Editorial Type:
Infectious Disease

Evaluation of sequential coinfection with Anaplasma phagocytophilum and Anaplasma marginale in cattle

Bruce R. HoarDepartment of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616.

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Nathan C. NietoDepartment of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616.

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Diane M. RhodesDepartment of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616.

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Janet E. FoleyDepartment of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616.

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DOI:
https://doi.org/10.2460/ajvr.69.9.1171
Volume/Issue:
Volume 69: Issue 9
Online Publication Date:
01 Sep 2008
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Abstract

Objective—To determine whether sequelae of infection differed among single versus double infection with Anaplasma phagocytophilum or Anaplasma marginale, with and without tick salivary extract, in cattle.

Animals—Eighteen 13-month old steers.

Procedures—Treatment groups of 3 cattle each included A marginale inoculated ID followed on day 35 by A phagocytophilum without tick saliva, A phagocytophilum followed on day 10 by A marginale without tick saliva, A marginale followed on day 35 by A phagocytophilum with tick saliva, A phagocytophilum followed on day 10 by A marginale with tick saliva, tissue culture control injection, and tick saliva control injection. Infection was monitored via clinical observations, CBC, serologic testing, and PCR analysis of blood and tissues.

Results—Infected cattle had significantly reduced weight gain. Anemia occurred 25 to 32 days after A marginale infection, which was attenuated by tick saliva. Parasitism was greater if cattle had not previously been inoculated with A phagocytophilum. Nine of the 12 treated cattle had positive results of PCR analysis for A phagocytophilum from at least 1 blood sample. Five tissue samples had positive results of PCR analysis for A phagocytophilum; PCR results for A marginale were positive in spleen, lung, lymph node, heart, and ear skin of infected cattle.

Conclusions and Clinical Relevance—Results indicated an important biological interaction between A marginale and A phagocytophilum infection as well as with tick saliva in disease kinetics and severity in cattle, which may be important for interpretation of diagnostic tests and management of disease in areas where both pathogens occur.

Abstract

Objective—To determine whether sequelae of infection differed among single versus double infection with Anaplasma phagocytophilum or Anaplasma marginale, with and without tick salivary extract, in cattle.

Animals—Eighteen 13-month old steers.

Procedures—Treatment groups of 3 cattle each included A marginale inoculated ID followed on day 35 by A phagocytophilum without tick saliva, A phagocytophilum followed on day 10 by A marginale without tick saliva, A marginale followed on day 35 by A phagocytophilum with tick saliva, A phagocytophilum followed on day 10 by A marginale with tick saliva, tissue culture control injection, and tick saliva control injection. Infection was monitored via clinical observations, CBC, serologic testing, and PCR analysis of blood and tissues.

Results—Infected cattle had significantly reduced weight gain. Anemia occurred 25 to 32 days after A marginale infection, which was attenuated by tick saliva. Parasitism was greater if cattle had not previously been inoculated with A phagocytophilum. Nine of the 12 treated cattle had positive results of PCR analysis for A phagocytophilum from at least 1 blood sample. Five tissue samples had positive results of PCR analysis for A phagocytophilum; PCR results for A marginale were positive in spleen, lung, lymph node, heart, and ear skin of infected cattle.

Conclusions and Clinical Relevance—Results indicated an important biological interaction between A marginale and A phagocytophilum infection as well as with tick saliva in disease kinetics and severity in cattle, which may be important for interpretation of diagnostic tests and management of disease in areas where both pathogens occur.

Cattle may be infected with 2 bacterial species in the genus Anaplasma: Anaplasma marginale and Anaplamsa phagocytophilum. Anaplasma marginale invades erythrocytes; leads to extravascular hemolysis; and can result in anemia, icterus, fever, and weakness. Primary infections are the most severe, after which infection persists for the life of the animal as cyclic, but often subclinical, bacteremia. The mechanism for persistence is the sequential expression of variants of the MSP2 and MSP3 antigens, with subsequent host antibody response.1 This disease causes major production loss in tropical, subtropical, and western American cattle.2 In the western United States, A marginale infection occurs primarily in cattle on the range. Disease also is reported in southern and central Europe.3 Transmission routes include ticks, particularly Dermacentor spp, as well as mechanical transmission by biting flies and fomites (iatrogenically).

Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila in European hoofstock; Ehrlichia equi in North American horses; and the human granulocytic ehrlichiosis, now human granulocytic anaplasmosis or HGA agent) infects granulocytes of humans, cattle, dogs, sheep, deer, horses, and rodents.4 Development of clinical signs of so-called pasture fever in cattle varies, depending on whether the cattle are infected with strains from the United States or Europe. In Europe, A phagocytophilum is transmitted by Ixodes ricinus ticks and is associated in cattle with fever as high as 40°C, reluctance to move, tachypnea, cough, nasal discharge, leukopenia, and thrombocytopenia.5 To the authors' knowledge, no reports of clinical illness in cattle infected with strains of A phagocytophilum from the United States have been published. Anaplasma phagocytophilum also causes disease in humans, along with nonspecific clinical signs and hematologic changes including fever, headache, and thrombocytopenia.6 Cases of human anaplasmosis are reported with increasing frequency,7 and infection is common in Californian horses and dogs.8,9

The pathogenesis of disease associated with Anaplasma spp infection is influenced by the tick vector as it attaches to host skin, feeds, and inoculates the animal with the bacteria. During feeding, hard ticks including Ixodes spp and Dermacentor spp secrete bioactive salivary molecules into the skin to promote host bleeding and reduce antitick inflammation.10 Saliva may have complement, cytokine, and antibody inhibitors; histamine-binding proteins; leukocyte modulators; and anti-hemostatics.11 Thus, inoculation of tick-borne pathogens directly into the skin in the presence of tick saliva is likely to induce local changes in the dermis, and these changes may modulate the early pathogenesis of infection. Anaplasma phagocytophilum infection is in itself immunosuppressive6; thus, coinfection with 2 Anaplasma spp may modulate immunopathologic sequelae of infection, resulting in either enhancement of morbidity, increased mortality rate, or a cross-protective effect.

In diverse host-pathogen systems, including trypanosomes and spirochetes, the original presentation of antigens by dermal dendritic cells can have an important influence on the nature of the subsequent immune response.12–14 Late events in anaplasmosis, when clinical disease occurs, depend critically on a cascade of interdependent host-pathogen interactions starting as early as the first few hours after the pathogen is inoculated into the skin and antigen is presented by dendritic cells. The reticuloendothelial system is important in the pathogenesis of erythrocytic anaplasmosis because infected cattle develop autoantibodies, and anemia is a result of splenic removal of antibody-tagged erythrocytes. Thus, splenectomized cattle, which are often used in studies of anaplasmosis in cattle, have an altered disease outcome, compared with natural disease. The present study used nonsplenectomized steers, ensuring that the important effects of interactions between rickettsial-infected cells and cells in the spleen were present.

Despite the high likelihood that cattle in Europe and western North America could be infected with both A marginale and A phagocytophilum, little is known about how coinfection with the 2 pathogens could change the risk and course of disease and how tick bites could influence the outcome of infection. In particular, coinfection with the 2 Anaplasma spp could increase severity of both infections. The objective of the study reported here was to evaluate responses to sequential inoculation of A marginale and A phagocytophilum in yearling cattle and compare the effect of inoculation with and without a tick salivary gland extract.

Materials and Methods

Animals—Eighteen 13-month old Black Angus steers were obtained from a site in Nevada from which anaplasmosis was reportedly absent and were screened for exposure to A marginale and A phagocytophilum by use of serologic and PCR testing; all cattle had negative results for both pathogens. Cattle were housed in a single pen in a cement-floored feedlot, and the experiment took place during late winter before ticks appeared. The study was reviewed and approved by the University of California, Davis, Animal Care and Use Committee.

Bacterial isolates—The A marginale strain, designated South Idaho, was derived from a naturally infected animal in Caldwell, Idaho.15 Bovine blood with a rickettsemia of 42% was collected into bottles containing 4 U of heparin/mL and centrifuged at 1,700 × g for 30 minutes. The plasma and buffy coat were removed, and the blood was washed 3 times with PBS solution.

To prepare stabilates, 10 mL of 31.2% dimethyl sulfoxide in filter-sterilized PBS solution was added to 10 mL of packed and washed erythrocytes, the tube was mixed briefly by inversion, and the sample was frozen in liquid nitrogen.16 The stabilate was thawed quickly prior to inoculation, and each steer received 3 mL of stabilate. The granulocytic strain was A phagocytophilum Webster, a human origin isolate from the blood of an infected patient in northwestern Wisconsin, which was cultured in human promyelocytic leukemia (HL-60) cells.17 The inoculum for each animal consisted of 1 mL of 1 × 107 HL-60 cells in 1 mL of tissue culture fluid, with 70% of cells infected with A phagocytophilum as judged on the basis of visual inspection of Wright-stained samples.

Tick salivary extracts—Adult Ixodes pacificus and Dermacentor occidentalis were collected by flagging vegetation in western Yolo County, Calif. Adult ticks were fed on New Zealand White rabbits for 4 days; removed with fine forceps; and washed sequentially in bleach, 70% ethanol, and PBS solution. The salivary glands of 9 live adult female D occidentalis were dissected by use of sterile techniques, homogenized, and suspended in 1 mL of PBS solution. Glands from 9 live adult female I pacificus were treated comparably. Gland extracts were used fresh. To make inoculum for infection experiments, 111 μL of I pacificus salivary extract was added to each 1 mL of A phagocytophilum inoculum, 111 μL of D occidentalis salivary extract was added to each A marginale inoculum, and a mixture of 55.5 μL of I pacificus extract and 55.5 μL of D occidentalis extract was added to 1 mL of HL-60 cell culture control.

Experimental inoculation—On day 0, cattle were inoculated ID in the right lateral cervical region with either A phagocytophilum, A marginale, an HL-60 cell negative control injection, or a tick salivary extract control injection. Cattle were randomly allocated into the 6 treatment groups of 3 cattle each as follows: Am/Ap NS, Ap/Am NS, Am/Ap S, Ap/Am S, TCC, and TSC. Work with cattle was overseen by the campus veterinarian of the University of California, Davis, and approved by the university's institutional animal care and use committee.

Clinical observations and sample collection— Clinical observations were made at the time of inoculation and at 3-day intervals until the end of the experiment on day 50. Observations consisted of determination of weight and a physical examination by a veterinarian for any lesions at the infection site, attitude, hydration, capillary refill time, rectal temperature, respiratory rate and character, and heart rate. On each of the observation days, 5 mL of blood was collected via the coccygeal vein into EDTA for CBC, serologic testing, and PCR assay. Cattle were slaughtered on day 50 by use of a captive bolt gun, and all internal organs were examined visually for abnormalities by a veterinarian. Samples of spleen, heart, lung, mesenteric lymph node, and liver were harvested within 1 hour after slaughter. Half of each tissue sample was placed directly onto ice for transfer to the laboratory and freezing at −20°C for PCR assay, and the other half was fixed in neutral-buffered 10% formalin for histologic examination.

The CBCs were evaluated by use of an automated instrument.a Differential cell counts were performed manually from thin Wright-stained blood smears. The PPE for each smear was calculated by examination for A marginale via oil immersion at 1,000 × magnification. Three independent observers each counted 100 cells in 3 fields, and the number of infected cells was recorded. The PPE was determined according to the following equation:

article image

Formalin-fixed samples were embedded in paraffin, thinly sectioned, and stained with H&E for evaluation via light microscopy.

Serologic testing—Plasma was separated from blood by centrifugation at 1,800 × g for 10 minutes. A commercially available cELISA kitb was used for determination of antibodies against the MSP5 protein of A marginale, according to manufacturer's instructions. Samples with ≥ 30% inhibition were considered to have positive results. Antibodies against A phagocytophilum were evaluated in an IFA test by use of A phagocytophilum strain Webster propagated in HL-60 cell culture as a substrate. The IFA test was performed and evaluated as reported.18

DNA extraction and PCR assay—The DNA was extracted from blood samples on day 6 after A phagocytophilum inoculation and day 21 after A marginale inoculation (except that cattle initially infected with A phagocytophilum had been slaughtered before 21 days had elapsed after the A marginale inoculation). The DNA was extracted from blood and tissue by use of a kit.c The PCR assay for the A phagocytophilum p44 gene was performed as described19 in a combined thermocycler-fluorometer.d Each 12-μL reaction contained 1 μL of DNA, 1× universal master mix,e 2 nmol of each primer, and 400 pmol of probe. The thermocycling conditions consisted of 50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles at 95°C for 15 seconds, followed by 60°C for 1 minute. Samples were considered to have positive results if they had a cycle threshold value < 40 and characteristic amplification plots. Anaplasma marginale PCR assay for the MSP4 gene was performed as described,20 with modifications. Each 50-μL reaction contained 10 pmol of primers MSP45 and MSP43, 1.5mM MgCl2, 0.2mM dNTPs, 1× Taq DNA reaction buffer, 1.25 units of Taq DNA polymerase, and 31.75 μL of water. Reaction conditions were as described.20 Samples were evaluated via UV transillumination of 1% agarose gels.

Statistical analysis—Statistical analysis was performed with a commercially available software program.f Average daily gain for each animal was calculated as final weight minus initial weight divided by days in trial. A 1-way ANOVA was used to compare gain among groups, with the method of least significant difference used for post hoc comparisons. Changes in Hct, MCV, RDW, and total WBC count were graphed, and differences among treatments were analyzed by use of ANOVA. The PPE was also graphed, and differences were analyzed by use of ANOVA. For all tests, P ≤ 0.05 was considered significant.

Results

Cattle in the control groups did not have any clinical changes, alterations in RBC or WBC values, seroconversion to either pathogen, or histopathologic abnormalities. They did not have positive PCR assay results in blood or tissue at any point during the study.

Cattle in the infected treatment groups did not have clinical evidence of morbidity; that is, there were no apparent signs of depression, weakness, ataxia, pallor, or increased reperfusion time of mucous membranes. However, cattle in all infected treatment groups had significantly lower average daily weight gain than cattle in the control groups (Table 1).

Table 1—

Mean ± SE daily gain (kg/d) of control steers and steers infected with Anaplasma marginale and Anaplasma phagocyto-philum.
Treatment groupDaily gain
Ap/Am NS0.99 ± 0.27
Am/Ap NS0.65 ± 0.27
Ap/Am S0.67 ± 0.07
Am/Ap S0.86 ± 0.38
TCC1.30 ± 0.16
TSC1.75 ± 0.17

Cattle in all infected treatment groups had alterations in a variety of blood values (Figure 1). The Hct decreased 25 to 32 days following A marginale infection in all A marginale–infected treatment groups. The onset and degree of anemia were significantly attenuated in cattle in infection groups that also included tick saliva, compared with those in treatment groups that did not include tick saliva. Increases in MCV and RDW occurred shortly after onset of anemia, approximately 35 days after A marginale challenge (Figures 2 and 3). The experiment was not conducted long enough to determine relative differences in magnitude between cattle pretreated and not pretreated with A phagocytophilum. The presence of tick saliva resulted in significantly lower MCV in cattle in both treatments, compared with that in cattle lacking saliva. There was a small peak in RDW in the Am/Ap NS group on day 8, followed by peaks in the Am/Ap NS and Am/Ap S groups on day 35. Peaks for the Ap/Am S and Ap/Am NS cattle were not fully within the time scale of the experiment, but their maximum values were significantly higher than those in the Am/Ap cattle. In both cases, cattle that were inoculated with saliva had significantly lower RDW than did the comparable treatment group without saliva. Platelet counts were transiently decreased in the Am/Ap NS and Am/Ap S groups on day 22, followed by rebound thrombocytosis (although not outside the reference range) on days 34 to 37 after inoculation (Figure 4). If cattle had been pretreated with A phagocytophilum, there was often greater thrombocytopenia, compared with nonpretreated groups, on day 42 (ie, 32 days after A marginale inoculation). No significant abnormalities were detected in leukocyte counts.

Figure 1—
Figure 1—

Changes in Hct in control cattle or following infection in cattle with Anaplasma marginale and Anaplasma phagocytophilum or control injections *Significant (P < 0.05) difference among groups.

Citation: American Journal of Veterinary Research 69, 9; 10.2460/ajvr.69.9.1171

Figure 2—
Figure 2—

Changes in MCV in control cattle or following infection in cattle with A marginale and A phagocytophilums. *Significant (P < 0.05) difference among groups.

Citation: American Journal of Veterinary Research 69, 9; 10.2460/ajvr.69.9.1171

Figure 3—
Figure 3—

Changes in RDW in control cattle or following infection in cattle with A marginale and A phagocytophilum. *Significant (P < 0.05) difference among groups.

Citation: American Journal of Veterinary Research 69, 9; 10.2460/ajvr.69.9.1171

Figure 4—
Figure 4—

Changes in platelet numbers in calves infected with A marginale and A phagocytophilum. *Significant (P < 0.05) difference among groups.

Citation: American Journal of Veterinary Research 69, 9; 10.2460/ajvr.69.9.1171

No A phagocytophilum morulae were observed during CBCs. In contrast, A marginale organisms were observed in cattle from 22 days after infection to the end of the experiment, with values ranging from 2% to 70% of erythrocytes infected (Figure 5). Cattle initially infected with A marginale had a significantly greater PPE of A marginale than cattle initially infected with A phagocytophilum.

Figure 5—
Figure 5—

Changes in A marginale PPE in calves infected with A marginale and A phagocytophilum. See Figure 1 for key.

Citation: American Journal of Veterinary Research 69, 9; 10.2460/ajvr.69.9.1171

At necropsy, no lesions were grossly visible in any animal. Histologically, the heart, lungs, spleen, and lymph nodes were all grossly normal. In 4 calves, the liver had mild periportal pyogranulomatous inflammation (all 3 calves in the Am/Ap S treatment and 1 calf in the Ap/Am S treatment group), and 1 calf (Am/Ap NS treatment group) had focal hepatic abscesses.

All cattle that initially received A marginale seroconverted on the basis of results of the A marginale cELISA, with cattle that were coinoculated with tick saliva typically seroconverting on day 6 and those lacking tick saliva seroconverting on day 8. There was serologic cross-reactivity between the ELISA for A marginale and the IFA for A phagocytophilum. Thus, cattle became seropositive to A phagocytophilum approximately 22 days after infection with A marginale, but before they had been inoculated with A phagocytophilum. Calves in the 2 treatment groups initially infected with A marginale remained seropositive throughout the trial.

When cattle were initially inoculated with A phagocytophilum, they consistently seroconverted (IFA test) on day 6, although titers were low. After these calves were infected with A marginale, they began to seroconvert to A marginale 6 days later. In this case, calves that were inoculated without tick saliva typically seroconverted before those inoculated with tick saliva. Calves in these 2 treatment groups also remained seropositive throughout the trial.

Nine of the 12 cattle had a positive PCR assay result for A phagocytophilum from at least 1 blood sample. The positive result was obtained at 3 days after infection (n = 1 calf), 6 days after infection (6), or 22 days after infection (2). Four calves had 3 or more blood samples with positive results of the PCR assay. Only 5 tissue samples had PCR assay positive results for A phagocytophilum: lung (n = 1), spleen (1), liver (1), and heart (2). All but one of the tissue samples came from a calf that had at least 1 blood sample with positive results.

Results of the A marginale PCR assay were positive in most spleen, lung, lymph node, heart, and ear skin samples from infected, but not control, cattle, with the following exceptions: heart and lymph node samples yielded negative results in 1 Am/Ap steer that received salivary extract and 1 that did not, whereas the ear skin sample also yielded negative results in these cattle as well as in 1 Ap/Am steer that did not receive saliva.

Discussion

This experiment revealed an important biological interaction between A marginale and A phagocytophilum infection as well as with tick saliva, with regard to disease kinetics and severity in cattle. These findings may be important for the interpretation of diagnostic tests and management of disease in cattle in areas where both pathogens occur, such as in the western United States and southern and central Europe. For example, if diagnostic testing for A marginale were to occur in an area where A phagocytophilum is present, falsepositive interpretation of test results could result, given the cross-reactivity observed with A phagocytophilum, which could lead to inappropriate disease management if clinical decisions were based on those test results. Infection with A marginale causes considerable clinical signs and production loss, although management strategies help mitigate against extensive mortality rates.21 These include movement of young cattle (which are more resistant to severe clinical disease) onto endemically infected rangeland and use of a state-licensed live vaccine and an experimental killed vaccine. Most severe signs are seen in cattle that have primary infection when > 2 years of age. Severely affected cattle develop hemolytic anemia; icterus; reduction in milk production and rumination; lethargy; and, possibly, aggressiveness because of cerebral hypoxia.21

Clinical granulocytic anaplasmosis appears to be common in Europe and is typically associated with moderate to severe clinical disease in sheep and cattle.22 In contrast, infection is uncommon in American cattle. Moreover, experimental infection with European cattle strains, but not European human or American horse or humanorigin strains, reproduces natural severe disease.5,23 In 6 cattle, in which 2 served as control animals, 2 received a Californian equine isolate, and 2 received a midwestern human isolate, none developed clinical signs or hematologic abnormalities or had positive results of PCR assay, although all seroconverted.23 Thus, we expected that any impact of granulocytic anaplasmosis on cattle in the present study would likely be via modulation of the more severe clinical impact of erythrocytic anaplasmosis.

Cattle in the present study were exposed to anaplasmosis via a simulated tick exposure. Some prior experimental studies of A marginale have relied on IV inoculation of splenectomized cattle, which causes reduced ability to clear the pathogen from circulation. Inoculation into the dermis could differ with regard to early pathogenesis, particularly in the presence of tick saliva. Ticks inject an array of anti-inflammatory and immunomodulatory chemicals during feeding to prevent host blood clotting, platelet aggregation, natural killer cell activity, and Th1 cytokine responses.11 Unfortunately, cattle that received A marginale followed by A phagocytophilum developed acute disease shortly before they were slaughtered, making it impossible to compare the recovery phase of the different treatment groups. Additionally, the use of a mixed salivary extract (ie, half from each D occidentalis and I pacificus) in the control cattle precluded us from being able to evaluate the effect of specific tick species on the outcome of exposure to the extract. Nevertheless, we could identify significant differences in some aspects of the outcomes of infection in cattle exposed to tick saliva and pathogens, compared with those that were not. In particular, changes in Hct, MCV, and RDW associated with A marginale infection were delayed and attenuated in cattle exposed to tick saliva. One possible mechanism could be that immunomodulatory effectors in the saliva created a milieu in which A marginale had reduced access to target cells. Alternatively, given that many clinical outcomes of erythrocytic anaplasmosis are immune mediated, early anti-inflammatory effects caused by tick saliva could have delayed the onset of clinical signs. Also, we used young cattle, and in older cattle, immunologic sequelae may be more pronounced. Thus, an important target of future research would be to evaluate short-term immunomodulation in the dermis after A marginale is inoculated in the presence of tick saliva in both older and younger cattle.

There were considerable hematologic but lesser clinical sequelae of A marginale infection in the present study. Cattle infected with either or both pathogens had failure of weight gain, and there was fever in A marginale–infected cattle, probably caused by hemolysis. Anemia was extensive, possibly because of a large inoculum dose, the effect of salivary mediators, or ID inoculation. Despite the extent of anemia and thrombocytopenia, cattle did not develop lethargy, changes in capillary refill time, or mucous membrane color. Concurrent with the anemia, erythrocyte parasitism was extensive, typically affecting as many as 70% of RBCs, but as many as 100% in a few cattle. These findings suggested more severe erythrocytic anaplasmosis than in some earlier studies. For example, in splenectomized cattle after 42 days, there was a peak parasitemia in 48% of the erythrocytes and a peak reduction in PCV by 74%.24

The main effect of A phagocytophilum infection seemed to be modulation in changes associated with A marginale infection. The mild granulocytic anaplasmosis observed in the present study was consistent with another report23 of infection with North American A phagocytophilum strains and many human cases25,26 in which hematologic abnormalities are often minor. Sequential coinfection was hypothesized to exacerbate single infection via immunomodulation or to reduce infection severity (a vaccine-like effect). However, the observed effect of the coinfection was exacerbated hematologic abnormalities, compared with those attributable to either pathogen alone. Autoimmune changes have been reported in cats and humans with granulocytic anaplasmosis,27 with development of anti-nuclear or antiplatelet antibodies in 80% of human patients in 1 study.28 Anaplasma phagocytophilum–infected cattle, goats, and sheep are more susceptible to staphylococcal pyemia, Pasteurella haemolytica and Chlamydia psittaci infections, and parainfluenza-3 viral pneumonia.29–32 Typically, cattle with so-called pasture fever are neutropenic with defective neutrophil migration (diapedesis) and activity, lymphocytopenic, have reduced CD4+ and CD8+ counts, and have reduced T-cell lymphoproliferation in response to mitogens.33–36 Anaplasma phagocytophilum infection also impairs the host's response to humoral challenge35 and prevents phagosome-lysosome fusion in neutrophils.37 Thus, in the present study, some immune-mediated sequelae of A marginale infection may have been exacerbated by A phagocytophilum preinfection (anemia and erythrocyte responses such as RDW and MCV); however, rates of A marginale parasitism in erythrocytes were reduced after A phagocytophilum infection, which suggested 2 differing interactive mechanisms between the pathogens.

To some extent, the immunologic interaction between the 2 pathogens also was reflected in the difficulties interpreting serologic results when cattle were coinfected. The MSP5 protein, which is the target of the cELISA licensed for the diagnosis of A marginale in cattle, is reportedly 63% identical in amino acid sequence to the paralog from A phagocytophilum, and some serologic cross-reaction by use of recombinant MSP5 has been reported among humans, cattle, sheep, and horses.38,39 Sheep, horses, and cattle that were experimentally infected with A phagocytophilum seroconverted by use of the A marginale cELISA, although the duration of seropositivity was limited, whereas cattle infected with A marginale developed A phagocytophilum IFA-positive results.39 However, dogs and horses naturally infected with A phagocytophilum were not seropositive by use of the A marginale cELISA.38 The value of serologic testing for A marginale probably depends on the magnitude of the titer, prevalence of pathogens, and clinical signs. In a field study40 of cattle in Switzerland in which both A marginale and A phagocytophilum were present, A marginale seropositivity was strongly statistically associated with anemia and there was excellent agreement between serologic and microscopic findings. These published results and our findings make interpretation of serologic results problematic for cattle with coinfection. In contrast, PCR assay may be more specific than serologic testing, but results may be positive only during active infection. The finding of positive results of PCR assay for A phagocytophilum was transient in blood and tissue, consistent with the lack of visible morulae in RBCs. However, almost all tissues yielded positive PCR assay results for A marginale terminally.

Erythrocytic anaplasmosis remains a devastating disease for cattle in the United States, and granulocytic anaplasmosis is an important disease of cattle in Europe as well as a potential zoonotic disease in the United States. Opportunities for coinfection are common, yet immunopathologic interactions, mechanisms, and implications have received little attention. Other techniques to induce anaplasmosis, both erythrocytic and granulocytic, often rely on needle inoculation methods to study kinetics of infection in cattle; however, results of the present study have provided evidence that natural-like ID inoculation with tick saliva represents a good method for ongoing evaluation of Anaplasma spp coinfection.

ABBREVIATIONS

Am/Ap

NS Infection with Anaplasma marginale followed on day 35 (at peak parasitemia) by Anaplasma phagocytophilum without tick saliva

Am/Ap S

Infection with A marginale followed by A phagocytophilum on day 35 with tick saliva

Ap/Am NS

Infection with A phagocytophilum followed on day 10 by A marginale without tick saliva

Ap/Am S

Infection with A phagocytophilum followed on day 10 by A marginale with tick saliva

cELISA

Competitive ELISA

IFA

Immunofluorescent antibody

MCV

Mean corpuscular volume

MSP

Major surface protein

PPE

Percentage of parasitized erythrocytes

RDW

Red cell distribution width

TCC

Injected with tissue culture (control group) on days 0 and 35

TSC

Injected with tick saliva (control group) on days 0 and 35
a.

Serono Baker 9000, Biochemical Immunosystems, Allentown, Pa.

b.

cELISA, VMRD, Pullman, Wash.

c.

DNeasy tissue kit, Qiagen, Valencia, Calif.

d.

ABI Prism 7700, Applied Biosystems, Foster City, Calif.

e.

Applied Biosystems, Foster City, Calif.

f.

SPSS, version 15.0 for Windows, SPSS Inc, Chicago, Ill.

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    Madigan J. Equine ehrlichiosis. Vet Clin North Am Equine Pract 1993;9:423–428.

  • 9.

    Foley JE, Foley P, Madigan JE. Spatial distribution of seropositivity to the causative agent of granulocytic ehrlichiosis in dogs in California. Am J Vet Res 2001;62:1599–1605.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.↑

    Nuttall PA, Labuda M. Tick-host interactions: saliva-activated transmission. Parasitology 2004;129(suppl 129):S177–S189.

  • 11.↑

    Brossard M, Wikel SK. Tick immunobiology. Parasitology 2004;129:S161–S176.

  • 12.

    Gajewski TF, Pinnas M, Wong T, et al. Murine Th1 and Th2 clones proliferate optimally in response to distinct antigen-presenting cell populations. J Immunol 1991;146:1750–1758.

    • Search Google Scholar
    • Export Citation
  • 13.

    Weaver CT, Hawrylowicz CM, Unanue ER. T helper cell subsets require the expression of distinct costimulatory signals by antigen-presenting cells. Proc Natl Acad Sci U S A 1988;85:8181–8185.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Altenschmidt U, Ricciardi-Castagnoli P, Modolell M, et al. Bone marrow-derived macrophage lines and immortalized cloned macrophage and dendritic cells support priming of Borrelia burgdorferi —specific T cell responses in vitro and/or in vivo. Immunol Lett 1996;50:41–49.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.↑

    McGuire TC, Palmer GH, Goff WL, et al. Common and isolaterestricted antigens of Anaplasma marginale detected with monoclonal antibodies. Infect Immun 1984;45:697–700.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16.↑

    Love JN. Cryogenic preservation of Anaplasma marginale with Dimethyl sulfoxide. Am J Vet Res 1972;33:2557–2560.

  • 17.↑

    Asanovich KM, Bakken JS, Madigan JE, et al. Antigenic diversity of granulocytic Ehrlichia isolates from humans in Wisconsin and New York and a horse in California. J Infect Dis 1997;176:1029–1034.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.↑

    Dumler SJ, Asanovich KM, Bakken JS, et al. Serologic crossreactions among Ehrlichia equi, Ehrlichia phagocytophila, and human granulocytic Ehrlichia. J Clin Microbiol 1995;33:1098–1103.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19.↑

    Drazenovich NL, Foley JE, Brown RN. Use of real-time quantitative PCR targeting the msp2 protein gene to identify cryptic Anaplasma phagocytophilum infections in wildlife and domestic animals. Vector Borne Zoonotic Dis 2006;6:83–90.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20.↑

    de la Fuente J, Van Den Bussche RA, Garcia-Garcia JC, et al. Phylogeography of New World isolates of Anaplasma marginale based on major surface protein sequences. Vet Microbiol 2002;88:275–285.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21.↑

    Palmer GH, Lincoln S. Anaplasmosis. In: Smith B, ed. Large animal internal medicine. 3rd ed. St Louis: Mosby, 2002;1049–1051.

  • 22.↑

    Blanco JR, Oteo JA. Human granulocytic ehrlichiosis in Europe. Clin Microbiol Infect 2002;8:763–772.

  • 23.↑

    Pusterla N, Anderson RJ, House JK, et al. Susceptibility of cattle to infection with Ehrlichia equi and the agent of human granulocytic ehrlichiosis. J Am Vet Med Assoc 2001;218:1160–1162.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24.↑

    Stich RW, Sauer JR, Bantle JA, et al. Detection of Anaplasma marginale (Rickettsiales: Anaplasmataceae) in secretagogue-induced oral secretions of Dermacentor andersoni (Acari: Ixodidae) with the polymerase chain reaction. J Med Entomol 1993;30:789–794.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    Foley JE, Crawford-Miksza L, Dumler JS, et al. Human granulocytic ehrlichiosis in northern California: two cases of human granulocytic ehrlichiosis in northern California. Clin Infect Dis 1999;29:388–392.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Dumler JS, Bakken JS. Human ehrlichioses: newly recognized infections transmitted by ticks. Annu Rev Med 1998;49:201–213.

  • 27.↑

    Foley J, Leutenegger C, Dumler J, et al. FIV-infection and AIDS modulate the severity of human granulocytic ehrlichiosis in a cat model. Comp Immunol Microbiol 2003;26:103–113.

    • Search Google Scholar
    • Export Citation
  • 28.↑

    Wong SJ, Thomas JA. Cytoplasmic, nuclear, and platelet autoantibodies in human granulocytic ehrlichiosis patients. J Clin Microbiol 1998;36:1959–1963.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29.

    Batungbacal MR, Scott GR. Tick-borne fever and concurrent parainfluenza-3 virus infection in sheep. J Comp Pathol 1982;92:415–428.

  • 30.

    Foggie A. Further experiments on the effect of tick-borne fever infection on the susceptibility of lambs to staphylococci. J Comp Pathol 1957;67:369–377.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31.

    Brodie TA, Holmes PH, Urquhart GM. Some aspects of tickborne diseases of British sheep. Vet Rec 1986;118:415–418.

  • 32.

    Munro R, Hunter AR, MacKenzie G, et al. Pulmonary lesions in sheep following experimental infection by Ehrlichia phagocytophila and Chlamydia psittaci. J Comp Pathol 1982;92:117–129.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33.

    Woldehiwet Z. Depression of lymphocyte response to mitogens in sheep infected with tick-borne fever. J Comp Pathol 1987;97:637–643.

  • 34.

    Woldehiwet Z. The effects of tick-borne fever on some functions of polymorphonuclear cells of sheep. J Comp Pathol 1987;97:481–485.

  • 35.↑

    Larsen HJ, Overnes G, Waldeland H, et al. Immunosuppression in sheep experimentally infected with Ehrlichia phagocytophila. Res Vet Sci 1994;56:216–224.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36.

    Foster WN, Cameron AE. Observations on the functional integrity of neutrophil leucocytes infected with tick-borne fever. J Comp Pathol 1970;80:487–491.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37.↑

    Gokce HI, Ross G, Woldehiwet Z. Inhibition of phagosome-lysosome fusion in ovine polymorphonuclear leucocytes by Ehrlichia (Cytoecetes) phagocytophila. J Comp Pathol 1999;120:369–381.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 38.↑

    Strik NI, Alleman AR, Barbet AF, et al. Characterization of Anaplasma phagocytophilum major surface protein 5 and the extent of its cross-reactivity with A. marginale. Clin Vaccine Immunol 2007;14:262–268.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 39.↑

    Dreher UM, de la Fuente J, Hofmann-Lehmann R, et al. Serologic cross-reactivity between Anaplasma marginale and Anaplasma phagocytophilum. Clin Diagn Lab Immunol 2005;12:1177–1183.

    • Search Google Scholar
    • Export Citation
  • 40.↑

    Hofmann-Lehmann R, Meli ML, Dreher UM, et al. Concurrent infections with vector-borne pathogens associated with fatal hemolytic anemia in a cattle herd in Switzerland. J Clin Microbiol 2004;42:3775–3780.

    • Crossref
    • Search Google Scholar
    • Export Citation

Contributor Notes

The authors thank Elizabeth Holmes, Edwin Saada, Tom Yuan, Veronica Villanueva, Jerry Johnson, and Niki Drazenovich for technical assistance; Dr. Steve Dumler for the Webster strain of Anaplasma phagocytophilum; and Dr. Anthony Barbet for Anaplasma marginale.

Supported by the UC Davis Center for Food Animal Health, and the Department of Veterinary Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, Calif.

Address correspondence to Dr. Hoar.
Cover American Journal of Veterinary Research
American Journal of Veterinary Research
ISSN:
0002-9645
Publication Date:
01 Sep 2008
  • Figure 1—
    View in gallery
    Figure 1—

    Changes in Hct in control cattle or following infection in cattle with Anaplasma marginale and Anaplasma phagocytophilum or control injections *Significant (P < 0.05) difference among groups.

  • Figure 2—
    View in gallery
    Figure 2—

    Changes in MCV in control cattle or following infection in cattle with A marginale and A phagocytophilums. *Significant (P < 0.05) difference among groups.

  • Figure 3—
    View in gallery
    Figure 3—

    Changes in RDW in control cattle or following infection in cattle with A marginale and A phagocytophilum. *Significant (P < 0.05) difference among groups.

  • Figure 4—
    View in gallery
    Figure 4—

    Changes in platelet numbers in calves infected with A marginale and A phagocytophilum. *Significant (P < 0.05) difference among groups.

  • Figure 5—
    View in gallery
    Figure 5—

    Changes in A marginale PPE in calves infected with A marginale and A phagocytophilum. See Figure 1 for key.

Figure 1—

Changes in Hct in control cattle or following infection in cattle with Anaplasma marginale and Anaplasma phagocytophilum or control injections *Significant (P < 0.05) difference among groups.
Figure 2—

Changes in MCV in control cattle or following infection in cattle with A marginale and A phagocytophilums. *Significant (P < 0.05) difference among groups.
Figure 3—

Changes in RDW in control cattle or following infection in cattle with A marginale and A phagocytophilum. *Significant (P < 0.05) difference among groups.
Figure 4—

Changes in platelet numbers in calves infected with A marginale and A phagocytophilum. *Significant (P < 0.05) difference among groups.
Figure 5—

Changes in A marginale PPE in calves infected with A marginale and A phagocytophilum. See Figure 1 for key.
  • 1.

    Brayton KA, Meeus PF, Barbet AF, et al. Simultaneous variation of the immunodominant outer membrane proteins, MSP2 and MSP3, during Anaplasma marginale persistence in vivo. Infect Immun 2003;71:6627–6632.

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  • 2.

    Maas J. Fact sheet No. 1: Anaplasmosis. University of California Cooperative Extension, 2003. Davis, Calif: University of California, 2003. http://www.vetmed.ucdavis.edu/vetext/INF-BE.html. Accessed July 25, 2007.

  • 3.

    Dreher UM, Hofmann-Lehmann R, Meli ML, et al. Seroprevalence of anaplasmosis among cattle in Switzerland in 1998 and 2003: no evidence of an emerging disease. Vet Microbiol 2005;107:71–79.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4.

    Dumler JS, Barbet AF, Bekker CPJ, et al. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and ‘HGE agent’ as subjective synonyms of Ehrlichia phagocytophila. Int J Syst Evol Microbiol 2001;51:2145–2165.

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    • Export Citation
  • 5.

    Pusterla N, Pusterla JB, Braun U, et al. Experimental cross-infections with Ehrlichia phagocytophila and human granulocytic ehrlichia-like agent in cows and horses. Vet Rec 1999;145:311–314.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6.

    Dumler JS, Choi KS, Garcia-Garcia JC, et al. Human granulocytic anaplasmosis and Anaplasma phagocytophilum. Emerg Infect Dis 2005;11:1828–1834.

  • 7.

    CDC. Summary of notifiable diseases—United States, 2004. MMWR Morb Mortal Wkly Rep 2006;53:1–79.

  • 8.

    Madigan J. Equine ehrlichiosis. Vet Clin North Am Equine Pract 1993;9:423–428.

  • 9.

    Foley JE, Foley P, Madigan JE. Spatial distribution of seropositivity to the causative agent of granulocytic ehrlichiosis in dogs in California. Am J Vet Res 2001;62:1599–1605.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    Nuttall PA, Labuda M. Tick-host interactions: saliva-activated transmission. Parasitology 2004;129(suppl 129):S177–S189.

  • 11.

    Brossard M, Wikel SK. Tick immunobiology. Parasitology 2004;129:S161–S176.

  • 12.

    Gajewski TF, Pinnas M, Wong T, et al. Murine Th1 and Th2 clones proliferate optimally in response to distinct antigen-presenting cell populations. J Immunol 1991;146:1750–1758.

    • Search Google Scholar
    • Export Citation
  • 13.

    Weaver CT, Hawrylowicz CM, Unanue ER. T helper cell subsets require the expression of distinct costimulatory signals by antigen-presenting cells. Proc Natl Acad Sci U S A 1988;85:8181–8185.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Altenschmidt U, Ricciardi-Castagnoli P, Modolell M, et al. Bone marrow-derived macrophage lines and immortalized cloned macrophage and dendritic cells support priming of Borrelia burgdorferi —specific T cell responses in vitro and/or in vivo. Immunol Lett 1996;50:41–49.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.

    McGuire TC, Palmer GH, Goff WL, et al. Common and isolaterestricted antigens of Anaplasma marginale detected with monoclonal antibodies. Infect Immun 1984;45:697–700.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16.

    Love JN. Cryogenic preservation of Anaplasma marginale with Dimethyl sulfoxide. Am J Vet Res 1972;33:2557–2560.

  • 17.

    Asanovich KM, Bakken JS, Madigan JE, et al. Antigenic diversity of granulocytic Ehrlichia isolates from humans in Wisconsin and New York and a horse in California. J Infect Dis 1997;176:1029–1034.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.

    Dumler SJ, Asanovich KM, Bakken JS, et al. Serologic crossreactions among Ehrlichia equi, Ehrlichia phagocytophila, and human granulocytic Ehrlichia. J Clin Microbiol 1995;33:1098–1103.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19.

    Drazenovich NL, Foley JE, Brown RN. Use of real-time quantitative PCR targeting the msp2 protein gene to identify cryptic Anaplasma phagocytophilum infections in wildlife and domestic animals. Vector Borne Zoonotic Dis 2006;6:83–90.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20.

    de la Fuente J, Van Den Bussche RA, Garcia-Garcia JC, et al. Phylogeography of New World isolates of Anaplasma marginale based on major surface protein sequences. Vet Microbiol 2002;88:275–285.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21.

    Palmer GH, Lincoln S. Anaplasmosis. In: Smith B, ed. Large animal internal medicine. 3rd ed. St Louis: Mosby, 2002;1049–1051.

  • 22.

    Blanco JR, Oteo JA. Human granulocytic ehrlichiosis in Europe. Clin Microbiol Infect 2002;8:763–772.

  • 23.

    Pusterla N, Anderson RJ, House JK, et al. Susceptibility of cattle to infection with Ehrlichia equi and the agent of human granulocytic ehrlichiosis. J Am Vet Med Assoc 2001;218:1160–1162.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24.

    Stich RW, Sauer JR, Bantle JA, et al. Detection of Anaplasma marginale (Rickettsiales: Anaplasmataceae) in secretagogue-induced oral secretions of Dermacentor andersoni (Acari: Ixodidae) with the polymerase chain reaction. J Med Entomol 1993;30:789–794.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    Foley JE, Crawford-Miksza L, Dumler JS, et al. Human granulocytic ehrlichiosis in northern California: two cases of human granulocytic ehrlichiosis in northern California. Clin Infect Dis 1999;29:388–392.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Dumler JS, Bakken JS. Human ehrlichioses: newly recognized infections transmitted by ticks. Annu Rev Med 1998;49:201–213.

  • 27.

    Foley J, Leutenegger C, Dumler J, et al. FIV-infection and AIDS modulate the severity of human granulocytic ehrlichiosis in a cat model. Comp Immunol Microbiol 2003;26:103–113.

    • Search Google Scholar
    • Export Citation
  • 28.

    Wong SJ, Thomas JA. Cytoplasmic, nuclear, and platelet autoantibodies in human granulocytic ehrlichiosis patients. J Clin Microbiol 1998;36:1959–1963.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29.

    Batungbacal MR, Scott GR. Tick-borne fever and concurrent parainfluenza-3 virus infection in sheep. J Comp Pathol 1982;92:415–428.

  • 30.

    Foggie A. Further experiments on the effect of tick-borne fever infection on the susceptibility of lambs to staphylococci. J Comp Pathol 1957;67:369–377.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31.

    Brodie TA, Holmes PH, Urquhart GM. Some aspects of tickborne diseases of British sheep. Vet Rec 1986;118:415–418.

  • 32.

    Munro R, Hunter AR, MacKenzie G, et al. Pulmonary lesions in sheep following experimental infection by Ehrlichia phagocytophila and Chlamydia psittaci. J Comp Pathol 1982;92:117–129.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33.

    Woldehiwet Z. Depression of lymphocyte response to mitogens in sheep infected with tick-borne fever. J Comp Pathol 1987;97:637–643.

  • 34.

    Woldehiwet Z. The effects of tick-borne fever on some functions of polymorphonuclear cells of sheep. J Comp Pathol 1987;97:481–485.

  • 35.

    Larsen HJ, Overnes G, Waldeland H, et al. Immunosuppression in sheep experimentally infected with Ehrlichia phagocytophila. Res Vet Sci 1994;56:216–224.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36.

    Foster WN, Cameron AE. Observations on the functional integrity of neutrophil leucocytes infected with tick-borne fever. J Comp Pathol 1970;80:487–491.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37.

    Gokce HI, Ross G, Woldehiwet Z. Inhibition of phagosome-lysosome fusion in ovine polymorphonuclear leucocytes by Ehrlichia (Cytoecetes) phagocytophila. J Comp Pathol 1999;120:369–381.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 38.

    Strik NI, Alleman AR, Barbet AF, et al. Characterization of Anaplasma phagocytophilum major surface protein 5 and the extent of its cross-reactivity with A. marginale. Clin Vaccine Immunol 2007;14:262–268.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 39.

    Dreher UM, de la Fuente J, Hofmann-Lehmann R, et al. Serologic cross-reactivity between Anaplasma marginale and Anaplasma phagocytophilum. Clin Diagn Lab Immunol 2005;12:1177–1183.

    • Search Google Scholar
    • Export Citation
  • 40.

    Hofmann-Lehmann R, Meli ML, Dreher UM, et al. Concurrent infections with vector-borne pathogens associated with fatal hemolytic anemia in a cattle herd in Switzerland. J Clin Microbiol 2004;42:3775–3780.

    • Crossref
    • Search Google Scholar
    • Export Citation

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