAAP). A complex of attraction, aggregation and attachment stimulating pheromone components are used by these ticks to detect hosts, mates and, perhaps, minimize interspecific breeding. Solvent extraction of pheromone emitting ticks followed by gas chromatography and mass spectrometry revealed little qualitative difference in the composition of the AAAP in these two species. However, subtle differences in the relative proportions of the pheromonal blend are noted suggesting that such differences in concentration may facilitate species-specific discrimination during aggregation and attachment. Differences in the relative abundance of benzaldehyde and methyl salicylate in the males of the two species were especially noteworthy. Possible means by which such differences in phenolic compound composition may affect the behavior of these ticks are discussed.

Detection of Cowdria ruminantium by means of a DNA probe, pCS20 in infected bont ticks, Amblyomma hebraeum, the major vector of heartwater in southern Africa.

A DNA probe, pCS20, previously described for use in detection of Cowdria ruminantium infections in Amblyomma variegatum (the principal vector of heartwater) hybridized with C. ruminantium DNA in organs of laboratory-infected A. hebraeum adult ticks (the major southern African vector of heartwater). The probe hybridized with C. ruminantium DNA in 46/49 midguts from male ticks and 26/29 from females, thus indicating infection. Corresponding salivary glands were less heavily infected, but infections were more numerous in glands from males. Infection in ticks was confirmed by transmission of the disease to susceptible goats. The probe did not hybridize with DNA from uninfected ticks or with DNA from a spotted fever group rickettsia commonly associated with A. hebraeum in Zimbabwe. The C. ruminantium specific pCS20 DNA probe can be applied to determine accurately the infection rates in the two major vectors of heartwater and the risk of exposure of ruminants in endemic areas.

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.

Olfactory responses of adult Amblyomma hebraeum and A. variegatum (Acari: Ixodidae) to attractant chemicals in laboratory tests.

Unfed adults of the African ticks, Amblyomma hebraeum Koch and A. variegatum (Fabricius), important vectors of human and animal diseases, were exposed to volatile compounds in an olfactometer in efforts to identify both tick-produced or synthetic chemicals capable of eliciting an attraction response. A formula, relative efficacy of attraction, was devised for comparison of responses between species and sexes to a particular test stimulus, or within a homogeneous population to different stimuli. Adults of both species responded strongly to known tick-pheromone constituents, nonanoic acid, methyl salicylate, 2.6-dichlorophenol and benzyl alcohol, as well as to a commercially produced antiseptic, TCP (Pfizer), and its major components, chlorinated and iodinated phenols. Benzaldehyde, a proposed tick-pheromone component, and heptadecane, not known from ticks, were markedly attractive to adults of A. hebraeum but not to those of A. variegatum. Males of the former species, but neither conspecific females not either sex of the latter species, responded significantly to salicylaldehyde (known from males of four species of ticks, including A. variegatum). o-nitrophenol, a major component of the aggregation-attachment pheromone of males of both A. variegatum and A hebraeum and a proven long-range attractant for them in the field, was only partially attractive to either species in the olfactometer. Neither species was attracted to 2-methylpropanoic acid, previously identified in volatile effluents form feeding male A. hebraeum. It is concluded that these important disease vectors respond positively to a variety of volatile chemicals, which may conceivably be used to attract them to traps, animals or acaricides in efforts to control ticks or the diseases they transmit.

Comparison of known and suspected pheromonal constituents in males of African ticks, Amblyomma hebraeum Koch and Amblyomma variegatum (Fabricius).

Three low molecular weight compounds were found in hexane:diethyl ether extracts of fed males of the African ticks, Amblyomma variegatum (tropical bont tick) and A. hebraeum (bont tick), namely, o-nitrophenol, methyl salicylate and 2,6-dichlorophenol. These same compounds were also fond in a rinse of fed A. variegatum males, but were absent or present in only trace amounts in a rinse of fed A. hebraeum males, o-Nitrophenol and methyl salicylate were present in much higher concentrations (i.e., amounts/tick) in A. variegatum than in A. hebraeum. 2,6-Dichlorophenol was also more abundant in A. variegatum than in A. hebraeum, but the differences were not as great as with the former two compounds. Extraction in hexane over a 3-week period revealed four additional compounds, benzaldehyde, benzyl alcohol, benzothiazole and nonanoic acid. The first three compounds were found in males of both species; nonanoic acid was found only in A. hebraeum males. Published reports consistently show strong attraction by o-nitrophenol and methyl salicylate for both sexes of the two bont tick species; 2,6-dichlorophenol and benzaldehyde have been reported to be attractive to both sexes of A. hebraeum. The possible roles of these compounds, as well as others occasionally reported from A. hebraeum and A. variegatum, as components of the aggregation/attachment pheromone or other pheromones is discussed.

A cloned DNA probe identifies Cowdria ruminantium in Amblyomma variegatum ticks.

Heartwater, caused by Cowdria ruminantium and transmitted by ticks of the genus Amblyomma, is a constraint to ruminant animal production in sub-Saharan Africa. This rickettsial disease could spread from endemically infected areas of sub-Saharan Africa and certain Caribbean islands to other countries, including the United States, in which Amblyomma ticks exist. To detect C. ruminantium in tick vectors and animals, we made DNA probes from C. ruminantium DNA isolated from endothelial cell cultures. Two clones were evaluated; pCS20 from Crystal Springs (Zimbabwe) strain DNA had a 1,306-bp insert, and pCR9 from Kiswani (Kenya) strain DNA had a 754-bp insert. Both DNA probes detected 1 ng of Crystal Springs DNA; however, the pCS20 probe had a 10-fold-greater ability to discriminate between C. ruminantium DNA and DNA from other organisms. Also, the pCS20 probe did not hybridize to 400 ng (highest amount tested) of DNA from bovine cells, 3 protozoa, 3 rickettsiae, and 12 bacteria. In all experiments, C. ruminantium DNA was detected in midguts from 99 of 160 Amblyomma variegatum nymphs infected as larvae and in midguts from 38 of 80 adult ticks infected as nymphs but not in midguts from control nymphs and adults. The presence of C. ruminantium in nymphs and adults was confirmed by transmission of heartwater to goats. The DNA sequences of both probes were determined; synthetic oligonucleotides from pCS20 are recommended as DNA probes for C. ruminantium.

Pheromone/acaricide mixtures in the control of the tick Amblyomma hebraeum: effects of acaricides on attraction and attachment.

Unfed adults and nymphs of the bont tick Amblyomma hebraeum Koch are attracted to hosts on which fed males, emitting an aggregation-attachment pheromone (AAP), are attached. Pheromone/acaricide mixtures have the potential to selectively attract and kill these ticks. We have investigated the effects of three acaricides, amitraz (an amidine), flumethrin (a synthetic pyrethroid) and chlorfenvinphos (an organophosphate), combined with AAP, on the attraction and attachment of the unfed adults. Attraction, measured in field assays involving AAP and CO2, was not inhibited by any of the acaricides. Attachment was measured over 24 h on the ears of rabbits which had been treated with AAP and mixtures of AAP and the acaricides. None of the acaricides inhibited initial attachment. Flumethrin caused rapid and high mortality in attached and unattached ticks. Chlorfenvinphos caused little mortality in the first 24 h on the ears of the rabbits, but 75% of the exposed ticks died over the next 14 days while held in an incubator. Amitraz caused the ticks to detach after an initial period of attachment; there was little mortality in the detached ticks over the next two weeks and they could later be induced to re-attach to other rabbits. Flumethrin was considered to be the compound of choice for use in pheromone/acaricide mixtures.

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.

Improved culture conditions for Cowdria ruminantium (Rickettsiales), the agent of heartwater disease of domestic ruminants.

The causal agent of heartwater disease of domestic ruminants, Cowdria ruminantium, can, with difficulty, be isolated and passaged in lines of bovine endothelial cells grown in the presence of the Glasgow modification of Eagle's minimal essential medium. However, when Leibovitz's L-15 medium supplemented with 0.45% glucose at pH 6.0-6.5 is used as maintenance medium for these cells, isolation and serial passage may routinely be achieved.

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.

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.

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.

Field sampling of unfed nymphs of Amblyomma hebraeum.

Unfed nymphs of Amblyomma hebraeum Koch, 1844 shelter under debris on the soil surface and become active in host-seeking when stimulated with carbon dioxide. The active nymphs are not attracted to sources of carbon dioxide and hence cannot be effectively sampled using static carbon dioxide traps. However, these nymphs will cling to flannelette and can be sampled by flagging. Larger numbers are recovered if the soil surface is sampled using a vacuum device.