Percutaneous infection of nymphal Dermacentor andersoni with Anaplasma marginale

Newly replete nymphal Dermacentor andersoni (principals) were percutaneously exposed to Anaplasma marginale by injection of either intact or lysed infected bovine erythrocytes. Control nymphs were fed on calves with anaplasmosis. The subsequently molted adults were examined for infection by light microscopy, and companion ticks were tested for infectivity by allowing them to feed on susceptible calves. When they fed as adults, both control ticks and percutaneously inoculated principals transmitted A marginale to susceptible calves. Prepatent periods in calves varied according to the method by which nymphs were infected. Colonies of A marginale were found in all ticks that acquired infection by feeding, but colonies were not observed in any ticks exposed percutaneously. The possible developmental cycle of A marginale in artificially infected ticks is discussed.     

Longevity of colonies of Anaplasma marginale in midgut epithelial cells of Dermacentor andersoni

Colonies of Anaplasma marginale in midgut epithelial cells of experimentally infected Dermacentor andersoni were studied in adult ticks 1, 3, and 6 months old. Longevity of the parasite in ticks was assessed by evaluating its infectivity for splenectomized calves; calves were exposed by feeding ticks and by inoculation of tick gut homogenates. Longevity was also evaluated by determining size, type, and density of colonies in male and female ticks. The effect of incubation (2.5 days at 37 C) on colony density was also examined for ticks at each age period. All methods used to assess longevity of A marginale in ticks (tick transmission, calf inoculation, and histologic studies) indicated a decrease of the numbers of organisms in 6-month-old ticks. Furthermore, when tick gut homogenates from 6-month-old nonincubated ticks were not infectious for susceptible calves, incubation of ticks before dissection restored infectivity of homogenates. Colonies of A marginale were detected in gut tissues of 6-month-old ticks that were not infective; therefore, infectivity of ticks could not be confirmed merely by presence of A marginale colonies.     

Intermediate site of development of Anaplasma marginale in feeding adult Dermacentor andersoni ticks that were infected as nymphs

The development of Anaplasma marginale in midgut epithelial cells was studied in feeding, transmitting adult Dermacentor andersoni ticks. Laboratory-reared ticks experimentally infected as nymphs were allowed to feed from 1 to 9 days on susceptible calves. Gut tissues from ticks were collected on each day they fed (total, 9 days) and were processed for light and transmission electron microscopy. Colonies of A marginale were abundant during the first 6 days of feeding, after which numbers decreased. Colonies were adherent to the basement membrane of gut cells early during feeding, with resultant flattening of the colonies. Colonies also were seen in muscle cells on the hemocoel side of the basement membrane. Morphologic features of A marginale within muscle cells varied and were similar to those observed in gut cells. In addition, however, a large reticulated form in the colonies was observed in muscle cells and appeared to give rise to small particles by budding. Development of A marginale in muscle cells appears to represent an intermediate site of development between those in gut and in salivary glands.     

INFECTIVITY FOR CATTLE OF ANAPLASMA MARGINALE EXTRACTED FROM BOOPHILUS MICROPLUS TICKS EXPOSED TO CERTAIN TEMPERATURES

SUMMARY: Boophilus microplus ticks collected from calves with patent Anaplasma marginale infections were incubated at either 4 to 5°C, 14°C, 22°C, 27°C or 37°C for up to 14 days. Extracts prepared either from larvae, nymphs, immature females, adult males or mixtures of both sexes were infective for 14 of the 16 splenectomised calves inoculated. Extracts either from nymphs or from adult ticks deriving from nymphs moulted in vitro were infective for 11 of 12 nonsplenectomised calves. Possible application of the findings to producing a vaccine strain of A. marginale is discussed.     

Transstadial and attempted transovarial transmission of Anaplasma marginale by Dermacentor variabilis

Transstadial and transovarial transmission of Anaplasma marginale by Dermacentor variabilis were attempted with with ticks exposed to the organism once by feeding as larvae or nymphs, and twice by feeding as larvae and nymphs. Typical colonies of A marginale were in gut tissues of adults that were infected as larvae, larvae and nymphs, and as nymphs; repeated exposure of ticks did not appear to result in an increase in the number of colonies in the gut of subsequently molted adults nor did it affect severity of the clinical disease that developed in cattle they fed on. In contrast, colonies of A marginale were not found in the midgut epithelium of unfed nymphs exposed as larvae, even though companion nymphs transmitted the parasite, causing severe clinical anaplasmosis in susceptible calves. The organism was not transmitted transovarially by F1 larvae or nymphs from the groups exposed as parent larvae, nymphs, larvae and nymphs, and as adults. Some of the calves fed on by F1 progeny had a few erythrocytic marginale bodies that looked suspiciously like A marginale, as well as postchallenge exposure prepatent periods that were longer than other calves in the transovarial transmission study. Sera from these calves were tested for antibody to A marginale, using a highly sensitive immunoblot technique. Antibodies were not detected in any of the sera.     

Development of Anaplasma marginale in male Dermacentor andersoni transferred from parasitemic to susceptible cattle.

The development and transmission of Anaplasma marginale was studied in Dermacentor andersoni males. Laboratory-reared male D andersoni were allowed to feed for 7 days on a calf with ascending A marginale parasitemia. The ticks were then held in a humidity chamber for 7 days before being placed on 2 susceptible calves. Anaplasmosis developed in the calves after incubation periods of 24 and 26 days. Gut and salivary glands were collected from ticks on each day of the 23-day experiment and examined with light and electron microscopy. Colonies of A marginale were first observed in midgut epithelial cells on the sixth day of feeding on infected calves, with the highest density of colonies found in gut cells while ticks were between feeding periods. The first colonies contained 1 large dense organism that subsequently gave rise to many reticulated organisms. Initially, these smaller organisms were electron-lucent and then became electron-dense. On the fifth day after ticks were transferred to susceptible calves for feeding, A marginale colonies were found in muscle cells on the hemocoel side of the gut basement membrane. A final site for development of A marginale was the salivary glands. Colonies were first seen in acinar cells on the first day that ticks fed on susceptible calves, with the highest percentage of infected host cells observed on days 7 to 9 of that feeding. Organisms within these colonies were initially electron-lucent, but became electron-dense.     

Infectivity and antigenicity of Anaplasma marginale from tick cell culture

The infectivity and immunogenicity of Anaplasma marginale grown in a tick cell culture from embryonic Dermacentor variabilis ticks were assessed in splenectomized and intact calves, respectively. Culture 1 consisted of the cell line inoculated with midguts of adult ticks infected with the Mississippi isolate of A marginale and dissected 5 to 10 days after repletion and detachment from an experimentally infected calf. Cultures 2 and 3 consisted of the cell line inoculated with midguts of ticks infected with the Virginia isolate of the organism. Inoculum for culture 2 was derived from nymphal ticks dissected 5 to 10 days after repletion and detachment from the infected calf; inoculum for culture 3 was midguts from adult ticks that were fed as nymphs, allowed to molt in the laboratory and dissected 21 to 24 days after molting. In trial 1, cultures 1, 2, and 3 were maintained at pH 6.9 and incubated at 28 C; in trial 2, cultures 1 and 3 were maintained at pH 7.4 and incubated at either 28 C or 37 C. Cultures 1, 2, and 3 failed to induce infection when injected IV and SC into 6 calves in 2 separate trials. Pre-challenge sera from these calves reacted with 2 purified Anaplasma antigens in the ELISA, but failed to react in the complement-fixation test. Results of a trial to use cultures 1 and 3 in combination with an oil-in-water adjuvant to immunize intact calves against A marginale were inconclusive. However, pre-challenge sera from immunized calves reacted with the 2 purified Anaplasma initial body antigens in the ELISA but failed to react in the complement-fixation text.(ABSTRACT TRUNCATED AT 250 WORDS)     

Infectivity of three Anaplasma marginale isolates for Dermacentor andersoni

Three isolates of Anaplasma marginale--Virginia (VAM), Illinois (IAM), and Florida (FAM)--were compared for infectivity for Dermacentor andersoni. The isolates were selected, in part, because of a tail-like appendage that has been demonstrated in the VAM and IAM, but not in the FAM. Ticks were exposed to the isolates as nymphs either naturally by feeding on a calf with anaplasmosis or artificially by percutaneous inoculation with infected bovine erythrocytes. They were examined for infectivity after molting to the adult stage by determining their capability to transmit the disease to susceptible calves and by demonstrating colonies in tick gut sections. Only those ticks exposed to the VAM proved to be infected with A marginale; ticks naturally exposed and those artificially infected with this isolate transmitted the disease to susceptible calves. Colonies of A marginale were observed only in gut tissues of ticks naturally infected with VAM. The IAM (appendage present) and FAM (appendage absent) could not be found in ticks exposed by either method, indicating that factors other than the presence of inclusion appendages may be involved in infection of ticks by A marginale.     

Detection of colonies of Anaplasma marginale in salivary glands of three Dermacentor spp infected as nymphs or adults

Salivary glands from males of 3 Dermacentor species (D andersoni, D variabilis and D occidentalis) that were infected with either the Virginia or Idaho isolate of Anaplasma marginale as nymphs or adults were examined for colonies of A marginale by use of light and electron microscopy. Prior to dissection of salivary glands, exposed ticks were held at 25 C for 15 to 18 days, followed by a 3-day incubation at 37 C. Ticks of 2 species transmitted A marginale to calves; the third tick species was confirmed infected by demonstration of typical colonies in tick gut cells, but transmission was not attempted; Colonies of A marginale were seen with light microscopy in salivary glands of all 3 species of ticks; they were located in acinar cells that contained simple granules. Colonies varied morphologically from small, compact ones to larger structures that contained distinct organisms and often were adjacent to the host cell nucleus. Electron microscopy confirmed that the colonies were rickettsial organisms. Morphologic features of A marginale varied and included reticulated forms, forms with electron-dense centers, and small particles; these various forms were similar to those described previously in midgut epithelial cells of ticks. We believe that the organism seen within tick salivary glands may replicate in the glands before its transmission to the vertebrate host.     

Lack of infectivity of a Brazilian Anaplasma marginale isolate for Boophilus microplus ticks

Previous studies have shown that one Brazilian Anaplasma marginale isolate presents an inclusion appendage (tail), while other isolates do not present such inclusion. Studies on tick transmission have been carried out with tailless isolates but little is known about transmission of tailed isolates by Boophilus microplus. Two splenectomized calves were experimentally inoculated with the tailed A. marginale isolate. During ascending rickettsemia, B. microplus larvae, free from hemoparasites, were fed on the calves and the resulting nymphs, adult males and engorged females were examined by optic and electronic microscopy. No A. marginale colonies were observed in the gut cells of engorged females and the larvae originated from them did not transmit A. marginale to susceptible calves. In addition, no colonies of A. marginale were seen in the gut cells or in salivary glands of adult males and nymphs. These results suggest that B. microplus is not the biological vector for this tailed isolate.     

Isolate of Anaplasma marginale not transmitted by ticks

The tick-borne transmissibility of 2 isolates of Anaplasma marginale was compared. Dermacentor variabilis were exposed to A marginale as nymphs by feeding on 1 of 4 splenectomized calves during periods of ascending parasitemia (maximum 49% to 81% parasitized erythrocytes) induced by injection of a stabilate. Tick-borne transmission was attempted, using 26 to 224 adult ticks within 30 to 220 days after molting. Adult D variabilis did not transmit an Illinois isolate of A marginale in 7 tick-borne transmission experiments (P = 0.0047), including 2 experiments in which calves were inoculated IV with homogenates of adult ticks. In contrast, a Virginia isolate of A marginale was readily transmitted by the same tick colony. Thus, previously reported morphologic and immunologic differences among A marginale isolates may extend to tick-borne transmissibility. The Virginia and Illinois A marginale isolates had an inclusion appendage that was not a marker for tick transmissibility.     

THE AUSTRALIAN BROWN DOG TICK RHIPICEPHALUS SANGUINEUS AS AN EXPERIMENTAL PARASITE OF CATTLE AND VECTOR OF ANAPLASMA MARGINALE

SUMMARY Experiments were done to explore the possible relationship between cattle, Australian dog ticks (Rhipicephalus sanguineus) and Anaplasma marginale. Calves' ears were exposed to larval, nymphal and adult ticks on 8, 9 and 7 occasions, respectively. The immature instars fed readily, but the adults attached very poorly to calves. Transtadial transmission of A. marginale was achieved on 6 occasions from 9 attempts: ticks infected as larvae or nymphs were able to transmit at the subsequent stage or stages. Transovarial transmission was not achieved. Six calves supported more than one infestation of ticks. Attached ticks caused the calves no apparent discomfort and calves developed no noticeable skin reactions. An abattoir survey of 200 hides detected no R. sanguineus.     

Persistence of colonies of Anaplasma marginale in overwintering Dermacentor variabilis

Dermacentor variabilis were infected as nymphs with Anaplasma marginale by allowing the ticks to feed on a single infected donor calf. Two weeks after molting to the adult stage, the ticks were allotted into 1 of 3 groups and were allowed to overwinter at room temperature (25 C) in the laboratory (group 1), cold storage (4.5 C) in the laboratory (group 2), or outdoors in leaf litter (group 3). Persistence of A marginale was assessed by determining density of colonies (number of colonies/0.1 mm2 of gut tissue examined) in tick gut specimens at 3, 5, 7, 9, and 12 months after molting to the adult stage. Colonies of A marginale were found in all groups at every density evaluation period. Highest colony densities were observed uniformly in specimens collected at month 7 (May); densities decreased at month 9 and were lowest at month 12. Statistical analysis indicated that ticks subjected to cold storage and to outdoor conditions had similar colony densities of A marginale; the density curve in these 2 groups indicated significant quadratic effects over time, with peak densities in May. Mean colony density in ticks kept at room temperature fit a different quadratic equation. The morphologic data indicated that A marginale overwinters in Dermacentor variabilis, and that increasing numbers of organisms are found from January to May.     

Demonstration of Anaplasma marginale in hemolymph of Dermacentor andersoni by animal inoculation and by fluorescent-antibody technique

Hemolymph was collected from adult Dermacentor andersoni Stiles that had been infected with Anaplasma marginale Theiler as nymphs. Before hemolymph was collected, the adult ticks were either incubated and unfed at 37 C for 2.5 days or fed for 6 days on sheep. Hemolymph collected from groups of 100 ticks was inoculated into susceptible splenectomized calves. Smears of hemolymph from the same groups of ticks were prepared for examination by fluorescent antibody technique. Hemolymph from incubated ticks caused anaplasmosis in 2 of 4 trials, and hemolymph from feeding ticks caused anaplasmosis in 4 of 4 trials. Moderately fluorescing bodies were demonstrated in some hemocytes from incubated ticks, whereas hemocytes from feeding ticks contained numerous clusters of brightly fluorescing bodies. Fluorescing bodies were not observed in hemocytes from control ticks.     

Transmission of Anaplasma marginale by adult Dermacentor andersoni during feeding on calves

Laboratory-reared Dermacentor andersoni ticks experimentally infected as nymphs with Anaplasma marginale were allowed to feed as adults from 1 to 9 days on susceptible, splenectomized calves to determine when, during feeding, the hematozoan was transmitted from ticks to cattle. In experiment 1, ticks were allowed to feed on calves for 1, 2, 3, 4, 5, or 6 days and anaplasmosis did not result. The same calves were used for experiment 2, and ticks were allowed to feed for 1, 3, 6, 7, 8, or 9 days and anaplasmosis occurred in all calves on which ticks fed for greater than or equal to 6 days. In 2 trials in experiment 3, ticks were allowed to feed on calves for 1 to 9 days. Anaplasmosis developed only in calves on which ticks fed for 7, 8, or 9 days. The prepatent periods shortened with longer tick feeding, and linear regression analysis of combined prepatent periods of both trials of experiment 3 indicated a significant (P = 0.05) slope with an estimated daily decrease of 7.75 days from day 7 to 9 of feeding. There was no apparent correlation between length of tick feeding and severity of clinical signs in those calves that developed anaplasmosis. Seemingly, A marginale can be transmitted to cattle by adult D andersoni ticks no earlier than the 6th or 7th day of feeding.     

Preliminary studies of the development of Anaplasma marginale in salivary glands of adult, feeding Dermacentor andersoni ticks

On each day of feeding on susceptible calves, salivary glands obtained from groups of adult ticks that transmitted Anaplasma marginale were examined for A marginale colonies by use of light microscopy and transmission electron microscopy. On day 8 of feeding, salivary glands were examined, using fluorescein-labeled antibody and methyl green-pyronine stain. Use of fluorescein-labeled antibody consistently revealed small numbers of fluorescent foci in salivary gland acinar cells obtained from ticks that had fed for 8 days. Colonies of A marginale were seen by transmission electron microscopy only in salivary gland acini of male ticks; these colonies could not be identified, using light microscopy, in companion 1-micron plastic sections stained with Mallory stain. Methyl green-pyronine stain, used commonly to detect theilerial parasites in tick salivary glands, did not differentiate A marginale from cytoplasmic inclusions normally found in salivary gland acinar cells.     

Demonstration of colonies of Anaplasma marginale in the midgut of Rhipicephalus simus

Rhipicephalus simus nymphs were allowed to feed on a cow experimentally infected with the BW-strain of Anaplasma marginale from Republic of South Africa, and they were studied as adults. Colonies were demonstrated by light microscopy in midgut epithelial cells of adult ticks that were unfed (as adults), incubated, or prefed for 72 hours on a cow. The colonies occurred in 5 different morphologic types (1 to 5) that were similar to those described previously for a Virginia isolate of A marginale in Dermacentor andersoni. The colony density (number of colonies/0.001 mm2 midgut tissue examined) ranged from 0 to 2.0 and was highest in unfed ticks that were not incubated (mean 0.566). Colonies observed by light microscopy were sectioned for study with the electron microscope. The colonies contained both electron-dense forms and reticulated forms. The organisms in type 2 and 3 colonies appeared to be attached to one another, and those in type 4 and 5 colonies occurred separately. Small particles were seen within the limiting membrane of some organisms. A few colonies contained a dense matrix and were surrounded by many small electron-dense particles.     

Demonstration of colonies of Cowdria ruminantium in midgut epithelial cells of Amblyomma variegatum

The development of colonies of Cowdria ruminantium was studied in midgut epithelial cells of adult Amblyomma variegatum that had become infected by feeding as nymphs on cattle with experimentally induced heart-water disease. Colonies were not observed in gut tissues obtained from nymphs during the feeding period, but were present in midgut epithelial cells of ticks obtained at 15 days after they were replete through molting to the adult stage. Colonies were small (1 to 10 micron) initially, but as tick development progressed, their diameter increased to as much as 60 micron. With electron microscopy, colonies were observed to be membrane bound and contained pleomorphic organisms that were reticulated. The organisms seemed to be dividing by binary fission. Many colonies contained a large, electron-dense inclusion that was morphologically similar to hemoglobin deposits found in the cytoplasm of midgut epithelial cells of recently fed ticks. Cowdria ruminantium was often observed adhered to these inclusions.     

Anaplasma marginale in tick cell culture

Anaplasma marginale was propagated in a tick cell line derived from Dermacentor variabilis embryos. The rickettsial organism was identified and monitored in culture by transmission electron microscopy and the indirect immunofluorescence technique, using specific monoclonal antibodies. Inoculation of the embryonic tick cell line with midguts of infected adult ticks (culture 1), nymphal ticks (culture 2) and adult ticks that were infected as nymphs and dissected as adults (culture 3) resulted in 3 continuous cultures of A marginale. Culture 1 had been maintained through 22 passages over a 11-month period; cultures 2 and 3 had been maintained for 18 passages over a 9-month period. Growth of A marginale in the cell line began in the area of the nuclear membrane at approximately 4 days after inoculation or transfer. Thereafter, the organisms were observed in inclusions scattered throughout the cytoplasm of the host cells. Maximal growth of the organism occurred at 7 to 14 days, after which numbers of inclusions rapidly decreased to minimal or undetectable levels. The organism began new cycles of growth with each 1:5 to 1:10 split and transfer of the host cells. Electron microscopy of recently infected cells revealed a morphology of the organism that closely resembled that observed in marginal bodies of infected erythrocytes. After several passages, A marginale organisms had a varied morphology and resembled the organism described in midgut cells of naturally infected ticks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental transmission of bovine anaplasmosis (caused by Anaplasma marginale) by means of Dermacentor variabilis and D. andersoni (Ixodidae) collected in western Canada

Canadian cattle are free of bovine anaplasmosis, with the exception of 4 isolated incursions since 1968, which were eradicated. It is not known why the disease has not become established in regions of Canada adjacent to the United States where it is endemic. To assess the vector competence of wild-caught ticks in cattle-rearing regions, Dermacentor variabilis and D. andersoni were collected in western Canada and fed on calves experimentally infected with Anaplasma marginale (St. Maries strain). The 2 tick species were equally competent in transmitting A. marginale to splenectomized calves, all 15 tick-exposed calves becoming infected. The prepatent periods in 13 calves ranged from 18 to 26 d and did not vary in relation to the numbers of ticks fed or the duration of transmission feedings. The unusually long prepatent periods in 2 calves (45 and 55 d) were probably due to concomitant Eperythrozoon infection. This study clearly demonstrated that tick species present in western Canada are competent vectors of bovine anaplasmosis. Potential barriers, including climate, must be considered in developing strategies to prevent A. marginale from becoming established in anaplasmosis-free regions.