Heartwater in the Caribbean: isolation of Cowdria ruminantium from Antigua

Adult Ambylomma variegatum ticks were collected from 184 cattle, 13 sheep and one goat in Antigua, and ground in phosphate buffered saline. The resultant supernates were cryopreserved in liquid nitrogen. Five supernate pools, each derived from approximately 100 ticks collected from different herds, were thawed and each was inoculated intravenously into a separate experimental goat. One goat exhibited a febrile response with Cowdria ruminantium demonstrable in brain biopsies; after recovery, this animal showed no reaction to a lethal challenge with a Guadeloupe isolate of C ruminantium.     

Heartwater. The morphology of Cowdria ruminantium and its staining characteristics in the vertebrate host and in vitro

The morphology of Cowdria ruminantium is described and its staining characteristics in the vertebrate host and in vitro are summarized. Morphologically, the organisms are characterized in the cytoplasm of endothelial cells, macrophages and reticulo-endothelial cells. Based on the morphology of the internal structure of the organisms, elementary (electron-dense), intermediate and reticulate bodies are identified. Each organism is surrounded by a double membrane and a "capsule" is evident around a few organisms in vitro. Usually, only organisms of the same form are found within a particular vacuole, although mixed colonies are described in the in vitro studies.     

Heartwater. The artificial transmission of Cowdria ruminantium in domestic ruminants and mice

The artificial transmission of Cowdria ruminantium with infected blood, organ homogenates, peritoneal macrophages, tick stabilate and tissue culture cells is discussed. Organ homogenates prepared from the myocardium, spleen, kidneys and liver of diseased animals are commonly used to infect mice. The efficacy of organ homogenates as a source of C. ruminantium depends on factors such as the route of inoculation and the heartwater isolate used. Heartwater is artificially transmitted with infected tick stabilate, haemocytes, rectal ampules and hypodermal homogenates. The infectivity of saliva collected from Amblyomma hebraeum female ticks was very low compared to the ground-up suspensions prepared from the same group of ticks.     

Heartwater. The development and life cycle of Cowdria ruminantium in the vertebrate host, ticks and cultured endothelial cells

Various aspects of the development and life cycle of Cowdria ruminantium are discussed. C. ruminantium is transmitted transstadially by certain Amblyomma species. Apparently organisms initially develop in the gut epithelial cells of ticks and subsequent stages of C. ruminantium invade and develop in the salivary gland acini cells of the vector. Stages at which transmission to the final host are attained appear to be coordinated with the feeding cycle of the ticks and the vertebrate host is infected via salivary glands of the tick. In the vertebrate host, ticks and cultured endothelial cells, different morphological forms of C. ruminantium (electron-dense and reticulated forms) are found. Organisms enter cells through a process resembling phagocytosis and reticulated forms of the organisms appear to be the main vegetative stage. In the vertebrate host, organisms proliferate in vascular endothelial cells, neutrophils, macrophages and reticulo-endothelial cells.     

In vitro cultivation of Cowdria ruminantium

Cowdria ruminantium was cultivated in a calf endothelial cell line after the cells had been irradiated at 45 & 90 GY. Another experiment in which the inoculum and non-irradiated cells were centrifuged together also yielded positive results. In some irradiated cultures, colonies of organisms could be demonstrated microscopically up to 70 days after the cultures had been inoculated with infected tick stabilate. The infectivity of cultures, even after 4 passages and 88 days post-inoculation, was demonstrated by their intravenous injection in sheep.     

Heartwater serology: some problems with the interpretation of results

Antibodies in the sera of domestic ruminants that have been infected with Ehrlichia bovis and other ehrlichial agents cross-react with the Kümm strain of Cowdria ruminantium used in the indirect fluorescent antibody test as antigen. These cross-reactions are also shown by the Elisa test in which the Ball 3 strain of the heartwater agent is used as antigen.     

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.     

Heartwater in Ghana: Implications for control of ticks

Tropical Animal Health and Production - Heartwater, an often fatal rickettsial disease of domestic ruminants transmitted byAmblyomma variegatum ticks, ranks with theA. variegatum-associated skin...     

Cowdria ruminantium infection in the mouse: a review

The current knowledge of the pathogenicity, clinical signs and mortality of artificial infections by syringe inoculation of Cowdria ruminantium in laboratory and wild strains of mice is reviewed. It is concluded that a wide spectrum of pathogenicity for mice exists in stocks of the organism.     

Reattachment of tooth fragment: an in vitro study

Canine tooth fracture is common in dogs. Application of an esthetic and durable restoration may be challenging in veterinary dental practice. This study used traditional human dental laboratory methods to evaluate fracture strength of intact dog canine teeth and fractured teeth that had been restored by reattachment of the tooth fragment. The results showed that the teeth restored by reattachment of the tooth fragment supported a test load equal to 45.4 % of the load necessary to fracture intact canine teeth.     

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.     

Core decompression of the equine navicular bone: an in vitro biomechanical study

Objective: To determine the effect of core decompression surgery and bone mineral density (BMD) on the mechanical properties of equine navicular bones. Study Design: Experimental, in vitro study. Sample Population: Fore limb navicular bones (n=36 pairs) from sound 2–5‐year‐old horses with no radiographic abnormalities of the distal aspect of the forelimbs. Methods: Navicular BMD was measured using dual energy X‐ray absorptiometry. One randomly assigned navicular bone from each pair served as control. The contralateral test specimen was allocated to 1 of 6 treatment groups defined by drill bit size (3.2 versus 2.5 mm diameter) and by the number of drill channels (1, 2, or 3) created in the proximal border of the bone. Bones were then tested until failure in 3‐point bending. Data were statistically analyzed using ANOVA and regression analysis. Results: There were significant (P<.001) positive correlations between BMD and biomechanical data. A significant (P<.001) reduction in breaking strength was noted between intact and drilled bone pairs; however, the diameter and number of decompression channels did not significantly (P>.05) influence the extent of the reduction in mechanical strength. Conclusion: In vitro core decompression significantly decreases the breaking strength of the equine navicular bone.     

Gender differences in the rat corpus callosum: An ultrastructure study

Sexual dimorphism exists at all levels of the nervous system, from genetic, anatomical and system levels. The sexual dimorphism in the axonal content of the corpus callosum (CC) has always been controversial; hence, the aim of this study was to analyse the differences in total, myelinated and unmyelinated axons density of various regions of the CC between male and female rats. To assess that, six pairs of adult male and female rats were perfused and the CC was removed and sectioned. Four sections from different subregions of the corpus callosum that represent the genu, anterior body, posterior body, and splenium, were stained, and electron microscopic images were captured using stereological guidelines. Later, the axons density for each subregion was calculated and compared between males and females. The findings of the present study indicated region‐specific differences in the myelinated, unmyelinated or the ratio of myelinated/total axons in the CC between male and female rats.     

Cranial cruciate stability in the rottweiler and racing greyhound: an in vitro study

An in vitro biomechanical study of cadaver stifles from rottweilers and racing greyhounds was undertaken to evaluate the contribution of the cranial cruciate ligament to stifle joint stability. This was performed at differing stifle joint angles, first with the joint capsules and ligaments intact and then with all structures removed except for the cranial cruciate ligament. Craniocaudal laxity increased in both breeds as stifle flexion increased. The rottweiler stifle showed greater craniocaudal joint laxity than the racing greyhound at all joint angles between 150 degrees and 110 degrees, but the actual increases in joint laxity between these joint angles were similar for both breeds. Tibial rotation during craniocaudal loading of the stifle increased craniocaudal laxity in both breeds during joint flexion. The relative contribution of the cranial cruciate ligament to cranial stability of the stifle joint increased as the joint flexed and was similar in both breeds.     

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.     

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.     

Time-dependent low-field MRI characteristics of canine blood: an in vitro study

This study was conducted to assess time-sensitive magnetic resonance (MR) changes in canine blood using low-field MR. Arterial and venous blood samples were collected from eight healthy beagle dogs. Samples were placed in 5-mL tubes and imaged within 3 hours of collection at 1 day intervals from day 1 to day 30. The following sequences were used: T1-weighted (T1W), T2-weighted (T2W), fluid-attenuated inversion recovery (FLAIR), short tau inversion recovery (STIR), and T2-star gradient-echo (T2^*-GRE). Visual comparison of the images revealed that four relatively homogenous blood clots and twelve heterogeneous blood clots developed. The margination of the clot and plasma changed significantly on day 2 and day 13. On day 2, heterogeneous blood clots were differentiated into 2 to 3 signal layers in the T2W, T1W, and especially the STIR images. Hypointense signal layers were also detected in the blood clots in STIR images, which have T2 hypo, FLAIR hypo, and T1 hyper intense signals. In all images, these signal layers remained relatively unchanged until day 13. Overall, the results suggest that hematomas are complex on low-field MRI. Accordingly, it may not be feasible to accurately characterize hemorrhages and predict clot age based on low-field MRI.