Abstract
Bovine anaplasmosis, caused by the rickettsia Anaplasma marginale, is an economically important tick-borne disease of cattle that is found worldwide. Its clinical effects of severe anemia, decreased growth, weight loss, and death negatively impact cattle welfare and create a significant economic burden for cattle producers. Despite availability of highly sensitive and specific assays for anti–A. marginale antibodies (competitive ELISA) and A. marginale genetic material (real-time PCR), the interpretation of test results for the diagnosis of clinical anaplasmosis in cattle remains challenging. Treatment and control usually consist of administration of oral and/or injectable tetracyclines; however, this approach is unlikely to be sustainable in the face of increasing scrutiny of antimicrobial usage in livestock. Statistically robust prospective studies are needed to characterize the prevalence, distribution, and transmission of bovine anaplasmosis under field conditions, as the epidemiology of this disease remains incompletely understood. Apart from minimizing exposure of naïve cattle to carriers (e.g., testing new introductions and interpreting in the context of herd-level seropositivity, changing needles between cattle), veterinarians and producers have few tools for prevention of bovine anaplasmosis based on data-driven risk assessment. A vaccine that is consistently safe and effective has proved elusive, but ongoing research into A. marginale vaccine candidates offers hope for a more effective means of protecting cattle from this costly disease.
The goal of this review is to summarize our current understanding of bovine anaplasmosis within its historical context. I sought to leave no claim unverified until it was tracked as closely to its original source as possible. As a result, some references are many decades old and difficult to find on the Internet; however, the full text of all references cited in this review is available through interlibrary loan. I began by searching PubMed for review articles with the keywords “bovine anaplasmosis” or “Anaplasma marginale” within the title (22 results in 2023). After studying these articles, I identified references that were cited by multiple reviews, on the assumption that these were pivotal works; I then searched the citation lists of those articles for commonalities, and so forth. In a few topic areas, such as the history of vaccine development in the United States, it was often necessary to rely on non-peer reviewed sources such as conference proceedings and newsletter articles.
This review is intended to be accessible to diagnosticians, practitioners, and those new to the field of bovine anaplasmosis research. To the many practitioners and researchers whose work has informed the progress summarized in this review, we owe a debt of gratitude.
The Anaplasma marginale organism
One of the earliest descriptions of A. marginale was published in 1893 by Daniel E. Salmon and Theobald Smith. Salmon and Smith were studying a hemolytic disease of cattle in the American South, “Texas fever”, which hindered the trade and shipment of cattle between endemic and non-endemic areas of the United States. Their morphologic description remains accurate (“a very minute roundish body which is stained blue [. . .] near the edge of the corpuscle”), however, Salmon and Smith misidentified it as a developmental stage of Babesia bovis. 237 Sir Arnold Theiler determined that the described organism was not Babesia and named it Anaplasma marginale for its distinctive location at the edge of the red blood cell. 272 Early researchers hypothesized that A. marginale might be a protozoan or a virus. It was not until the 1960s that the organism was identified by electron microscopy as a rickettsia. 229
Anaplasma marginale (order Rickettsiales, family Anaplasmataceae) is an obligate intracellular parasite that replicates within host cell membrane-derived vacuoles, 81 specifically within the RBCs of cattle and the tissues of tick hosts. Its inability to survive outside a host cell hindered in vitro studies until a method was devised to propagate the organism in tick cell cultures. 189 Anaplasma marginale infects cattle, along with various domestic and wild ruminants, and is found worldwide (summarized in the “Epidemiology” section). It has also been detected in capybaras 9 and giant anteaters 115 in Argentina, although the significance of these findings is yet to be characterized.
Other Anaplasma species include A. centrale, A. ovis, A. phagocytophilum, A. bovis, and A. platys. Several of these organisms, most notably A. phagocytophilum, were formerly known as members of the genus Ehrlichia before re-classification in 2001 based on genetic sequencing. 81 Other genera within the family Anaplasmataceae are Ehrlichia, Wolbachia, and Neorickettsia, all of which include other vector-borne pathogens of medical and/or veterinary importance.
A. marginale has a small DNA genome of ~1.2 Mbp. 20 Six major surface proteins have been characterized:212,286 MSP1a, MSP1b, and MSP2-5. In older literature, MSP1 may be referred to as Am105 or AmF105, with the “105” suffix referring to the protein’s molecular weight in kilodaltons (kDa). Specifically, MSP1a was designated Am105U and MSP1b was designated Am105L. Similarly, MSP2 may be referred to as AmF36. MSP3 may be referred to as an 86-kDa protein; MSP4 as a 31-kDa protein; MSP5 as a 19-kDa protein.
The MSP1 complex is composed of 2 subunits: MSP1a, which is encoded by a single gene, msp1a, and MSP1b, which is encoded by at least 2 genes, msp1b1 and msp1b2. 289 Both MSP1a and MSP1b facilitate A. marginale adhesion and entry into tick midgut epithelial cells63,195 and bovine erythrocytes.62,182,194 Variable numbers of tandem repeats in msp1a are useful for distinguishing between strains of A. marginale. 66 The msp1b gene contains variable regions and regions that are highly conserved between isolates 31 ; the highly conserved sequences within msp1b make it a useful target for detection of A. marginale via PCR-based assays. 32
MSP2 and MSP3 are encoded by multigene families and exhibit antigenic variation within a single isolate over time, which is critical to the ability of A. marginale to establish persistent infection in the bovine host.5,21,99 MSP5 is highly conserved among all known Anaplasma spp., and is the target of a commercial antibody test widely used to detect seropositive cattle.145,291
Pathogenesis of infection
The only known site of replication of A. marginale in cattle is within erythrocytes, 63 in which the organism develops within host cell membrane-derived vacuoles. Once cattle are infected, the incubation period varies widely but is typically 3–6 wk; the actual length of the incubation period is inversely proportional to the infective dose. 226 The number of infected erythrocytes increases exponentially until an effective immune response can be mounted. At this point, macrophages in the spleen phagocytize infected erythrocytes, which leads to anemia without hemoglobinemia or hemoglobinuria. Cattle that recover enter a lifelong state of subclinical persistent infection in which rickettsemia levels are maintained at ≤106 infected erythrocytes/mL of blood. 87
The ability of A. marginale to establish persistent infection in the bovine host depends on the variability of its immunodominant surface proteins MSP2 and MSP3; MSP2 has been studied in more detail.21,99,209 MSP2 has a central hypervariable region flanked by highly conserved regions, 21 with B-cell epitopes located predominantly in the hypervariable region. 1 Recombination of the hypervariable region allows repeated emergence of antigenically distinct variants in a persistently infected animal, and each new variant provokes a specific antibody response. 98 This mechanism of immune evasion allows A. marginale to infect a new cohort of RBCs every 5–8 wk,87,99 leading to waxing and waning levels of the organism (“cyclic rickettsemia”) characteristic of the subclinical carrier state. Rickettsemia reaches cyclic peaks of ~106 organisms/mL of blood; each peak is followed by a rapid decline to ~102 organisms/mL, as an immune response is launched against each successive variant. 99
Host immune response
Although the bovine immune response to A. marginale has been relatively well studied, large knowledge gaps remain in our understanding of protective immunity to bovine anaplasmosis. Disease protection depends on both humoral and cell-mediated immunity.
Cattle immunized with purified outer membranes of A. marginale are protected against homologous strain challenge; however, antigenic variation in surface proteins between isolates makes resistance to heterologous strain challenge (cross-protective immunity) more difficult to achieve, 200 and outer membrane extracts are not easily standardized. 193 Specific antibodies are developed by the host against individual major surface proteins, and variable degrees of protection against clinical disease can be induced by immunizing cattle with purified MSP1,207,208 MSP2, 213 and MSP4. 211 Although naturally infected cattle develop antibodies against MSP5,145,291 immunization with this protein does not protect against experimental challenge. 211 MSP2 and MSP3 are the immunodominant surface proteins of A. marginale (i.e., the proteins toward which antibody production is skewed), 193 but there is inconsistent correlation between antibody titers and the degree of protection from challenge.29,193,196 Native outer membrane proteins are noncovalently cross-linked with distinct spatial relationships 286 and this may help to explain why purified and/or recombinant protein subunits often fail to recapitulate the protective immunity observed in cattle vaccinated with whole outer membranes of A. marginale. 193 In one experiment, immunization with recombinant outer membrane proteins (AM854, AM936) stimulated IgG responses similar to those that occurred in calves immunized with native outer membranes; however, the recombinant proteins failed to confer protection from challenge, and actually exacerbated clinical disease. 80 Additionally, T-cell and B-cell responses can be directed against different regions of the same protein, as has been demonstrated in the case of MSP2.1,196
Despite the importance of A. marginale–specific antibodies to protection from clinical disease, passive transfer of A. marginale–specific antibody does not protect against experimental challenge. 102 Various studies have demonstrated the importance of CD4+ Th1 cell responses to the ability of the host to control A. marginale infection1,27,28,30; loss of A. marginale–specific T-cell responses (“T-cell exhaustion”) may contribute to the ability of this organism to maintain persistent infection.2,119,278 Evidence for the importance of CD4+ Th1 cell responses is further demonstrated by the rapid recrudescence of rickettsemia in splenectomized calves, which occurs before a significant drop in antibody levels.11,138,161 A similar phenomenon is well-documented in humans with babesiosis 285 and malaria. 124 Much, if not most, of A. marginale antigen presentation is thought to occur in the spleen, as with other blood-borne pathogens26,169; however, the specific mechanisms by which the spleen contributes to the immune response against A. marginale are not fully understood.
Maternal antibodies do not prevent A. marginale infection in nursing calves, although they may reduce the severity of clinical disease. 302 Both the parity and antibody level (measured as optical density) of cows have been shown to predict seropositivity of their calves at ~7 weeks of age. 164 In areas with low levels of calfhood exposure, maternal antibodies may wane prior to natural exposure. 134 The complex nature of the immune response to A. marginale is also illustrated by the difficulty of vaccine development (summarized in the “Control Methods” section).
Finally, host resistance to clinical anaplasmosis is genetically determined, at least in part.34,58 Researchers in Australia found Bos indicus cattle were less susceptible to clinical anaplasmosis than were Bos taurus cattle 15 ; however, these results have not been consistently replicated by other researchers, 206 and the susceptibility of B. indicus cattle to the infection itself does not appear to be different. 295 Additional work is necessary to determine whether the benefits of using B. indicus cattle and/or crossbreds are sufficient when balanced with other factors such as reproductive performance and carcass quality.
Strain diversity
More than 130 strains of A. marginale are known to circulate worldwide, with at least 40 in the United States.66,68 Strains can be identified by variations in the number of tandem repeats in the msp1a gene, with further characterization of strain diversity (i.e., genotyping) via analysis of the unique sequences of the individual tandem repeats. A. marginale strains have also been referred to as “geographic isolates”, although extensive cattle movement detracts from the usefulness of this term. 69
It was once thought that cattle could only be infected with a single strain of A. marginale at a time.64,214 However, individual cattle have been found to be naturally infected with up to 9 unique strains at the time of testing.88,130,156,210,281 Individual cattle may also harbor different strains sequentially over time. 105 Superinfection – also referred to as coinfection or complex infection – occurs when the strains have distinct variants of MSP2100,232 and is more common in tropical regions with high transmission rates, often associated with vector transmission by the one-host ticks Rhipicephalus microplus and R. annulatus. 281
Superinfection of cattle with A. marginale that were previously vaccinated with the A. centrale live vaccine is well documented.104,249 In addition to the use of A. centrale as a live vaccine in many countries, wild-type A. centrale also circulates in some cattle populations. 130
Some strains of A. marginale are more readily tick-transmitted than others. Such strains have a competitive advantage when ticks feed on superinfected cattle, and strain-specific differences in tick transmissibility are thought to determine strain predominance in the host population.104,279 Although it was previously thought that ticks could only become infected with one strain of A. marginale at a time, 60 simultaneous acquisition of 2 strains by D. andersoni fed on superinfected calves has been demonstrated, particularly when ticks were allowed to feed for longer periods.104,168 The phenomenon of strain-specific “infection exclusion” does occur in D. andersoni ticks, but is dependent on the interval between exposures; when feedings were several weeks apart, ticks became infected with both strains. 192
In herds with a high prevalence of A. marginale infection, within-herd strain transmission appears to be determined randomly. In a herd of 261 cows with 29% infection prevalence, followed from 1998 through 2003, there was no significant difference in genotype frequency between the “overall herd” (the 75 cows characterized in 1998) and the 20 cows born and infected between 1998 and 2003. 210 The authors concluded there was no evidence for preferential transmission of a given strain and that strain transmission was stochastic. These conclusions might not be generalizable to anaplasmosis outbreaks in populations of naïve animals, or to the introduction of a novel strain into a herd. Other authors found that multiple genotypes are also involved in acute outbreaks; however, in that study, only a single genotype was found per individual animal. 7
Finally, although strain-dependent differences in virulence have been documented in at least one study, 207 this phenomenon has not been well studied. Experimental studies of strain virulence are challenging given that factors such as dose, route of exposure, and even genetic determinants of host immune response 271 would need to be controlled rigorously. Even well-designed experiments would not recapitulate the complexity of natural disease in the field, which further depends on factors such as prior exposure to A. marginale, plane of nutrition, and comorbidities.
Transmission
Biological transmission
Approximately 20 species of ticks have been incriminated, or implicated, as vectors of A. marginale worldwide.75,89,150 In the United States, biological transmission occurs primarily via Dermacentor spp. ticks, which include D. variabilis, D. andersoni, D. occidentalis, and D. albipictus.116,153,258,261,262 D. variabilis populations west of the Rocky Mountains have been proposed to represent a novel species, D. similis, which may prompt re-evaluation of vector competence of these populations for A. marginale and other pathogens. 163 D. variabilis and D. andersoni are also competent vectors of A. marginale in Canada, although bovine anaplasmosis is historically rare this far north.18,166,298 Experimental work has shown D. hunteri to be a competent vector of A. marginale 260 ; however, this tick feeds almost exclusively on desert bighorn sheep (Ovis canadensis nelson) 53 and is therefore unlikely to play a significant role in the natural transmission of anaplasmosis to domestic cattle.
Among Dermacentor spp. ticks, adult males are the main drivers of biological transmission of A. marginale, given their well-documented behavior of feeding on multiple hosts.40,173,259 Females, although susceptible, 154 typically feed on a single host and drop off once they are engorged. In nature, larvae and nymphs of 3-host Dermacentor spp. feed almost exclusively on small mammals, 13 which are refractory to infection with A. marginale73,82,83,266; therefore, their importance to the field epidemiology of bovine anaplasmosis appears to be minimal.
Although the one-host tick D. albipictus is capable of transmitting A. marginale, it is less well-studied than the 3-host ticks D. andersoni and D. variabilis.90,116 D. albipictus preferentially feeds on cervids, including moose, elk, and white-tailed deer,36,157 although it is also well-documented on horses and cattle.13,79 D. albipictus can be an important vector in certain circumstances, particularly during the winter months when other vectors are less active or inactive.
In tropical and subtropical parts of the world, A. marginale is efficiently transmitted by the one-host ticks Rhipicephalus (Boophilus) microplus and R. annulatus.49,101,238,243 These ticks are considered to be the primary biological vectors of anaplasmosis in Mexico and South America.114,190 R. microplus was also an important vector of bovine anaplasmosis in the United States before its eradication in the 1940s, and its potential reintroduction via Mexico and/or the Caribbean is a constant concern, especially given its role as a vector of bovine babesiosis. 101 The brown dog tick, R. sanguineus, can also transmit A. marginale.215,248
Two other common North American ticks, the lone star tick 110 (Amblyomma americanum) and the blacklegged tick 151 (Ixodes scapularis), have not been shown to transmit A. marginale. Likewise, the Asian longhorned tick (Haemaphysalis longicornis) is not a competent vector of A. marginale.48,122
The 2 main determinants of vector competence for bovine anaplasmosis are 1) the ability of a tick to be infected by A. marginale at the level of the midgut, and 2) the ability of A. marginale to replicate within and be transmitted from the tick salivary gland to the next ruminant host.87,101,280 The feeding events associated with these developments are typically referred to as “acquisition feeding” (a susceptible tick feeding on an infected host) and “transmission feeding” (an infected tick feeding on a susceptible host).
The degree to which A. marginale will persist in tick populations depends on the frequency of exposure of ticks to infected reservoir hosts and whether the infection can be transmitted between stages (i.e., transstadial transmission from nymphs to adults) and/or between generations of ticks (i.e., transovarial transmission from adult females to their offspring). Transovarial transmission of A. marginale has not been demonstrated in Dermacentor spp. ticks,258,263 but has been demonstrated in R. microplus. 59 In most of the United States, then, the maintenance of an infective tick population depends on continuous re-exposure to persistently infected cattle (reservoir hosts).
Development of A. marginale in the tick begins in the midgut, followed by migration to and replication in the salivary glands.87,101,146,155 This cycle is complex and coordinated with tick feeding behavior. For example, replication of A. marginale in the tick midgut can be stimulated experimentally by incubation at 37°C for 2–3 d, which is thought to recapitulate the conditions experienced by ticks attached to a warm-blooded host. 149 Replication within the salivary glands occurs during the tick’s subsequent meal (transmission feeding). This process was successfully stimulated by in vitro incubation in one study 152 ; another study did not identify salivary gland infection until ticks were allowed to take a second blood meal. 106 Intriguingly, ticks fed A. marginale–infected blood in vitro via capillary tube developed infection of the midgut, but did not develop infection of the salivary gland when fed on a subsequent host, indicating that host attachment during the acquisition feed is necessary for completion of the A. marginale developmental cycle. 147
Acquisition of A. marginale infection by competent tick vectors is efficient, even when ticks feed on cattle with low level rickettsemia. Adult male D. andersoni ticks can acquire A. marginale infection in as little as 2 d.87,103 Adult male Dermacentor spp. ticks fed for 6–7 d on infected calves acquire infection 27–96% of the time, depending on the level of rickettsemia.101,240 Replication within the salivary gland occurred whether the ticks were initially fed at high or low levels of rickettsemia. Adult male R. microplus ticks, after acquisition feeding for 1 wk on persistently infected calves with microscopically undetectable levels of rickettsemia, acquired infection 92% of the time. 101
Dermacentor spp. ticks that are actively infected have a high efficiency of transmission. As few as 1–5 infected Dermacentor spp. ticks are sufficient to transmit anaplasmosis when fed on a naïve calf.87,242 Transmission feeding by adult D. andersoni males has been found to require 6–7 d, 148 with the result that most subsequent transmission studies have used a transmission feeding period of at least 6 d. Adult D. andersoni fed via a silicone membrane–based in vitro system began secreting A. marginale on day 5 of transmission feeding, provided they were exposed to a high level of rickettsemia during acquisition feeding, 288 which may suggest the possibility of in vivo transmission with <6 d of tick attachment under conditions of high pathogen load.
Most vector competence experiments have involved the use of splenectomized recipient calves; however, lab-raised adult male D. andersoni ticks acquired and transmitted A. marginale from a mature, spleen-intact, persistently infected cow in Idaho to 2 mature, spleen-intact, naïve herdmates. 301 In another study, 100% of 35 wild-type adult male D. andersoni ticks collected from a naturally infected cow with clinical disease were infected with A. marginale at the level of the midgut, and transmitted disease to a splenectomized calf after feeding for 5 d. 86
Not all strains of A. marginale are readily transmissible by ticks.241,254,293 Some, such as Florida and Mississippi isolates, are unable to establish infection at the level of the tick midgut.63,280 A. centrale was shown to infect the midgut and replicate to high levels within the salivary gland, but was not transmitted when 100 infected D. andersoni ticks were fed on a naïve calf 280 ; interestingly, this barrier was overcome when 425 A. centrale–infected ticks were fed. 279 In contrast, the St. Maries strain of A. marginale can be transmitted by a single infected D. andersoni tick. 242 The molecular basis for these strain-specific differences in tick transmissibility remains to be elucidated. In the case of the Florida strain, the inability of A. marginale to infect the midgut epithelium has been attributed to a defect in the structure of MSP1a.61,63 However, A. centrale readily infects the tick midgut, 280 even though its msp1a homolog is significantly different from that of A. marginale. 144 Survival of A. marginale within midgut epithelium depends on its ability to escape digestion by altering the characteristics of its host-derived vacuole (e.g., by maintaining a neutral pH, preventing fusion with lysosomes, and altering the expression of surface markers associated with endosome maturation). 177 This ability to create and maintain a safe replicative niche may help to explain strain-specific differences in ability to infect the midgut.
R. microplus ticks infected with “tropical” (Puerto Rico) and “temperate” (St. Maries) strains of A. marginale showed no significant differences in infection level within the midgut; however, there was significantly enhanced replication of the Puerto Rico strain within the salivary gland, which was considered strong evidence of strain adaptation to its local vector. 101 Even when the A. marginale strain is held constant, tick susceptibility at the level of the midgut has also been shown to vary among geographically separated subpopulations of D. andersoni ticks. 244
Finally, the tick midgut microbiome (e.g., presence of certain endosymbiotic Rickettsia spp.) has been shown to affect the susceptibility of ticks to infection with A. marginale.10,103 Clearly, much remains to be learned about the complex interactions between A. marginale and its tick vectors.
Mechanical transmission
In circumstances in which the predominant strain of A. marginale is not capable of infecting ticks, or when there is little tick activity, 253 mechanical transfer of infected erythrocytes is a major route of transmission. Anaplasma marginale can be transmitted mechanically by blood-contaminated needles54,223 or veterinary instruments, 127 and is thought to be transmitted by various biting flies when conditions are favorable.
Sham vaccination of a heavily infected steer (2% of erythrocytes parasitized), followed by sham vaccination of a naïve steer immediately afterward, resulted in iatrogenic transmission of A. marginale in 6 of 10 trials. 223 While this clearly demonstrates the risk of iatrogenic transmission from an acutely infected animal with a high level of parasitemia, the actual risk in field conditions will depend on a variety of factors, including the proportion of acutely vs. persistently infected cattle in the herd and the level of rickettsemia at the time of vaccination. In the same study, the use of a needle-free pneumatic injection device was associated with significantly decreased risk of iatrogenic transmission. 223
Biting flies, particularly horse flies, have been implicated as mechanical vectors of bovine anaplasmosis. 235 Unlike biological transmission by ticks, however, the efficiency of mechanical transmission is highly dependent on the level of rickettsemia in the infected host. Rigorous studies demonstrated that mechanical transmission by stable flies (Stomoxys calcitrans) 240 and horse flies (Tabanus fuscicostatus) 242 were each at least 100 times less efficient than biological transmission by D. andersoni ticks. While these results prompt skepticism regarding the relative importance of biting flies to the field epidemiology of bovine anaplasmosis, it is worth noting that the flies were transmission fed in groups of ≤30 flies per host, whereas in nature cattle may be attacked by hundreds or even thousands of flies daily. In such conditions, it seems plausible that the inefficiency of fly-borne transmission could sometimes be overcome by the sheer number of bites.
Vertical transmission to calves, and its relation to abortion
In utero transmission of A. marginale has been documented269,299; however, its role appears to be relatively minor at the herd level. 216 In utero infection has been identified in 6–17% of calves born to A. marginale–infected cows.52,83,113,218,302 Infected calves were frequently subclinical, and infection was confirmed by splenectomy and/or subinoculation of blood into recipient calves. In most reports, infected calves were born to dams that experienced acute infection during pregnancy and subsequently recovered. In one report, 15 of 60 calves (25%) were positive for A. marginale via PCR at birth; 3 of these newborn calves had microscopically detectable parasitemia of 0.1–2%, and each was markedly anemic and died within 24 h of birth. 52
While abortion is frequently cited as a feature of bovine anaplasmosis outbreaks, abortion and/or stillbirth directly attributable to in utero infection with A. marginale has been documented only rarely.51,123,225 Most published reports involve single-digit sample sizes, and the specific identity of the organisms as A. marginale was not confirmed in all reports. It therefore seems likely that most abortions associated with bovine anaplasmosis are the result of maternal disease rather than direct infection of the fetus. Diagnosis usually relies on clinical signs of hemolytic anemia in the dams and an absence of other explanatory findings in the fetuses. 94 I did not find any population-based studies comparing the incidence of abortion between cattle with and without anaplasmosis, whether determined via serology or PCR.
Epidemiology
Estimates of bovine anaplasmosis incidence, prevalence, morbidity, and mortality are challenging to glean from the literature. The practical consequences of “incidence” will change depending on whether first-time exposures are occurring in young calves or naïve adult cattle. Few prospective herd-level studies have been conducted.
Incidence has been estimated using survey responses from producers and veterinarians, but since this relies on recall of clinically affected cattle, these estimates are prone to bias and tend to underestimate incidence among young calves, in which clinical signs are unlikely to be noticeable. Hence, these estimates of “incidence” are in reality closer to estimates of morbidity (e.g., California, 1.85% 112 ; Texas, 0.28% 3 ; Louisiana, 0.31% 187 ).
Prevalence is comparatively easy to estimate with cross-sectional studies, but this will generally reflect prevalence of persistently infected animals (i.e., the subclinical carrier state). Most such animals are apparently healthy and protected against subsequent disease if re-exposed. Finally, mortality estimates should be interpreted with caution, as it is not always clear whether the authors are referring to anaplasmosis-attributable mortality at the herd level (e.g., 0.75% 112 ) or case fatality rate (e.g., 35.9% 3 ; 53.0% 187 ).
A. marginale principally infects cattle, along with various wild ruminants, including bison (Bison bison), elk (Cervus canadensis), white-tailed deer (Odocoileus virginianus), and mule deer (Odocoileus hemionus). American bison appear to be capable of serving as reservoirs. 65 Although wild cervids are susceptible to infection with A. marginale and mount an effective antibody response,175,255 the level of parasitemia is extremely low in experimentally infected animals and their role as reservoirs for infection of cattle appears to be minimal,143,300 with the possible exception of black-tailed deer (Odocoileus hemionus columbianus). 159 In a survey of free-ranging elk in Tennessee, all 27 elk were seropositive for A. marginale via competitive ELISA (cELISA); however, none of these elk were positive via real-time PCR (rtPCR), 158 suggesting that their infections were below detectable levels, had been transient, or the detected antibodies were against a different Anaplasma sp.
Sheep inoculated with A. marginale become subclinically infected, and inoculation of blood from infected sheep into susceptible cattle will produce disease;74,228 however, the role of sheep in field conditions has not been well studied and is thought to be minimal.
Young calves, while equally susceptible to infection with A. marginale, are much less likely to develop clinical signs than are adult and aged cattle; prepubertal calves reached similar nadirs of PCV but had a significantly lower percentage of parasitized erythrocytes and recovered more quickly. 140 Older cattle are more likely to be seropositive, which is likely due to increased likelihood of eventual exposure with age.188,201 -204
Anaplasmosis occurs with a markedly higher frequency in beef cattle than in dairy cattle.134,188,204 This is generally thought to be the result of dairy cattle spending most of their time in confinement, where tick exposure is presumptively minimal. Outbreaks of anaplasmosis have been reported in dairy herds, associated with shared needles. 54 In the United States, most detections in beef cattle are in late summer through late fall.201,203,204,287
Transmission is suggested to increase after periods of increased rainfall.136,216 Whether this is due to increased tick survival, the presence of other arthropods such as biting flies, or whether season happens to coincide with management practices that favor transmission remains to be investigated.
Prevalence and geographic distribution
Bovine anaplasmosis is distributed globally, with overall country-level prevalence estimates of 33–42%. 197 It is endemic in Latin America and the Caribbean, where R. microplus and R. annulatus are major vectors.136,164 Seroprevalence estimates include 56–77% in Mexico, 95 75–88% in Costa Rica,126,247 and 27–30% in Puerto Rico.97,283 A meta-analysis estimated an overall prevalence in South America of 48.9% (95% CI: 30–68%), with a particularly high prevalence in northern Brazil (71.9%). 95
Bovine anaplasmosis as a disease does not appear to have been recognized in the United States before the early 1920s, as clinical cases were conflated with bovine babesiosis (“Texas cattle fever”). As eradication efforts progressed and cases of babesiosis decreased, veterinarians recognized sporadic outbreaks of hemolytic anemia in herds free of cattle fever ticks, and which were not accompanied by hemoglobinuria. Confirmed reports of bovine anaplasmosis began to appear in the United States veterinary literature in the mid-1920s.57,73,109
No national survey of bovine anaplasmosis in the United States has been reported since 1972. 180 Regional and statewide surveys must be compared with caution, given differences in time period, sampling strategy, and test method(s) used. Many of the following studies are based on samples that are selected non-randomly. Changes in test methodology also demand cautious interpretation of the literature. Although the following paragraphs do not represent statistically robust estimates of prevalence, they may provide valuable points of reference and illustrate the volume of work that has been done in an attempt to understand the impact of bovine anaplasmosis in the United States. Studies are summarized and listed alphabetically by state to facilitate easy reference (Table 1).
Summary of bovine anaplasmosis prevalence estimates from studies conducted in the United States, listed alphabetically by state.
CAT = card agglutination test; cELISA = competitive ELISA; CFT = complement fixation test; IFAT = indirect fluorescent antibody test; Se = sensitivity; Sp = specificity.
In a retrospective study of over 65,000 serum samples submitted to AAVLD-accredited veterinary diagnostic laboratories in the southern United States from 2002–2012, apparent seroprevalence was 18.8% overall, 5.1–56% by state. The same authors conducted a cross-sectional sampling of 977 beef cows at slaughter from 7 southeastern states and found a seroprevalence of 13% overall, 2.4–35.2% by state. 292 In a 2006–2007 serosurvey of California beef herds, prevalence was lowest in the Central Valley, which the authors attributed to a lack of vectors (presumably the result of the arid climate). 129
In a 1982–1984 serosurvey of bovine anaplasmosis in Idaho, between-herd seroprevalence in the 10 northernmost counties was lower (7.8%) than in the 11 southernmost counties (43.3%). Seroprevalence also tended to decrease as elevation increased. While this could suggest that higher elevations tend to support lesser tick populations, it could also be a proxy for other unmeasured variables, such as stocking density of cattle or likelihood of new introductions. 303
Using diagnostic laboratory records of anaplasmosis cases, steady increases of anaplasmosis were found in Kansas since 2005. 120 Prospective sampling of 925 Kansas cow-calf operations 2016–2017 found an overall between-herd prevalence of 52.5%, with higher proportions of positive herds in the eastern portion of the state. 257 Two serosurveys in Louisiana, each including beef and dairy cattle, found at least 1 positive animal in >50% of herds sampled.133,188
In 2 surveys of Texas beef cattle,116,117 seroprevalence tended to be higher in the western portions of the state. Interestingly, in a 1980 survey of producers and veterinarians, clinical cases of anaplasmosis were most often reported in the eastern and northeastern portions of the state. 3 This may reflect actual changes in distribution over space and time, or it may be that higher proportions of seronegative (i.e., susceptible) cattle in these regions led to a higher frequency of clinical disease.
Endemic stability
Endemic stability refers to a state of tick-pathogen-host equilibrium in which the cattle population has a low incidence of clinical disease, despite a high prevalence of persistent infection. A sufficiently high exposure rate in young calves, which have innate resistance to clinical disease, creates a population in which a large proportion of adult cattle are carriers with protective immunity. 178 This balance can be upset when carrier cattle and naïve cattle are co-mingled; when calfhood exposure decreases; or, uncommonly, when antimicrobial treatment of carrier cattle causes reversion to a susceptible state. 222 Calving season may also influence the rate at which young calves are exposed to ticks and become infected with A. marginale. 216 Finally, strain-dependent differences in tick transmissibility and, potentially, virulence 207 contribute to the difficulty of estimating risk related to movement of cattle between endemic and non-endemic areas.
Although it has been applied to the management of bovine anaplasmosis risk, the concept of endemic stability was first developed based on studies of Babesia bovis transmission by R. microplus. 178 The model depended on the number of ticks, the proportion of ticks infected with the organism of interest, and the proportion of tick bites that result in successful transmission to the host. The validity of direct extrapolation of this model to A. marginale has been questioned. 141
Researchers in Brazil provided evidence to support the usefulness of the endemic stability model with regard to bovine anaplasmosis. Thirty-six cow-calf farms were classified as “cases” (n = 13) or “controls” (n = 23) based on the presence or absence, respectively, of confirmed clinical anaplasmosis cases in the preceding 2 y. Testing of 20 cattle on each farm found that 590 of 720 (81%) of individual cattle were seropositive, and within-herd seroprevalence was 25–100%. Farms with >75% seroprevalence were significantly less likely to be classified as “cases” (i.e., to have observed clinical cases in the preceding 2 y). Twelve months after the initial testing, the researchers conducted a follow-up survey to identify clinical cases of anaplasmosis. Farms with <75% seroprevalence were significantly more likely to have experienced an outbreak in the year following the initial testing (odds ratio 7.5; 95% CI: 1.27–44.1). 165
In California, a prospective study of 143 cattle intentionally moved to tick-infested pastures for exposure of young cattle to A. marginale found that seroprevalence among heifers was significantly lower than that of cows at the beginning of the study (heifers, 22.5–42.4%; cows, 86.5%) but not at study completion (heifers, 67.5–89.1%; cows, 91.9%). 277 No illness consistent with anaplasmosis was reported in any of the study cattle.
Given that a tick is actively infected at the time that it feeds on a susceptible host, the efficiency of transmission is very high.87,242 Therefore, the frequency of competent vector ticks feeding on cattle and A. marginale prevalence among ticks are expected to explain most of the variation in biological transmission rates in the field.
Contemporary estimates of tick activity on cattle are limited. In a survey of 3,087 ticks collected from cattle in Arkansas in 2020–2022, A. americanum accounted for >95% of specimens; D. variabilis accounted for ~1%. 22 In a survey of 740 ticks collected from cattle in Tennessee in 2015–2016, D. variabilis accounted for 4.7% of specimens. 273 At least in the southern United States, Amblyomma spp. appear to greatly outnumber D. variabilis ticks on cattle, although the degree to which this affects anaplasmosis transmission is not known.
The prevalence of A. marginale among vector ticks is not well described, despite its importance to the epizootiology of bovine anaplasmosis. In a 1976 study in Oregon, wild-type adult D. andersoni ticks were collected from pastures of an anaplasmosis-endemic cattle herd. Ticks were immediately placed on 2 spleen-intact calves from May–July 1975. One of the calves became infected with anaplasmosis, proving the possibility of transmission by D. andersoni in field conditions; however, a cumulative 237 adult ticks of both sexes were allowed to feed on the calf; hence, the actual proportion of infected ticks was not determined. 252
Many studies have failed to detect A. marginale by PCR in host-seeking Dermacentor spp. ticks collected from pastures in Tennessee, 217 Oklahoma, 191 the Intermountain West region of the United States, 244 and southern Canada,37,71,244,298 including regions where anaplasmosis is known to be endemic. As other authors have noted, this supports the prevailing theory that adult male ticks acquire and transmit A. marginale 244 ; if ticks became infected as nymphs, one would expect to find at least a few naturally infected adults. Clearance of A. marginale by infected male ticks (i.e. transient infection in the vector) also seems unlikely since experimentally infected D. andersoni adult males remained capable of transmitting A. marginale for 120 d, provided the humidity was sufficient to ensure their survival. 39 The lack of A. marginale detection in host-seeking adult males collected from vegetation by flagging or dragging further supports the idea that male ticks are likely to transfer between cattle through direct host-to-host contact (e.g., social behaviors such as grooming or huddling), rather than dropping off and questing for a new host. While this mode of direct host-to-host transfer has been speculated,40,259 it has not been demonstrated conclusively.
Changes in distribution of tick vectors
Climate change is expected to have significant impacts on the distribution of arthropod vectors and vector-borne diseases in both animals and humans. 142 The recent incursion of bovine anaplasmosis into Canada, where the disease was virtually nonexistent historically,18,132 is a possible example. Since the 1950s–1960s, D. variabilis has expanded its northern and western distribution limits in southern Canada, while the range of D. andersoni has remained relatively stable. 72 In Canada, bovine anaplasmosis was an “immediately notifiable” disease before 2014. Imported cattle from the United States were subject to stringent requirements 284 and local outbreaks were immediately investigated and followed with mandatory culling of infected cattle. The Canadian Food Inspection Agency removed anaplasmosis from its list of immediately reportable diseases in 2014, on the premise that continued attempts at eradication were no longer cost-effective and that climate change made additional incursions nearly inevitable. 256
In addition to the expanding distributions of Dermacentor spp. ticks,4,17,167,183 ticks may become active earlier in the calendar year and remain active later when temperatures are warmer than historical levels, 220 creating additional opportunities for disease transmission. In Canada, 39% and 20% unfed adult D. variabilis collected in May were capable of surviving the Manitoba winter until January and April, respectively. 297 In a few small experiments, D. andersoni males have overwintered on cattle in Canada, remaining attached and capable of successful mating for up to 178 d. 294 In another study, field-collected overwintered D. andersoni adult males failed to transmit A. marginale when fed on splenectomized calves, and A. marginale was thought to be unlikely to overwinter in the tick vector 239 ; however, given the apparently low prevalence of A. marginale in host-seeking Dermacentor adults, it seems likely that these ticks were uninfected to begin with. Ultimately, the ability of A. marginale to overwinter in unfed ticks has not been demonstrated.
R. microplus and R. annulatus, the cattle fever ticks, have been largely excluded from the United States since 1943 due to rigorous eradication effort. A narrow “quarantine zone” along the Texas–Mexico border is regularly patrolled by USDA and Texas Animal Health Commission (TAHC) employees on horseback—“tick riders”—to search for stray or smuggled livestock that may be carrying the ticks.108,234 Although the USDA’s primary interest in cattle fever ticks is related to their role as the vectors of bovine babesiosis, 282 these ticks are also efficient vectors of bovine anaplasmosis. The difficulty of excluding cattle fever ticks from the United States is increased by climate change, political instability, and growing numbers of alternative hosts in Texas such as nilgai (an introduced species of Asian antelope) and feral swine.205,274
Economic impact
Anaplasmosis is widely quoted to cost the U.S. cattle industry >$300 million annually, although this estimate is based on expert opinion rather than empirical data. 180 Accurate estimates of economic impact are extremely difficult to obtain. Accurate data on the incidence and distribution of clinical cases, and detailed records within individual herds, would be required to accurately estimate economic impact. Many studies are based on interviews with farmers and ranchers, whose responses are used to calculate dollar amounts based on current market prices. Such studies are prone to recall bias and may quickly become outdated. Still, it is generally accepted that production losses due to bovine anaplasmosis represent a significant economic burden for cattle producers in the United States and around the world.
Financial costs attributed to bovine anaplasmosis have included deaths of adult cattle, devaluation of cattle (e.g., culling due to weight loss and/or reproductive failure), abortions, and the expenses associated with treatment and control. In 1976, anaplasmosis was estimated to cost the California beef cattle industry >$5.2 million annually, 112 based on an anaplasmosis-specific death rate of 0.75% in beef cattle, a 3.7% decrease in calf crop, and a 30% increase in culling. These estimates are still used in contemporary models. 221 Additional costs included veterinary fees, treatment costs, vaccination, and vector control.
A survey of 499 Texas cattle producers in 1980 found an average estimated cost of $424 per clinical case, which was extrapolated to an estimated statewide loss of >$6.3 million annually. 3 Producers were asked to recall the frequency of deaths, weight loss, abortions, and treatment of cattle for anaplasmosis. Economic impact was estimated by applying 1980 market prices to cattle lost (deaths, abortions) or devalued (weight loss) due to anaplasmosis. Control methods, including low-level chlortetracycline in the feed, vector control, and vaccination, yielded a higher return on investment in areas where the incidence of clinical cases was high.
Based on survey responses from 427 beef cow-calf producers in 1983, anaplasmosis-attributable losses in Louisiana were calculated from an annual incidence of 0.31% in cows, a case fatality rate of 53% in cows, and a survivor culling rate of 6.4%186,187; 1983 market prices were then applied to these figures to estimate an overall loss of nearly $500,000. Additionally, the model included an estimated 26% reduction in milk production by lactating dairy cows 181 and the costs of antimicrobial treatment, vaccination, and various insecticides used for vector control.
An average loss of $660 per clinical case of anaplasmosis 221 was estimated using a stochastic computer model that incorporated parameters for morbidity, mortality, and treatment costs based on earlier published reports,3,112,186 based on market prices and U.S. dollar values from 2016. Additional studies are warranted to determine whether these economic cost estimates are accurate.
Most studies of economic impact have focused on the effects of clinical cases of anaplasmosis. Fewer studies have explored the possible economic impact of subclinical infections. A study of 659 feedlot calves in Iowa found no significant associations between A. marginale seropositivity and production variables (e.g., days on feed, average daily gain), morbidity, mortality, treatment costs, or carcass traits 47 ; however, this study lacked sufficient sample size to achieve a statistical power of 0.80.
Bulls with acute clinical anaplasmosis have testicular degeneration, loss of libido, 268 and sperm abnormalities, leading to inability to pass a breeding soundness examination (BSE) for up to 16 wk. 171 A follow-up study of 535 client-owned bulls in Kansas found no significant association between A. marginale subclinical infection and unsatisfactory BSE results, 135 which could suggest that the bulls were infected during calfhood, or that most bulls that recover from infection as adults will return to breeding soundness. Nevertheless, given the high investment involved in bull purchases, the economic impact of introducing a naïve bull into an endemic herd can be significant.
Investigation of an Iowa dairy herd following an anaplasmosis outbreak in 2010 found that cows seropositive in 2011 (38% of the herd) produced, on average, 10–15% less milk than seronegative cows in 2012 and 2013 lactations. 54 Data on the effects of anaplasmosis on milk production in beef cows is not available, but potential effects on calf weaning weights could be significant. Additional studies are needed to determine the impact of subclinical anaplasmosis on production outcomes.
Diagnosis
In the United States, a presumptive diagnosis of bovine anaplasmosis is often made on the basis of clinical signs: lethargy, labored breathing, aggressive behavior, and/or death with pallor and icterus of the mucous membranes. 50 Hemoglobinuria is not a feature of anaplasmosis. Anemia can be confirmed by measurement of the animal’s PCV. In acute cases, A. marginale organisms are identifiable on blood smears, 77 although, in practice, blood smears are not often obtained.
This empirical approach to anaplasmosis diagnosis is less reliable in parts of the world where other hemolytic diseases, such as babesiosis and theileriosis, are also endemic. Following the recent emergence of Theileria orientalis Ikeda genotype in the United States,76,198,199 laboratory confirmation is likely to become more important given that anaplasmosis and Theileria-associated bovine anemia are clinically indistinguishable in most animals.
In the setting of experimental research, the subinoculation of whole blood from infected cattle into splenectomized cattle served as the gold standard for the determination of A. marginale infection status prior to the development of highly sensitive nucleic acid–based tests. 224 Determination of A. marginale infection status is now based on specific PCR tests.
Accurate diagnosis of clinical bovine anaplasmosis cases remains difficult in many situations. Safe collection of blood samples from beef cattle generally requires handling and restraint in a squeeze chute, which may not be tolerated by severely affected animals. Postmortem diagnosis is usually based on a combination of characteristic gross lesions (e.g., pallor and/or icterus, splenomegaly), an absence of other explanatory findings, and a rtPCR result with a low Ct value. Except perhaps in cases of exceptionally low Ct values, a positive PCR result alone is not sufficient evidence that an animal died of anaplasmosis, given that the proportion of subclinical carriers is high in many populations.
Antibody-based tests
Antibody testing is an important tool in bovine anaplasmosis diagnosis and management. For most diseases, an antibody titer is an indication of prior exposure but does not necessarily indicate current infection. In the case of anaplasmosis, however, the great majority of infected cattle do not clear the infection but remain persistently infected. Hence, antibody testing is a reliable method of identifying persistently infected carrier cattle.
A commercial and widely used cELISA (VMRD; https://vmrd.com/test-kits/detail/anaplasma-antibody-test-kit-celisa-v2) uses a monoclonal antibody against MSP5, which is conserved in all known Anaplasma species.145,291 The cELISA has a reported sensitivity of 94.8–99.2% and specificity of 95–99.5%.38,46,185 Cross-reactivity occurs between A. marginale and A. phagocytophilum,78,265 although this is usually of minor concern because A. phagocytophilum infection is rare in U.S. cattle. 84 Similarly, this assay does not distinguish between A. marginale and A. centrale. 12
The card agglutination test (CAT) is another serologic test that has been widely used. Under tightly controlled conditions, the CAT can achieve sensitivity and specificity comparable to the cELISA 185 ; however, significant technical challenges to its reproducibility have led to the CAT being largely supplanted by the cELISA. 296
The complement fixation test (CFT) was introduced in the early 1950s and was used extensively for many years. 235 However, after the advent of nucleic acid–based methods, this test was found to have a sensitivity of just 20–26.5%.19,46 The CFT is no longer recommended.
Although the cELISA is a reliable test for the identification of carrier animals, practitioners and diagnosticians should keep in mind that clinical signs are not always consistent with a positive antibody test. Especially for postmortem cases in which clinical anaplasmosis is suspected, a rtPCR test would be more useful, so that the Ct value can be correlated with an approximate degree of parasitemia (i.e., to determine whether the animal likely “died of” or “died with” the infection).
Also, cattle in the acute stage of infection may be PCR positive without having developed a detectable antibody response. One study of experimentally infected steers found that the cELISA had a sensitivity of 47.5% at 9 d post-infection, which increased to 100% at 13 and 20 d post-infection. 46 In another study, reverse-transcription quantitative real-time PCR (RT-qPCR) detected Anaplasma marginale in experimentally infected calves up to 15 d before cELISA. 116
Nucleic acid–based tests
The earliest nucleic acid–based assays published for the detection and quantification of A. marginale were southern blot techniques using cloned DNA probes.111,290 Assays based on the msp1b gene successfully detected A. marginale in ticks 111 and bovine blood,85,107 with vastly improved sensitivity relative to that of light microscopy.
Detection and quantification of A. marginale rickettsemia was later accomplished using PCR based on the msp5 gene.99,118 However, since msp5 is highly conserved among all Anaplasma species, this assay does not distinguish between A. marginale and other members of the Anaplasma genus. 249 Other broad targets such the msp4 gene 67 and the heat-shock protein gene groEL70,267 are useful for screening and sequence comparison, as they are fairly well conserved across the Anaplasma genus, but are less ideal if detection of A. marginale is the primary goal. 251
A reverse-transcription PCR (RT-PCR) assay was later described which targets 16S rRNA. This assay offers high sensitivity because rRNA is present in higher copy numbers than DNA; however, this assay will amplify both A. marginale and A. phagocytophilum. 224
A rtPCR assay designed to detect the msp1b gene of A. marginale is highly sensitive and specific for the detection of A. marginale in infected bovine blood samples. 32 It does not cross-react with A. centrale, A. bovis, A. ovis, A. phagocytophilum, B. bovis, B. bigemina, T. annulata, or T. buffeli. This assay has also been incorporated into a duplex rtPCR assay designed to detect and distinguish between both A. marginale and A. centrale in countries where A. centrale is used as a live vaccine, 35 as well as in a duplex rtPCR assay to simultaneously detect A. marginale and T. orientalis. 199
Finally, nested PCRs have been developed against msp1b and msp5.184,276 Nested PCR is highly sensitive but is not recommended for routine diagnostic applications as the risk of cross-contamination is greatly increased. 296
The decision to use serologic vs. nucleic acid–based assays for A. marginale testing will depend on the diagnostic and/or research questions to be answered. The cELISA is far less sensitive than PCR in acutely infected cattle,46,223 whereas the sensitivity of PCR tends to decline in animals that have been persistently infected for long periods. 126 If warranted, the use of both tests can increase the accuracy of classification of individual animals and/or herds. 116
Control methods
Antimicrobials
Injectable formulations of oxytetracycline are the only antimicrobials currently approved for the treatment of bovine anaplasmosis in the United States (https://animaldrugsatfda.fda.gov). These drugs are effective treatments that ameliorate clinical signs and improve survival when given early enough in the course of disease,91,246 but none will completely eliminate A. marginale infections when administered at label doses, nor will they prevent infection when administered prophylactically.42,43,45,55,170
Injectable enrofloxacin (Baytril 100-CA1; Bayer) received conditional approval from the FDA in 2020 for the treatment of clinical anaplasmosis in replacement dairy heifers <20-mo-old and all beef cattle except calves <2-mo-old and bulls intended for breeding (regardless of age). However, the application for full approval was eventually withdrawn by the sponsor and the product was taken off the market by early 2023. While studies show that enrofloxacin is an effective treatment for bovine anaplasmosis,42,91,246 it is unclear whether it is meaningfully superior to oxytetracycline (which is less expensive), and this may have resulted in disappointing sales for this product.
Oral chlortetracycline in medicated feed and mineral is approved for the “control of active infection” with A. marginale in cattle and has been regulated by a Veterinary Feed Directive since January 2017. These products are not intended to eliminate infection, but rather to minimize clinical disease in at-risk cattle. Oral chlortetracycline administered at the label dose (1.1 mg/kg bodyweight) in hand-fed medicated feed for 60 consecutive days had no significant effect on rickettsemia. 55 Proposed explanations for this included strain-dependent variability in susceptibility to chlortetracycline, inconsistent delivery of the drug, and/or the development of antimicrobial resistance (AMR).
Similarly, although injectable oxytetracycline administered subcutaneously at the label dose (19.8 mg/kg bodyweight) 3 times at 3-wk intervals caused statistically significant decreases in rickettsemia compared to untreated controls, these decreases were transient, and levels quickly rebounded after each treatment. At the end of the 60-d study period, rickettsemia had either returned to or exceeded baseline levels before treatment. 55
Although several studies in the older literature apparently demonstrated clearance of A. marginale with injectable oxytetracycline at doses of 11 – 22 mg/kg bodyweight given at various dosing intervals,176,230,233,270 these studies are not easily compared given differences in methodology, study populations, and strain(s) of A. marginale tested. Additionally, the effectiveness of antimicrobial treatment is lessened when cattle are continually re-exposed.160,222
Successful chemosterilization of steers occurred with in-feed chlortetracycline at doses of 4.4 mg/kg/d, 11 mg/kg/d, and 22 mg/kg/d when top-dressed over the ration twice daily for 80 consecutive days. 222 Chemosterilization was confirmed with a negative RT-PCR assay designed to detect 16S rRNA, a negative cELISA, and lack of disease transmission via subinoculation of blood into splenectomized calves. Although this study demonstrates the possibility of eliminating persistent infection from valuable individual animals, such a feeding regimen would be impractical for implementation at the herd level.
Older studies25,227 which define “elimination” or “prevention” of persistent infection with oral chlortetracycline as a negative response in the CFT should be interpreted with skepticism given the low sensitivity of the CFT. 19 Subinoculation of blood from treated cattle into splenectomized calves was and still is considered to be the “gold standard”; however, for ethical and practical reasons, the sample sizes were usually quite small and thus the probability of inadequate statistical power should also be considered.
Based on conflicting reports of treatment effectiveness in different geographic regions of the United States, differences in antimicrobial susceptibility between isolates have been hypothesized, but this has never been demonstrated conclusively. Researchers in Argentina found that chemosterilization with oxytetracycline eliminated some msp1a genotypes but not others, suggesting genotype-dependent differences in antimicrobial susceptibility. 219 Several genes associated with AMR have been identified in various isolates of A. marginale; however, the significance of these findings is not well characterized.20,245 Besides the hypothetical development of AMR among A. marginale isolates, however, producers and veterinarians should consider the possibility of off-target effects, as there is evidence to suggest that long-term administration of chlortetracycline can increase the prevalence of tetracycline resistance among fecal E. coli isolates from treated cattle. 275
Finally, studies of antimicrobial susceptibility in A. marginale have historically proved difficult, as the obligate intracellular nature of this organism precludes most standard in vitro culture techniques. 44 In vitro treatment of A. marginale–infected Ixodes scapularis cell cultures has been proposed as a method of screening for strain-dependent differences in antimicrobial susceptibility, 8 but the degree to which this technique can be extrapolated to in vivo treatment of live animals is debatable.
Vaccination
In tropical and subtropical parts of the world, A. centrale has been used as a live vaccine for many years with relative success.23,236 In most cattle, A. centrale causes mild signs and, although it does not prevent infection with A. marginale, it protects against severe disease.249,250 However, A. centrale can occasionally cause severe disease, and vaccine failures have been reported.33,236 Because the A. centrale vaccine is a blood-derived product, it is not approved in the United States or European Union due to the risk of contamination with other blood-borne pathogens. 118 A subunit vaccine derived from the hypervariable region of A. centrale MSP2 was tested, but failed to confer protection comparable to the live vaccine. 92
The degree and consistency of protection provided by the A. centrale vaccine has been reported to vary widely by geographic region, which likely reflects antigenic variation in the strains to which vaccinees are exposed. 14 A. centrale causes lifelong infection and, presumptively, immunity does not wane.
In the United States, a vaccine derived from the RBC membranes of infected cattle (Anaplaz; Fort Dodge) was developed in the mid-1960s from an Oklahoma isolate and subsequently used widely.23,24 Although the vaccine was moderately effective at preventing clinical disease in A. marginale–challenged cattle, it was associated with an increased risk of neonatal isoerythrolysis (NI) in the calves of vaccinated dams, to a level that was deemed unacceptable. 264 Modifications were made to the vaccine in 1975 that appear to have alleviated, but not eliminated, this problem.6,231 Furthermore, protection varied according to the challenge strain, with vaccine failure occurring in one study when Anaplaz-vaccinated cattle were exposed to a Florida isolate. 162 Anaplaz was withdrawn from the market in 1998. 174
Schering-Plough Animal Health released a vaccine (AM-VAX) in September 1990, but withdrew it from the market in 1992. 231 A modified live vaccine (Anavac; Poultry Health Laboratories), 128 was originally developed in the 1960s and was derived from a Florida strain of A. marginale attenuated via numerous passages through sheep.125,228 Although effective, it was often associated with adverse reactions in cattle >24-mo-old. 128 Anavac was licensed for use only in the state of California at least until the early 2000s, after which manufacture was discontinued due to changes in licensing requirements. 179
At the time of this writing, the only vaccine available in the United States is a conditionally approved vaccine manufactured by University Products (Baton Rouge, LA). It was originally developed in the late 1980s and was briefly marketed as Plazvax before its manufacture under that brand was discontinued in 1998 following a series of company mergers.16,174 Like Anaplaz, the “Louisiana vaccine” is blood-derived, albeit with additional purification steps to separate marginal bodies from erythrocyte membrane fragments. 121 The manufacturer claims that no adverse events have been reported (www.anaplasmosis.com). In a non–peer-reviewed field trial conducted in 1987–1990, 1,661 cows in 3 herds received the experimental vaccine; none developed clinical anaplasmosis, and none of the newborn calves had signs of neonatal isoerythrolysis. 172 No non-vaccinated controls were included, and it is unclear whether vaccinated cows were randomly selected. To my knowledge, only one independent study of this vaccine’s effectiveness has been performed. That study, performed in Mexico, found no significant difference in clinical outcomes between vaccinated and control cattle and all vaccinated animals required treatment, indicating that the vaccine was ineffective under local field conditions. 96
Several other vaccine candidates have been proposed,56,118 and studies of a genetically modified live A. marginale vaccine appear promising.93,131 For more details on the state of vaccine development, the reader is directed to an excellent review. 236
Conclusion
Bovine anaplasmosis is a highly prevalent and economically significant disease affecting domestic cattle throughout North America and the rest of the world. It is transmitted biologically by various tick species and mechanically by blood-contaminated fomites. Other biting arthropods may also be involved in transmission, although experimental evidence suggests that this is a minor means of transmission. The epidemiology of bovine anaplasmosis in North America remains incompletely understood, as few statistically robust prospective studies have been done to estimate its prevalence and distribution under field conditions.
Despite availability of highly sensitive and specific assays for anti–A. marginale antibodies (cELISA) and A. marginale genetic material (qPCR), the interpretation of test results for the diagnosis of clinical anaplasmosis in cattle remains challenging. Diagnosticians and practitioners should remain mindful of these pitfalls. More accurate epidemiologic data on the prevalence, distribution, and transmission modes of bovine anaplasmosis in a given area would be helpful for optimal decision-making based on diagnostic results. Apart from careful selection of new introductions, veterinarians and producers have few tools for prevention of bovine anaplasmosis based on data-driven risk assessment.
Control of bovine anaplasmosis continues to rely heavily on administration of antimicrobials, which is unlikely to be sustainable. Continuing research into A. marginale vaccine candidates offers hope for a more effective means of protecting cattle from this costly disease.
Footnotes
Acknowledgements
I sincerely thank Rae Thudium and all of the staff at the Zalk Veterinary Medical Library of the University of Missouri College of Veterinary Medicine, for their unfailingly cheerful assistance in locating and scanning references. I also thank Dr. William J. Mitchell for his careful review of the manuscript and helpful suggestions.
Declaration of conflicting interests
The author declares no potential conflicts of interest with respect to the research, authorship and/or publication of this article.
Funding
The author received no financial support for the research, authorship and/or publication of this article.
