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.     

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.     

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.     

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.     

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.     

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.     

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.     

Experimental transmission of field Anaplasma marginale and the A. centrale vaccine strain by Hyalomma excavatum, Rhipicephalus sanguineus and Rhipicephalus (Boophilus) annulatus ticks

The cattle rickettsia Anaplasma marginale is distributed worldwide and is transmitted by about 20 tick species, but only Rhipicephalus simus, a strictly African tick species, has been shown to transmit the vaccine strain of A. centrale. The aim of the present study was to examine transmission of field strains of A. marginale and of the vaccine strain of A. centrale by three tick species -Hyalomma excavatum, Rhipicephalus sanguineus and Rhipicephalus (Boophilus) annulatus - to susceptible calves. Two genetically distinct Israeli field strains of A. marginale, tailed and non-tailed (AmIsT and AmIsNT, respectively), were efficiently transmitted by R. sanguineus, whereas H. excavatum transmitted only the tailed isolate, and R. (Boophilus) annulatus did not transmit A. marginale. None of the three tick species transmitted A. centrale. By means of msp1a primers in PCR assays, amplicons of similar sizes were obtained from either A. marginale-infected calves that were used for acquisition feeding, from R. sanguineus fed on the infected calves, or from calves to which anaplasmosis had been successfully transmitted by these ticks. Although an A. centrale-specific fragment was amplified from salivary glands of R. sanguineus, no transmission to susceptible cattle occurred during 3 months of observation, and anaplasmosis was not induced in splenectomized calves that were subinoculated with blood from calves on which R. sanguineus had fed.     

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.     

Applications of a cell culture system for studying the interaction of Anaplasma marginale with tick cells

A cell culture system for the tick-borne rickettsia Anaplasma marginale offers new opportunities for research on this economically important pathogen of cattle. A. marginale multiplies in membrane-bound inclusions in host cells. Whereas erythrocytes appear to be the only site of infection in cattle, A. marginale undergoes a complex developmental cycle in ticks and transmission occurs via the salivary glands during feeding. We recently developed a cell culture system for A. marginale using a cell line derived from embryos of Ixodes scapularis. Here we review the use of this cell culture system for studying the interaction of A. marginale with tick cells. Several assays were developed using the A. marginale/tick cell system. An adhesion assay was developed for the identification of proteins required by A. marginale for adhesion to tick cells. The effect of antibodies against selected major surface proteins in inhibiting A. marginale infection was tested in an assay that allowed further confirmation of the role of surface proteins in the infection of tick cells. A drug screening assay for A. marginale was developed and provides a method of initial drug selection without the use of cattle. The culture system was used to test for enhancing effects of tick saliva and saliva components on A. marginale infection. The tick cell culture system has proved to be a good model for studying A. marginale-tick interactions. Information gained from these studies may be applicable to other closely related tick-borne pathogens that have been propagated in the same tick cell line.     

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.     

Transmission of Anaplasma marginale Theiler by males of Dermacentor andersoni Stiles fed on an Idaho field-infected, chronic carrier cow

The role of ticks and carrier cattle in epizootics of bovine anaplasmosis was further clarified by demonstrating unequivocally, for the first time, that male ticks fed on a chronic carrier cow naturally infected with Anaplasma marginale can transmit this parasite intrastadially and biologically when subsequently fed on susceptible cattle. These data indicate that field epizootics of acute anaplasmosis may be initiated by males of tick vector species that feed on carrier cattle and subsequently transfer to susceptible cattle.     

Hybridization of DNA probes to A. marginale isolates from different sources and detection in Dermacentor andersoni ticks

DNA from the Washington, South-Idaho, Virginia and Florida isolates of Anaplasma marginale was hybridized to probes specific for Anaplasma centrale and A. marginale. The A. centrale probes AC-2 and AC-4 hybridized to identical bands on all of these isolates. The hybridization patterns suggests that the Virginia, Florida and the South African isolates are similar. A number of bands were obtained with the Washington isolate which differed from those obtained with the other isolates. Probe AC-2 could be developed to identify relatedness among Anaplasma isolates. Probe AC-2 detected A. marginale DNA in midgut material from infected Dermacentor andersoni ticks. No hybridization was obtained with DNA from salivary gland tissues from these infected ticks.     

Epidemiologic aspects of bovine anaplasmosis in semiarid range conditions of south central Idaho

The prevalance of Anaplasma marginale-infected cows, as determined by use of the modified rapid card agglutination (MRCA) test, was measured during a 4-year period (1980-1983). The prevalence of A marginale-infected cows, defined as positive reactors on the MRCA test, remained constant (31%-37%). The apparent incidence of A marginale transmission to susceptible cows was approximately 7% from 1980 to 1981, 8% from 1981 to 1982, and no transmission from 1982 to 1983. The occasional MRCA-positive cow became negative on the MRCA test, and 1 cow was determined to be free of A marginale infection by subinoculation of 100 ml of the cow's blood into a susceptible, splenectomized calf. Dermacentor andersoni, a known vector of A marginale, was often found on the cattle and in their environment. However, A marginale was not transmitted to susceptible, splenectomized calves, using collected ticks. Of 56 calves born to MRCA-positive cows, 82% were MRCA-positive within the first 3 months of life. These calves converted to MRCA-negative status and were determined to be free of A marginale infection by subinoculation of their blood into susceptible, splenectomized calves, indicating the passive transfer of colostral antibodies.     

The maturation of Theileria annulata in Hyalomma anatolicum anatolicum stimulated by incubation or feeding to produce sporozoites

Adult Hyalomma anatolicum anatolicum ticks infected with Theileria annulata (Hissar strain) were incubated at 36 degrees C or fed on rabbits. Tick salivary glands were stained whole with methyl green pyronin or ground up and deposited on microscope slides and stained with Giemsa's solution. Separate batches of ticks from both treatments were ground up, centrifuged and filtered to produce sporozoite suspensions. The suspensions were examined as deposits on microscope slides stained with Giemsa's solution. The Theileria in the salivary glands of the fed ticks matured more completely and rapidly than in the incubated ticks. The peak numbers of sporozoites from the fed ticks was greater by at least tenfold than the peak from the incubated ticks. This peak was on the third day of feeding or on the fourth day of incubation. It was confirmed that fed ticks will be more suitable for sporozoite production for infection of cattle and production of stabilates.     

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.     

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.     

Natural transmission of heartwater

Heartwater has been transmitted experimentally by 12 Amblyomma species. Their importance depends on the extent of their distribution, adaptation to domestic stock and their efficacy as vectors. Except for one report of transovarial transmission, transmission is transstadial. Ticks may obtain the infection while feeding on reacting animals, subclinically infected hosts or perhaps on immune animals after reinfection. There is a marked increase in the infectivity of infected ticks during feeding but this decreases before and during moulting. The demonstration of Cowdria ruminantium in salivary glands of ticks suggests that transmission takes place via the saliva and that regurgitation from the gut may not be as important as previously thought. Transmission takes place on the 2nd day from the time infected nymphae were placed on the animals and on the 4th-day in the case of adult ticks.     

Infectivity rate and transmission potential of Hyalomma anatolicum anatolicum ticks for Babesia equi infection

The infectivity rate of Babesia equi in the salivary glands of Hyalomma anatolicum anatolicum was assessed. The hungry nymphs were fed on a donkey experimentally infected with B. equi. The engorged dropped-off nymphs were collected at different levels of parasitaemia and kept in BOD incubator. After ecdysis, the hungry adults were prefed on rabbits for different time intervals, thereafter the salivary glands were dissected out and acini were examined after methyl green pyronin (MGP) staining. A total of 134 male and 139 female ticks were dissected out. Average infected acini per tick were found to be significantly higher (p<0.05) in male as compared to the female ticks. Further, maximum infected acini in both male and female ticks were found at 24h of prefeeding on rabbits and overall infected acini per tick increased with rise in parasitaemia. The release of infected ticks on susceptible donkeys resulted in development of clinical babesiosis.     

Development and infectivity of Anaplasma marginale in Dermacentor andersoni nymphs

The development of Anaplasma marginale was studied in Dermacentor andersoni nymphs after they had fed on a calf with ascending Anaplasma infection. Gut tissues were collected on day 4 of tick feeding, from newly replete (fed) nymphs and on postfeeding days (PFD) 5, 10, 15, 20, and were processed for light and electron microscopy to determine density of A marginale colonies. Homogenates of gut tissues were prepared from nymphs collected on the same days and inoculated into susceptible, splenectomized calves to test for infectivity. Anaplasma colonies were detected in gut cells on PFD 5, 10, 15, and 20. Although colony density appeared to be higher on PFD 10 and 15, differences were not significant. Nymphal type-1 colonies were detected in highest numbers on PFD 5 and 10, transitional colonies were seen in highest numbers at PFD 10 and 15, and nymphal type-2 colonies were observed only on PFD 20. Gut homogenates that were collected from ticks at 4 days of feeding, when newly replete, and on PFD 20 caused anaplasmosis when injected into susceptible calves, but homogenates made from ticks collected on PFD 5, 10, and 15 were not infective. The data indicate that of the colony types of A marginale that develop in replete nymphs, nymphal type-1 and transitional colonies may contain organisms that are not infective for cattle.