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.     

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.     

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.     

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 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.     

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.     

Attempted transmission to cattle of Anaplasma marginale from overwintered Dermacentor andersoni ticks.

Since the 1983 summer outbreak of anaplasmosis in southern Saskatchewan, the role of the tick, Dermacentor andersoni as an overwintering reservoir for Anaplasma marginale has been questioned. The purpose of this study was to determine if spring-collected ticks carried virulent A. marginale. Sixteen splenectomized calves were assigned randomly to two groups of 14 principals and two controls. Adult D. andersoni, collected in April from areas having high transmission rates of A. marginale, were confined to the ears of the principals by special bags and allowed to feed for eight days. The two control calves were subsequently challenged intravenously with blood from a calf infected with the Virginia strain of A. marginale. Principals and controls were monitored for 60 and 50 days postexposure respectively for signs of infection by clinical, hematological and serological procedures. None of the principals developed anaplasmosis but both control calves developed signs of disease.     

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.     

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.     

Immune responses of calves antigenically stimulated and challenge exposed with Anaplasma marginale during tick infestation or treatment with dexamethasone

Similar anamnestic antibody responses to a 2nd injection of a Anaplasma marginale vaccinal antigen were observed in calves infested with the tick Dermacentor albipictus and in tick-free calves. When challenge exposure of these calves to virulent A marginale was done, infestation with the tick Boophilus microplus increased anemia (P less than 0.01), but did not suppress antibody production to A marginale or increase parasitemia. None of the vaccinated calves, regardless of infestation, experienced clinical anaplasmosis. Mitogenic responses of lymphocytes from infested animals were unaltered by either infestation. Tick infestations did not cause immune suppression in the calves, whereas use of dexamethasone resulted in a significantly lower antibody response (P less than 0.05) after a 2nd injection of vaccinal antigen. After challenge with virulent A marginale, dexamethasone-treated calves showed more pronounced parasitemia (P less than 0.01) and anemia (P less than 0.01) than did control calves. Anaplasmosis did not prevent the calves from developing resistance to reinfestation, which was accompanied by immediate, but not delayed, hypersensitivity reactions against homologous tick extracts. Dermacentor albipictus did not seem to share common antigenic determinants with B microplus, since extracts from the latter did not elicit immediate hypersensitivity responses in calves sensitive to D albipictus extracts. Calves were somewhat resistant to reinfestation as evidenced by reduced numbers of adult fed ticks, decreased weights of ticks after feeding, and smaller egg masses.     

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.     

Relative infectivity of Theileria annulata (Dchunkovsky and Luhs, 1904) stabilates derived from female and male Hyalomma ecavatum (Koch, 1844) ticks

Partially engorged female and male Hyalomma excavatum ticks infected with Theileria annulata were triturated separately in Eagle's minimum essential medium complemented with fetal calf serum. Glycerol was added to the supernatant obtained after centrifugation and the material was frozen at—79°C. Infectivity of the stabilate was titrated by inoculating susceptible calves with dilutions of the material.Stabilate prepared from female ticks was considerably more infective on a per tick basis than stabilate from males. All calves inoculated with an estimated equivalent of 0.1 or more female ticks, or 3.3 or more male ticks, became infected.Time to fever, prepatent period, and mortality did not appear to be dose-dependent in the range of tick equivalents employed.     

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.     

Vaccination of cattle with Anaplasma marginale derived from tick cell culture and bovine erythrocytes followed by challenge-exposure with infected ticks

Anaplasmosis, a hemolytic disease of cattle caused by the tick-borne pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) has been controlled using killed vaccines made with antigen harvested from infected bovine erythrocytes. We recently developed a cell culture system for propagation of A. marginale in a continuous tick cell line. In this study, we performed a cattle trial to compare the bovine response to vaccination with A. marginale harvested from tick cell culture or bovine erythrocytes. All immunized and control cattle were then challenge-exposed by allowing male Dermacentor variabilis infected with A. marginale to feed and transmit the pathogen. Nine yearling cattle (three per group) were used for this study and were immunized with cell culture-derived A. marginale, erythrocyte-derived A. marginale or received adjuvant only to serve as controls. Each vaccine dose contained approximately 2 x 10(10) A. marginale and three immunizations were administered at weeks 1, 4 and 6. At week 8, cattle were challenge-exposed by allowing 60 D. variabilis male that were infected with A. marginale as adults to feed on the cattle. Antibody responses of cattle against major surface proteins (MSP) 1a, 1b and 5, as determined by ELISAs, peaked 2 weeks after the last immunization. Cattle immunized with infected IDE8 cell-derived antigens had a preferential recognition for MSP1b while cattle immunized with erythrocyte-derived antigens had a preferential recognition for MSP1a. Protection efficacy was evaluated using the percent infected erythrocytes (PPE), the packed cell volume (PCV), and the prepatent period. A. marginale-immunized cattle showed lower PPE and higher PCV values when compared to control animals and did not display clinical anaplasmosis. The cell culture-derived A. marginale shows promise for use as antigen in development of a new killed vaccine for anaplasmosis.     

Molecular phylogeny and biogeography of North American isolates of Anaplasma marginale (Rickettsiaceae: Ehrlichieae)

Anaplasma marginale (A. marginale) is a tick-borne ehrlichial pathogen of cattle that causes the disease anaplasmosis. Six major surface proteins (MSPs) have been identified on A. marginale from cattle and ticks of which three, MSP1a, MSP4 and MSP5, are from single genes and do not vary within isolates. The other three, MSP1b, MSP2 and MSP3, are from multigene families and may vary antigenically in persistently infected cattle. Several geographic isolates have been identified in the United States which differ in morphology, protein sequence and antigenic properties. An identifying characteristic of A. marginale isolates is the molecular weight of MSP1a which varies in size among isolates due to different numbers of tandemly repeated 28-29 amino acid peptides. For these studies, genes coding for A. marginale MSP1a and MSP4, msp1alpha and msp4, respectively, from nine North American isolates were sequenced for phylogenetic analysis. The phylogenetic analysis strongly supports the existence of a south-eastern clade of A. marginale comprised of Virginia and Florida isolates. Analysis of 16S rDNA fragment sequences from the A. marginale tick vector, Dermacentor variabilis, from various areas of the United States was used to evaluate possible vector-parasite co-evolution. Our phylogenetic analysis supports identity between the most parsimonious tree from the A. marginale MSP gene data and the tree that reflected the western and eastern clades of D. variabilis. These phylogenetic analyses provide information that may be important to consider when developing control strategies for anaplasmosis in the United States.     

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)     

Targeting the tick/pathogen interface for developing new anaplasmosis vaccine strategies

Bovine anaplasmosis is a tick-borne hemolytic disease of cattle that occurs worldwide caused by the intraerythrocytic rickettsiae Anaplasma marginale. Control measures, including use of acaricides, administration of antibiotics and vaccines, have varied with geographic location. Our research is focused on the tick-pathogen interface for development of new vaccine strategies with the goal of reducing anaplasmosis, tick infestations and the vectorial capacity of ticks. Toward this approach, we have targeted (1) development of an A. marginale cell culture system to provide a non-bovine antigen source, (2) characterization of an A. marginale adhesion protein, and (3) identification of key tick protective antigens for reduction of tick infestations. A cell culture system for propagation of A. marginale was developed and provided a non-bovine source of A. marginale vaccine antigen. The A. marginale adhesion protein, MSP1a, was characterized and use of recombinant MSP1a in vaccine formulations reduced clinical anaplasmosis and infection levels in ticks that acquired infection on immunized cattle. Most recently, we identified a tick-protective antigen, subolesin, that reduced tick infestations, as well as the vectorial capacity of ticks for acquisition and transmission of A marginale. This integrated approach to vaccine development shows promise for developing new strategies for control of bovine anaplasmosis.     

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.     

Novel genotypes of Anaplasma bovis, "Candidatus Midichloria" sp. and Ignatzschineria sp. in the Rocky Mountain wood tick, Dermacentor andersoni

Bovine anaplasmosis, caused by Anaplasma marginale, is a vector-borne disease that is enzootic in many parts of the USA. Although Dermacentor andersoni, a major vector of A. marginale, occurs in Canada, the Canadian cattle herds are currently considered free of bovine anaplasmosis. There have been two outbreaks of the disease in the province of Saskatchewan, but these have been linked to the importation of infected animals. However, the distribution of bovine anaplasmosis may alter with range expansion of the vectors. The aim of the present study was to use molecular techniques to determine if Anaplasma were present in D. andersoni at a locality near its northeastern distributional limit in Saskatchewan. Nested PCR analyses of the bacterial 16S rRNA gene were conducted on the total genomic DNA of 105 individual ticks. Single strand conformation polymorphism analysis and DNA sequencing of the 11 PCR-positive amplicons revealed the presence of three species of bacteria, none of which have been previously reported in D. andersoni. Although no ticks were infected with A. marginale, a novel genotype of A. bovis was detected in eight individuals. This discovery represents the first report of A. bovis in Canada. The potential implications of this finding with respect to animal health and anaplasmosis surveillance in Canada are discussed. The other two bacterial species detected were genetically similar to "Candidatus Midichloria mitochondrii" and Ignatzschineria larvae, the latter of which has been associated with human disease in Europe. Further investigations are needed to determine the prevalence, reservoir hosts, and pathogenicity of the Canadian genotype of A. bovis.     

Productivity and health effects of anaplasmosis and babesiosis on Bos indicus cattle and their crosses, and the effects of differing intensity of tick control in Australia

Tick fever is an important disease of cattle where Rhipicephalus (Boophilus) microplus acts as a vector for the three causal organisms Babesia bovis, Babesia bigemina and Anaplasma marginale. Bos indicus cattle and their crosses are more resistant to the clinical effects of infection with B. bovis and B. bigemina than are Bos taurus cattle. Resistance is not complete, however, and herds of B. indicus-cross cattle are still at risk of babesiosis in environments where exposure to B. bovis is light in most years but occasionally high. The susceptibility of B. indicus cattle and their crosses to infection with A. marginale is similar to that of B. taurus cattle. In herds of B. indicus cattle and their crosses the infection rate of Babesia spp. and A. marginale is lowered because fewer ticks are likely to attach per day due to reduced numbers of ticks in the field (long-term effect on population, arising from high host resistance) and because a smaller proportion of ticks that do develop to feed on infected cattle will in turn be infected (due to lower parasitaemia). As a consequence, herds of B. indicus cattle are less likely than herds of B. taurus cattle to have high levels of population immunity to babesiosis or anaplasmosis. The effects of acaricide application on the probability of clinical disease due to anaplasmosis and babesiosis are unpredictable and dependent on the prevalence of infection in ticks and in cattle at the time of application. Attempting to manipulate population immunity through the toleration of specific threshold numbers of ticks with the aim of controlling tick fever is not reliable and the justification for acaricide application should be for the control of ticks rather than for tick fever. Vaccination of B. indicus cattle and their crosses is advisable in all areas where ticks exist, although vaccination against B. bigemina is probably not essential in pure B. indicus animals.