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.     

Serologic and clinical responses of premunized, vaccinated, and previously infected cattle to challenge exposure by two different Anaplasma marginale isolates

Two Anaplasma marginale isolates, one originating in Florida (FAM) and the other from Virginia (VAM), were compared immunologically by cross-challenge exposure of 14 Anaplasma carrier cattle, 8 previously infected cattle, and 6 splenectomized carrier calves. In addition, 28 cattle vaccinated with a commercially available adjuvant killed vaccine and 22 nonvaccinated cattle were challenge exposed with either FAM or VAM. A detectable clinical response was not produced by either FAM or VAM challenge exposure in carrier and previously infected cattle; however, evidence of A marginale growth as characterized by low percentages of parasitemia and increased serum complement-fixation titers was seen in carrier cattle given a heterologous challenge organism and in previously infected cattle inoculated with either homologous or heterologous organisms. Among splenectomized calves, there was virtually no cross protection to the heterologous challenge exposure, whereas a homologous challenge failed to elicit any detectable response. Vaccinated cattle were resistant to VAM exposure, but the clinical response to FAM exposure was severe with a 47% mortality. Most of these cattle displayed typical acute anaplasmosis that was only marginally less severe than that encountered in nonvaccinated cattle.     

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.     

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.     

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.     

Serologic comparisons of 4 Anaplasma isolates as measured by the complement-fixation test

Anaplasma marginale isolated from Virginia ( VAM ), from North Texas ( NTAM ), from Florida (FAM), and Anaplasma ovis from Idaho were used in these trials. Complement-fixation antigens from each of the 4 isolates were used to compare complement-fixing antibody titers of 10 cattle infected with VAM , 17 with FAM, and 6 with NTAM . Strong cross-reactions occurred with all antigens and sera. The homologous system generally showed higher average antibody titers. The serum antibody titers occurring with the A. ovis antigen were significantly lower than those seen with A. marginale antigens. Serum antibody titer differences as measured by FAM and NTAM antigens on sera from VAM , NTAM and FAM failed to reach significance. Serum antibody titer comparisons using VAM antigen were significantly different from those occurring with FAM and NTAM in most instances.     

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.     

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.     

Flow cytometric evaluation of selected antimicrobial efficacy for clearance of Anaplasma marginale in short-term erythrocyte cultures

The tick-borne rickettsia, Anaplasma marginale, causes the economically important cattle disease anaplasmosis. Once infected, cattle remain lifelong carriers. Herein, we used flow cytometry to test the efficacy of three antimicrobials; oxytetracycline, imidocarb and enrofloxacin against Virginia (VGN) or Oklahoma (OK) A. marginale isolates in short-term erythrocyte cultures. Parasite viability was assessed using the vital dye hydroethidine (HE), which is detectable when living organisms convert HE to ethidium bromide. Viability of A. marginale in selected cultures was determined by subinoculation into susceptible calves. Data were analyzed by MANOVA, Tukey-Kramer honest significant difference and Wilcoxon rank sum tests. Receiver operating characteristic (ROC) analysis was used to correlate results with culture infectivity. Enrofloxacin inhibited A. marginale in a dose dependent manner. Surprisingly, higher concentrations of imidocarb were less effective than lower concentrations against A. marginale with significant differences (P < 0.05) observed between the two isolates. Oxytetracycline was the least active drug tested. Cultures infected with the OK isolate exposed to 4.0 microg/mL enrofloxacin and those of the VGN and OK isolates exposed to 1.0 microg/mL imidocarb were sterilized. This is the first in vitro study demonstrating the efficacy of enrofloxacin against A. marginale. Furthermore, these data indicate that flow cytometry is a useful assay for screening antimicrobials against A. marginale.     

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.     

Identification of antigens of two isolates of Anaplasma marginale, using a western blot technique

Antigens of the Illinois (IAM) and Florida (FAM) isolates of Anaplasma marginale were analyzed, using the western blot technique and antiserum from A marginale-infected calves. Crude antigens were prepared from the parasitemic blood of each. Antiserum was collected after the primary and recrudescent parasitemias. Antigens were separated, using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Antigens were then transferred onto nitrocellulose membranes and exposed to test sera. Antibodies attached to the membrane-bound antigens were detected, using an avidin/biotin peroxidase assay and biotinylated rabbit anti-goat immunoglobulin G. Antigens detected were of a high molecular weight group (108 to 91 kilodaltons [kd]) or of a low molecular weight group (47 to 27 kd). The IAM antigens were 100 kd, 96 kd, 47 kd, 38 to 43 kd, and 27 kd; these antigens were detected, using anti-IAM and anti-FAM antibodies, but the anti-FAM antibodies had a strong reaction to only the 100-kd and 38- to 43-kd antigens of IAM. The FAM antigens were 108 kd, 91 kd, 47 kd, 38 to 43 kd, and 27 kd; these antigens were detected, using anti-FAM antibodies and, except the 91 kd antigen, anti-IAM antibodies. Because the 91-kd antigen was detected only in the FAM antigen and detected only by sera from FAM-infected calves, this isolate-specific antigen may be associated with the ability of FAM to induce disease in an IAM-immune animal. Sheep anti-A ovis antibodies reacted only to the 38- to 43-kd antigens of each isolate, indicating that these antigens may be genus-specific.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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)     

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.     

Antibodies to Anaplasma marginale major surface proteins 1a and 1b inhibit infectivity for cultured tick cells

Major surface protein 1 (MSP1) of the cattle pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) is a complex of two proteins, MSP1a and MSP1b. Previous studies demonstrated that MSP1a and MSP1b are adhesins for bovine erythrocytes, while only MSP1a proved to be an adhesin for tick cells. In this study, a tick cell culture system for propagation of A. marginale was used to develop an infection inhibition assay for testing the ability of antisera to block infection of A. marginale for cultured tick cells. A. marginale derived from cell culture was incubated with various antisera prior to inoculation onto cell monolayers. The monolayers were harvested 7 days post-inoculation and A. marginale in the cultures was quantified using an antigen detection ELISA. Antisera tested in the infection inhibition assay were derived from persistently infected cattle, from cattle immunized with A. marginale purified from bovine erythrocytes, and from rabbits and cattle that were immunized with the recombinant MSP1a, MSP1b and MSP1 complex. Antibodies from cattle persistently infected with A. marginale, cattle immunized with A. marginale from bovine erythrocytes or cattle immunized with the recombinant MSP1 complex did not inhibit the infectivity of A. marginale for tick cells. Antiserum from rabbits immunized with MSP1a and MSP1b (individually or combined) reduced infection of both the Virginia and Oklahoma isolates of A. marginale for tick cells by 25-70%. Likewise, antisera from cattle immunized with recombinant MSP1a or MSP1b inhibited infection of tick cells by 26-37%. These results further confirm the role of MSP1 complex proteins in infection of tick cells. Lack of inhibition of infection by antisera from naturally infected cattle or cattle immunized with whole organisms suggests that the bovine immune response is not directed toward blocking infection of A. marginale for tick cells and may contribute to the continued infectivity of the pathogen for ticks.     

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.     

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.     

Co-feeding studies of ticks infected with Anaplasma marginale

Ticks often cluster at preferred feeding sites on hosts, and the co-feeding of ticks at the same site has been shown to increase feeding success and the transmission of some pathogens. While the major route of infection of ticks with pathogens is via the bloodmeal during feeding on a parasitemic host, non-systemic transmission of viruses and spirochetes has been shown to occur from infected to uninfected ticks at common feeding sites on uninfected hosts. In this research, two separate studies were done using the tick-borne rickettsial pathogen of cattle, Anaplasma marginale. In one study we tested whether A. marginale could be transmitted non-systemically from infected to uninfected Dermacentor variabilis males while co-feeding on rabbits. Infection of ticks was determined by allowing them to transmission feed on susceptible cattle and by DNA probe and microscopy studies on salivary glands. In the second study, we tested whether the co-feeding of male and female ticks on parasitemic cattle would increase the acquisition and development of A. marginale in males. A. marginale infections in salivary glands were determined by quantitative PCR after the ticks were allowed to transmission feed on susceptible cattle. Non-systemic transmission of A. marginale did not occur from infected and uninfected ticks that fed at the same site on rabbits and, therefore, does not appear to be a means of A. marginale transmission. A. marginale infections in male ticks were not increased while co-feeding with females. Thus, co-feeding of adult Dermacentor spp. does not appear to influence the dynamics of A. marginale transmission.     

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.