Preliminary observations on the use of the capillary flocculation test for the diagnosis of heartwater (Cowdria ruminantium infection).

A capillary flocculation test was developed to diagnose heartwater disease of ruminants. Antigen was prepared from the brains of cattle and goats highly infected with Cowdria ruminantium. Sera were obtained from experimentally infected ruminants which either recovered naturally or with the aid of oxytetracycline treatment. Antibodies were first detected one to two weeks after clinical recovery or after treatment, and persisted for periods varying between one and four weeks. Control sera collected from cattle (sheep) and goats in the Netherlands where heartwater does not occur, or from animals serologically positive for Anaplasma marginale or Eperythrozoon ovis infections, did not react to the test.     

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.     

An assessment of the economic impact of heartwater (Cowdria ruminantium infection) and its control in Zimbabwe.

Heartwater, caused by the rickettsial organism Cowdria ruminantium, is a serious constraint to livestock development in much of sub-Saharan Africa. Traditionally, the disease has been controlled by the use of chemical acaricides to control the vector tick. The University of Florida/USAID-supported heartwater research project (based in Zimbabwe) is developing a new inactivated vaccine to control the disease. In order that the vaccine is used effectively, the project has been studying the epidemiology of the disease in different livestock production systems of Zimbabwe, and evaluating the economic impact of the disease and of its future control using a vaccine such as the one under development. Initially, field studies were conducted to characterise the communal and commercial livestock-productions systems at risk from heartwater and to understand the epidemiology of the disease. The data from these studies were then applied to an infection-dynamics model of heartwater, which was used to provide estimates of disease incidence and impact under various scenarios over a period of 10 yr. Two principal outputs of the epidemiological model (cumulative annual heartwater incidence and infection-fatality proportion) were key inputs into an economics model. The estimated total annual national losses amount to Z$ 61.3 million (US$ 5.6 million) in discounted value terms over 10 yr. Annual economic losses per animal in the commercial production system (Z$ 56 discounted values) are 25 times greater than the losses in the communal system (Z$ 2.2). The greatest component of economic loss is acaricide cost (76%), followed by milk loss (18%) and treatment cost (5%). Losses in outputs other than milk (beef, traction and manure) appear to be minimal. A new vaccine has the promise of a benefit: cost ratio of about 2.4:1 in the communal and 7.6:1 in the commercial system. A control strategy based on a new vaccine would yield additional non-financial benefits to farmers and the government resulting from reductions in the use of chemical acaricides.     

Diagnosis of heartwater at post-mortem in ruminants and the confirmation of Cowdria ruminantium in mice

Criteria that are required to confirm heartwater in ruminants and mice are discussed. In ruminants it entails the macroscopical and microscopical lesions as well as the identification of Cowdria ruminantium in brain smears or histological sections. Macroscopical lesions in the majority of animals that die of the disease include effusion of body cavities, hydropericardium, oedema of the lungs, brain, mediastinum, and its associated lymph nodes, and splenomegaly. The effect of specific chemotherapy on the morphology of heartwater organisms in vivo is outlined. A severe nephrosis in heartwater-infected Angora goats, treated after the first day of the febrile reaction, is described.     

The role of males of the bont tick (Amblyomma hebraeum) in the transmission of Cowdria ruminantium (heartwater)

The role of males of the bont tick (Amblyomma hebraeum) in the transmission of Cowdria ruminantium (heartwater) was investigated. Transstadial (nymph to adult) and intrastadial transmission were demonstrated. Males transferred from live or dead hosts to live hosts were shown to transmit C. ruminantium repeatedly. It was concluded that male transmission is of importance in the epidemiology of heartwater.     

The pathology of heartwater. I. A study of mice infected with the Welgevonden strain of Cowdria ruminantium

Gross and microscopical lesions in mice intravenously infected with the Welgevonden strain of Cowdria ruminantium closely resembled the lesions described in cattle, sheep and goats. A high concentration of organisms was present in alveolar endothelial cells. Cytopathic changes in parasitized and non-parasitized endothelial cells and the morphology of the organisms are described and compared with the Ball3 strain of C. ruminantium. Possible mechanisms in the development of the lung oedema are considered and the role of mice as animal model is discussed.     

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.     

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.     

Colostrum-derived antibodies to Cowdria ruminantium in the serum of calves and lambs

Antibodies to Cowdria ruminantium were detected in the serum of calves born from artificially immunized heifers, by means of the indirect fluorescent antibody test, only for as long as 4 weeks after birth. Lambs born from artificially immunized ewes, however, were still serologically positive at 8-12 weeks of age. Much higher antibody titres were recorded in the sera of ewes and their lambs than in that of heifers and their calves.     

Morphology and development of Cowdria ruminantium in Amblyomma ticks

The morphology and development of Cowdria ruminantium have been studied in Amblyomma hebraeum and A. variegatum. Colonies of C. ruminantium have so far been demonstrated microscopically in gut, salivary gland cells, haemocytes and malphighian tubules of infected Amblyomma ticks. Colonies in gut cells were seen in both unfed and feeding ticks but colonies in salivary gland acini were observed only in nymphs that had fed for 4 days. Although the predominant type seen in both tick stages was the reticulated form that appeared to divide by binary fission, electron dense forms were also present. The latter are similar to those forms documented in endothelial cells of the vertebrate host as well as in cell culture. The presence of colonies of C. ruminantium in salivary glands of feeding ticks, along with the demonstration of different morphologic forms of the organism, suggests that a developmental cycle of the organism occurs in its invertebrate host. It is thought that organisms first infect and develop within gut cells. From there subsequent stages continue their development in haemolymph and salivary glands and are then transferred to the vertebrate host during tick feeding. Further studies are needed to completely understand the development of C. ruminantium in ticks and its subsequent transmission by these parasites.     

The efficacy of alternative routes for the infection or vaccination of animals with Cowdria ruminantium

The efficacy of subcutaneous, intramuscular and intraperitoneal inoculation of heartwater infective blood or nymph suspensions was tested in 5 experiments involving a total of 199 sheep. The success rate of subcutaneous injections varied greatly (0-100%) in the different groups. However, it was found that certain additives to the inoculum, such as dimethyl sulphoxide or uninfective brain tissue, increased the efficacy of the subcutaneous route. Indications are that the site of inoculation and especially the dose volume are important factors in the success rate of such inoculations. Intramuscular injection with infective nymph suspensions containing bradikynin or hyaluronidase produced heartwater reactions in 11 out of 14 sheep. In 2 experiments, involving 25 cattle, it was found that, although very few animals showed definite reactions after the subcutaneous or intramuscular injection of nymph suspension and additives, the majority were afterwards immune to challenge. This phenomenon, which was also present in some sheep, needs further investigation.     

The natural resistance of cattle to artificial infection with Cowdria ruminantium: the role played by conglutinin

The conglutinin titres of year-old Bonsmara-cross cattle infected with Cowdria ruminantium were inversely proportional to the severity of the reactions elicited by the infection. There was no correlation, however, between conglutinin levels of 8-month-old calves of the same breed, sex and origin and their susceptibility to heartwater. The role possibly played by conglutinin in the non-specific resistance of cattle to heartwater and in the epidemiology of the disease is discussed.     

Experimental transmission of Cowdria ruminantium by the Gulf coast tick Amblyomma maculatum: danger of introducing heartwater and benign African theileriasis onto the American mainland

The tick Amblyomma maculatum proved to be an experimental vector of Cowdria ruminantium, the cause of heartwater of ruminants. Transstadial transmission from larva to nymph and from larva through nymph to adult was successful; however, an experiment on transovarial transmission gave negative results. Neither A americanum nor A cajennense transmitted the infection. There is a potential danger of the disease gaining access to the American mainland from the Caribbean where it is known to occur and of maintaining itself in areas where A maculatum occurs. Transstadial transmission experiments, using 3 American Amblyomma spp and an African strain of Theileria mutans, which is thought to occur in the Caribbean, were unsuccessful.     

The effect of prolonged oral administration of oxytetracycline on the course of heartwater (Cowdria Ruminantium) infection in sheep

Summary The effect of the prolonged oral administration of tetracycline on the course of artificially-induced heartwater in sheep was investigated. It was shown that the course of the disease could be greatly modified by this treatment, only one sheep in thirty showing typical heartwater during a 38-day period post inoculation. All the remaining sheep were immune when challenged with heartwater after 38 days. The practical implications of these findings and the possible development of drug-resistance are discussed. Attention is drawn to the absence of significant side effects following high-level feeding of oxytetracycline to sheep for a prolonged period.

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Sumario El efecto de la administración oral prolongada de la tetraciclina sobre el curso del corazón de agua artificialmente inducido en ovinos fue investigado. So demostró que el curso de la enfermedad podía ser grandemente modificado mediante este tratamiento, mostrando solamente un ovino de treinta, típico corazón de agua durante un periodo de 38 dias despues de la inoculación. Todo el remanente de los ovinos fueron inmunes cuando se les desafió con corazón de agua despues de 38 dias. Se discuten las implicaciones práticas de estos hallazgos y el posible desarrollo de resistencia a las drogas. Se hace notar la ausencia de efectos secundariosde significación despues de la alimentación prolongada de altos niveles de oxitetraciclina en ovinos.

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Résumé L’effet de l’administration orale de longue durée de tétracycline sur l’évolution de la heartwater expérimentale chez le mouton a été étudié. Il a été montré que l’évolution de la maladie pouvait être grandement modifiée par ce traitement, étant donné qu’un seul mouton sur trente a montré la heartwater typique pendant une période de 38 jours suivant l’inoculation. Tous les autres moutons étaient immunisés contre l’épreuve par la heartwater après 38 jours. La signification pratique de ces résultats et le développement possible d’une résistance au médicament sont discutés. L’attention est attirée sur l’absence d’effets toxiques notables de l’administration orale à haute dose d’oxytétracycline au mouton pendant une longue période.

     

Lack of cross-protection between Cowdria ruminantium and Ehrlichia phagocytophila.

Antigenically distinct stocks of Cowdria ruminatium from Senegal and South Africa were compared with a Dutch isolate of Ehrlichia phagocytophila in cross-immunity trials in goats. There was a complete absence of cross-immunity between E. phagocytophila and C. ruminantium, despite previous observations that both rickettsial organisms have certain antigenic determinants in common.     

The pathology of heartwater. II. A study of the lung lesions in sheep and goats infected with the Ball3 strain of Cowdria ruminantium

Lung lesions in sheep and goats infected with the Ball3 strain of Cowdria ruminantium corresponded with those reported in mice infected with the Welgevonden strain of Cowdria ruminantium. Ultrastructural changes in the alveolar endothelial and epithelial cells are described and the pathogenesis of the lung oedema is briefly discussed.