Bovine anaplasmosis

Previous authors:F T POTGIETER AND W H STOLTSZ

Current authors:
SM Noh - Research Veterinary Medical Officer, DVM, PhD, DACVP, USDA-ARS Animal Disease Research Unit, Washington State University, Pullman, Washington, USA
KA Brayton - USDA-ARS Animal Disease Research Unit, Washington State University, Pullman, Washington, USA

GENERAL INTRODUCTION: ANAPLASMOSES

Introduction

Bovine anaplasmosis is a tick-borne disease caused predominantly by Anaplasma marginale, though a few reports indicate that Anaplasma centrale can also cause clinical disease. This pathogen has a worldwide distribution and is endemic in temperate, subtropical and tropical regions throughout the world. Bovine anaplasmosis is generally characterized by fever and progressive anaemia. In some instances, rapid destruction of erythrocytes by the reticuloendothelial system results in icterus, though this is not consistent. Infections can vary from clinically inapparent to severe, with clinical disease ranging from mild, self-limiting disease in small numbers of animals to larger herd outbreaks characterized by severe anaemia and unexpected death. Disease severity tends to increase with age and is typically inapparent in cattle less than nine months of age, although other, unknown factors, likely play a role in determining disease severity. Once infected, if animals survive initial infection, they become lifelong inapparent carriers and thus serve as a pathogen reservoir for ongoing transmission.

Figure 1 Anaplasma marginale : blood smear

Historical perspective

‘Peripheral coccus-like bodies’ and ‘marginal points’ were observed in erythrocytes of cattle by Sir Arnold Theiler in 1910, which was long before he identified these inclusion bodies as being the causative organism of the specific disease known by local farmers as gallsickness^{168, 169, 170}.  Mixed infections of Babesia bigemina and A. marginale, which frequently occur, led earlier workers to believe that these ‘coccus-like bodies’ represented a stage in the life cycle of B. bigemina¹⁵⁸. In 1911, Theiler¹⁷¹  referred to the following serendipitous discovery as a ‘lucky coincidence’. In 1908, cattle due to be imported from England to South Africa were vaccinated with a South African isolate of B. bigemina. On their arrival in the province then known as Transvaal, Theiler challenged them with blood from immune local cattle. Fifty per cent of these animals died, having shown fever, icterus, severe anaemia, ‘marginal bodies or points’ in the erythrocytes but no haemoglobinuria, and this led him to believe that these deaths were due to an agent other than B. bigemina. He concluded that the ‘marginal points’ were independent organisms responsible for the acute signs observed and called them anaplasmata because of their lack of cytoplasm, and the disease caused by them anaplasmosis. The specific organisms were called A. marginale because most resided near the margin (Figure 1) of the erythrocyte⁹.

Figure 2 Anaplasma centrale : blood smear

In 1911 Arnold Theiler¹⁷¹ discovered a second, similar bacterium, which proved to be invaluable in the control of anaplasmosis. He named this related organism A. marginale variety centrale (Figure 2). By virtue of an erroneous report by Ristic who stated that Theiler had named this organism as a separate species, the name A. centrale was taxonomically validated, but it was only in 2018 that evidence was published establishing A. centrale was indeed a separate species from A. marginale^{67, 74, 143}.  Theiler based the distinction between A. marginale and A. centrale on: the different position taken up by the two organisms within the erythrocyte, the lower virulence of A. centrale, and the incomplete cross-immunity to A. marginale demonstrated in animals that recovered from A. centrale infections.

Theiler immediately realized the tremendous potential of A. centrale for use in a vaccine, and the difference in the lengths of the incubation periods of B. bigemina and A. centrale made it possible and practical to vaccinate imported cattle with a combined B. bigemina (redwater) and anaplasmosis vaccine, which was sold for a shilling¹⁷².

The A. centrale isolate that Theiler made has been used extensively in a live-blood based vaccine in southern Africa, Australia, Israel and South America. Therefore, the distinct possibility exists that any subsequent isolations of A. centrale in these countries may represent reisolation of the original vaccine strain. A more detailed account of the history of anaplasmosis in the southern African region and elsewhere is given by Henning⁶¹.  

Aetiology and life cycle

Anaplasma marginale is a Gram negative, obligate intracellular bacteria in the family Anaplasmataceae, which includes the genera Anaplasma, Ehrlichia, Neorickettsia and Wolbachia⁴⁴.  Anaplasma spp. and Ehrlichia spp. are tick transmitted pathogens that cause disease in a variety of animals and humans. Less is known about Neorickettsia spp, which have a complex life cycle that involves multiple invertebrates. Disease in domestic animals or humans, when it occurs, tends to be due to inadvertent infection, rather than a natural component of the pathogen life cycle. Wolbachia are found in many arthropods, and have a complex and variable relationship with the host, often having an endosymbiotic relationship. Interestingly, organisms in this family are often associated with invertebrate hosts or vectors and are thought to have a long co-evolutionary relationship with these invertebrates.

The formal classification of the genus Anaplasma includes A. marginale, A. centrale, A. ovis, A. bovis, A. caudatum, A. phagocytophilum and A. platys. With the advent of deep sequencing new Anaplasma species are being detected, some being named prematurely, and their relevance indeterminant.  One putative species with mounting evidence as a zoonotic agent is A. capra, but this name has not been validated yet. Anaplasma caudatum is thought to be a tailed strain of A. marginale, and not a distinct species. Anaplasma marginale and A. centrale are the two organisms that primarily infect bovine erythrocytes and cause bovine anaplasmosis. Anaplasma marginale is the primary pathogen, while A. centrale can cause mild disease, and is best known for its capacity to provide protective immunity against A. marginale as a live, blood-based vaccine.

Upon tick transmission, A. marginale enters and replicates in erythrocytes, though endothelial cells may also be infected²⁷. In Giemsa-stained blood smears, the intra-erythrocytic organisms are dense, deeply purple, roundish inclusions 0,3 to 1,0 μm in diameter (Figure 1) and (Figure 2). Anaplasma marginale and A. centrale are morphologically indistinguishable, though A. centrale is typically located in a more central position in the erythrocyte, while A. marginale tends to remain in the periphery as their names indicate. Ultrastructurally, Anaplasma inclusions are bound by a membrane and can contain up to eight bacteria.  Typical of the Anaplasmataceae, the individual bacteria have outer and inner cell membranes^{50, 146}  (Figure 3).

Figure 3 Ultrastructural morphology of Anaplasma marginale

Ixodid ticks are the principal biological vectors of A. marginale, and primarily include Rhipicephalus, Dermacentor and Hyalomma species^{77, 131, 132, 156, 178}. Amblyomma spp. are not competent vectors for A. marginale, nor are Argasid ticks. It is important to note that detection of pathogen DNA within a tick is insufficient evidence for vector competence.

The life cycle of A. marginale in adult D. andersoni ticks has been well-characterized^{78, 80, 81, 125, 160, 162}. Within the midgut lumen, the blood meal is lysed and A. marginale enter the midgut epithelium using an as yet to be determined mechanism and replicates via binary fission to form large colonies. During ingestion of a second blood meal, the A. marginale travel to the salivary glands, where they enter the acinar cells, replicate to form large colonies and are released to the mammalian host along with the salivary gland secretions. Though not experimentally examined in all competent vectors, A. marginale is intrastadially and transtadially transmitted.

Only short-term erythrocyte cultures have been successful, and cell-to cell spread does not occur⁷³. However, A. marginale can be maintained in vitro in several tick cell culture lines derived from embryonic cells including DAE100T (D. andersoni), DVE1 (Dermacentor variabilis), IDE8, IDE12 and ISE6 (I. scapularis), and BME26 (R. microplus). In addition, A. marginale can be propagated in Vero cells derived from kidney epithelial cells of an African Green monkey, Cercopithecus aethiops, as well as RF/6A cells derived from retina choroid endothelium of a rhesus monkey, Macaca mutatta^{31, 62, 111}. Importantly the morphology and development cycle of A. marginale in tick cell culture are similar to those in naturally infected Dermacentor spp. ticks^{18, 76}.

Epidemiology

Bovine anaplasmosis has a world-wide distribution and is endemic in many temperate, sub-tropical and tropical regions. Although bovine anaplasmosis is reportable to the World Organization for Animal Health (OIE), the amount of surveillance and reporting varies widely depending on national priorities of affected countries. Consequently, the availability of prevalence data is largely dependent on local research efforts. Data regarding incidence and economic impact of the disease in most regions is nearly absent.

The prevalence of A. marginale tends to be higher in tropical and subtropical regions than in temperate regions, but prevalence varies based on local and regional conditions and management practices. For example, Ghana is a tropical country, however, due to its north - south orientation, it has three vegetative zones: sub-humid dry, sub-humid humid, and humid. Accordingly, the highest within herd prevalence based on PCR to detect A. marginale is in the humid region of the coastal savannah with 75 per cent prevalence. The lowest prevalence (45 per cent) is in the sub-humid humid region of central Ghana¹⁵. Similarly, in Benin, also a tropical west African country, the more humid regions have a higher A. marginale prevalence of 77 per cent, based on PCR. While than the drier, more northern regions have a  prevalence of approximately 35 per cent⁵.             

In east Africa, reports from Ethiopia, Kenya and Tanzania, the prevalence based on either PCR or serology, is 5 per cent, 58 per cent, and 20-37 per cent, respectively^{1, 53, 165}. In cattle rearing areas of South Africa, PCR or serology indicates prevalence between 26-98 per cent^{64, 101, 110} and 25 per cent in Zambia¹⁶⁷.    

There are a number of studies reporting the prevalence of A. marginale in Central and South America. The reported prevalence in Puerto Rico is 24 per cent¹⁷⁹, while between 0-75 per cent of animals within herds in regions of Brazil^{35, 56} are seropositive for A. marginale.

In temperate regions of North America, herd prevalence in outbreaks in Manitoba, Canada, long considered free of A. marginale, was 0.04 to 66 per cent in a 2009 outbreak⁶⁶. In one herd in California, USA, seroprevalence was between 90 to95 per cent in mature cows, and 31to37 per cent in cows in the high desert of south central Idaho^{98, 176}. Recent studies indicate the seroprevalence in the Southeast United States, including Texas, Mississippi, Georgia and Kentucky varies from 4 per cent in Georgia to 88 per cent in some herds in Texas^{58, 121, 122, 123, 124}. In the Midwest, 15 per cent of animals were seropositive in two feedlots in Iowa³³. In Kansas herd prevalence was reported at 52.5 per cent¹⁵⁹.    

Anaplasma marginale can be transmitted by ticks, biting flies, iatrogenically via needles and equipment contaminated with blood, and transplacentally. Tick transmission is considered the most efficient means of transmission as A. marginale replicate within the tick and one to three ticks is sufficient to transmit the pathogen^{151, 153}.           

The ability of particular strains or isolates of A. marginale to be tick transmitted is a stable trait. For example, the St. Maries strain of A. marginale isolated in a temperate region of North America, is readily transmitted by R. microplus, a tropical tick. Similarly an isolate of A. marginale from Puerto Rico is readily transmissible by Dermacentor andersoni, a tick found in temperate regions of the western United States⁵². In contrast, a couple of strains of A. marginale have been isolated that are seemingly not tick transmissible.  The best described are a strain from Florida and an isolate of A. marginale from bison in Saskatchewan, Canada^{152, 157}. The A. centrale isolate contained in the commercial vaccine produced by the Onderstepoort Veterinary Institute can be transmitted by R. simus, but not by B. microplus or B. decoloratus¹³⁵, whereas both the latter species are capable of transmitting A. marginale. D. andersoni is capable of transmission of both A. marginale and A. centrale, though transmission of A. centrale is markedly less efficient¹⁷⁷.         

Blood feeding diptera in the genera Tabanus and Stomoxys can also transmit A. marginale mechanically. Based on the repeated detection of non-tick transmissible strains of A. marginale, and the presence of large numbers of biting flies in some environments, biting flies can play an important role in the epidemiology of A. marginale. Instruments used in veterinary procedures, such as castration, dehorning and ear-tagging operations, which are contaminated with fresh blood, may also be responsible for the mechanical transfer of the infection^{42, 139}. Circumstantial evidence indicates that some anaplasmosis outbreaks have followed vaccination programmes in South Africa, incriminating needle transmission between animals.

Transplacental transmission may also play a role in the maintenance of A. marginale within a herd.  It has been estimated that 10 to 15 per cent of calves born to A. marginale infected cows will be infected with A. marginale though the development of clinical signs in neonates is unusual^{55, 134, 184}. Overall the route of transmission that predominates in a particular herd or area depends on a variety of environmental conditions and management practices. 

Once infected, animals typically remain infected for life and serve as reservoirs for ongoing transmission, though not all animals develop clinically apparent disease^{28, 29}. Calves under six months old, irrespective of the immune status of their dams, tend to be resistant to severe clinical disease, but the risk of clinical anaplasmosis increases with age^{69, 113, 128}. In endemic areas with high transmission rates, natural infection involving all animals in a herd during calfhood will help ensure minimal disease, often termed endemic stability.

The risk factors for the development of disease, including herd outbreaks, are poorly understood and likely multifactorial. Introduction of naïve, adult animals into an infected herd, variation in tick burdens through time leading to episodic periods of high transmission, variation in virulence of A. marginale strains, concurrent infections with other pathogens, and efficiency at which particular vector populations transmit the pathogen may all influence the likelihood that individual animals or groups of animals develop clinical disease. 

Other domestic ruminants including sheep and goats can harbour A. marginale, though the ability to cause disease in small ruminants is unknown³⁷. Additionally, little is known about the role wildlife species play in the epidemiology of bovine anaplasmosis. It is important to differentiate between the susceptibility of a particular species to infection in laboratory conditions, presence of the pathogen in wild populations, and likelihood of serving as a reservoir for domestic animals. An additional consideration is that the results of serologic surveys to detect Anaplasma spp. are difficult to interpret as antibody cross reactivity among all Anaplasma spp. and Ehrlichia spp. is common.

 In South Africa, blesbok (Damaliscus dorcas phillipsi), common duiker (Sylvicapra grimmia L), and black wildebeest (Connochaetes gnou) are susceptible to experimental infection with A. marginale, but infections are subclinical¹¹². In north America, Bison (Bison bison), elk (Cervus elaphus), pronghorn antelope (Antelocapra americana), and bighorn sheep (Ovis canadensis) are susceptible to infection.^{11, 60}         

In the Americas, A. marginale has been identified in a number of species of deer, including Andean deer (Hippocamelus antisensis) in Argentina and Pampas deer (Ozotoceros bezoarticus leucogaster) in the Brazilian Pantanal^{57, 129}. Mule deer (Odocoileus hemionus) and black tailed deer (Odocoileus hemionus culumbianus) populations in the US are naturally infected with A. marginale, though white tailed deer (Odocoileus virginianus) seem to be resistant to infection¹¹.           

Pathogenesis

After primary infection, there is a prepatent period that terminates when the first infected erythrocytes are detected in blood smears. This is followed by a phase during which there is increasing parasitaemia and anaemia, and that ends either in death (preceded by evidence of severe anaemia and other clinical signs) or convalescence and the development of a carrier state.

The duration of the prepatent period seems to be directly related to the number of organisms in the inoculum, body weight of the host and route of infection. Generally, prepatent periods are shortest after intravenous inoculation, longer after intramuscular inoculation, and even longer following subcutaneous injection. The prepatent period can be as long as three to five weeks¹⁴⁶. The course of the infection is not affected by the size of the inoculum⁸², but prepatency may vary even between animals which receive the same infective doses¹³². In spite of this, the number of organisms in live-blood vaccines is manipulated in such a way that the desired prepatency is achieved so that a reasonable synchronization of reactions results in those animals that will react clinically to the vaccine. Similarly, in the manufacture of frozen vaccine, adjustments can be made to the number of organisms per vaccine dose to compensate for loss in viability after freezing and thawing of the infected blood¹³⁰. Prepatent periods may be prolonged after administration of tetracyclines⁶⁸.         

The intensity and duration of parasitaemia and the resulting anaemia are directly related to the age of the host⁶⁹. Increasing anaemia may develop over 4 to 15 days, and animals may lose up to 70 per cent of their circulating erythrocytes. Maximal anaemia usually occurs one to six days after the peak parasitaemia. In animals that survive the patent phase, convalescence begins with evidence of erythropoiesis. The time of convalescence is related to the ability of the animal to produce new erythrocytes.

Although the manner by which erythrocytes become infected is not clear, an electron microscopic study has revealed how initial bodies penetrate the erythrocytic membrane¹⁴⁷. It has been suggested that a direct transfer of initial bodies between erythrocytes takes place¹⁴⁷. Increased osmotic fragility, preceded by a decrease in erythrocytic acetylcholinesterase has been observed in bovine anaplasmosis^{155, 180}. This indicates that changes in the acetylcholinesterase activity may be associated with increased cell membrane permeability, which in turn facilitates the entry of A. marginale organisms into red blood cells¹⁵⁵.      

The pathogenesis of the anaemia in anaplasmosis was originally thought to be due to destruction of the erythrocytes by the parasites. However, the absence of haemoglobinaemia and haemoglobinuria indicates that intravascular haemolysis is not a feature of the disease⁶⁸. The anaemia is caused by extensive erythrophagocytosis initiated by parasite- induced red blood cell damage¹². Erythrocytes that become physically and chemically altered during the course of the disease⁴³ are removed by the reticuloendothelial cells in the spleen, liver, lungs and associated lymph nodes¹⁵⁴.       

The rate of erythrocyte loss and the level of parasitaemia during the period of increasing anaemia prior to the detection of reticulocytes in the circulation are usually closely related. Evidence of a haemopoietic response in the peripheral circulation, which is a favourable prognostic sign, is typically first apparent just before the occurrence of maximum anaemia⁶⁹.

Clinical signs

The prepatent or subclinical incubation period generally varies between 15 and 36 days (with an average of 26 days), although it may be longer. Disease manifestations can vary from from peracute disease leading to unexpected death to minimal disease.

Peracute anaplasmosis is the most severe form of the disease. It is rare but usually fatal. Animals may be found dead or succumb within a few hours of the onset of clinical signs. In addition to anaemia, milk production ceases, and there is excessive salivation, rapid respiration, irrational behaviour and signs of nervousness.

The most outstanding features of the acute bovine anaplasmosis are due to relatively rapid onset of anaemia and include pallor of mucous membranes, depression, inappetence, a decrease in milk production, general weakness, and a rapidly rising parasitaemia. In some animals, fever provides initial and persistent clinical evidence of anaplasmosis, but this is not consistent¹²⁶, as in others the rectal temperature may remain below 40 °C²⁶. As the disease progresses, constipation, rumen stasis, weight loss, dehydration, icterus, laboured respiration, a thumping heartbeat and muscle tremors are observed in severely affected animals. Diarrhoea may be manifested in the initial stages of the disease⁷⁰. Abortions may occur in severe cases^{17, 34, 49, 127, 181}. Abnormally aggressive behaviour, probably resulting from the anaemia and resultant cerebral hypoxia, is also manifested by some animals. The course of the disease may be protracted depending on the severity of anaemia and the capacity of the animal for erythropoiesis.

Once acute disease is controlled, most persistently infected animals are asymptomatic. However, chronic disease can occur and is manifested by slow recovery following acute disease. It may persist for a period of two weeks to several months¹⁴⁴. It is characterized by poor appetite, loss of weight and varying degrees of dehydration, anaemia and icterus which are usually milder than in the acute disease.

Anaplasma marginale infections may induce temporary infertility in bulls⁷⁰, anoestrus in heifers¹⁶⁶, and abortions and neonatal anaplasmosis following foetal infection^{17, 119}.

Pathology

The primary insult is extravascular haemolysis, which is the removal of A. marginale-infected RBCs by the reticuloendothelium system, particularly in the spleen, leading to anaemia. The rate at which the anaemia develops and the severity of anaemia will dictate the severity of disease and clinicopathological abnormalities. Initially, total and unconjugated bilirubin will be elevated if the capacity of the liver to take up and conjugate bilirubin is exceeded. If RBC destruction is rapid and severe, icterus may develop, which indicates a bilirubinaemia of >1.5 mg/dL. In some cases, particularly later in the course of infection, conjugated bilirubin can also be elevated suggesting impairment of the hepatocytes to excrete the conjugated bilirubin into the biliary system. Bilirubinuria may develop if sufficient amounts of conjugated bilirubin are released into circulation. Alkaline phosphatase (ALP), aspartate aminotransferase (AST) and serum urea nitrogen (SUN) can also be elevated, likely due to hypoxic injury to hepatocytes and the kidney^{8, 9, 10}.          

Gross and histopathologic lesions are primarily referrable to erythrophagocytosis and anaemia. On a post mortem examination, all tissues will be pale tan, indicating anaemia and icterus. The urine often has a brownish colour due to bilirubinuria. Splenomegaly is often marked. If hypoxia is severe and prolonged enough, the liver may be mottled due to centrilobular degeneration¹⁶⁹.

There are few reports on the histopathology of anaplasmosis. Histologically there is evidence of haemosiderosis, erythrophagocytosis by the RE system and hyperplasia of erythropoietic elements in the bone marrow. Typically there is hyperplasia of the splenic red pulp, and an increase in the number of plasma cells and haemosiderin-bearing RE cells^{154, 175}. Depending on severity, centribular hepatic necrosis and acute renal tubular necrosis, due to hypoxia may be apparent^{8, 68, 154}. Bile stasis, and proliferation and haemosiderin pigmentation of Kupffer cells have also been reported. Haemosiderin may be present in macrophages of the lungs and lymph nodes¹⁷⁵.

Diagnosis

It is important to understand the goal of the diagnostic testing for A. marginale to ensure selection of the appropriate test and accurate interpretation of results. In general, the primary purposes of diagnostic testing are to: 1) diagnosis of clinical cases of bovine anaplasmosis and; 2) to identify persistently infected animals. Robust laboratory-based diagnostic tests have been validated for both purposes. However, there remains a need for commercially available, low input or pen-side tests.

Ideally, a diagnosis of clinical bovine anaplasmosis requires documentation of anaemia in conjunction with the presence of infected erythrocytes. Infected erythrocytes can be readily visualized on a Diff-Quick or 10 per cent Giemsa- stained blood smear using light microscopy. If only post mortem samples are available, air-dried thin smears from the liver, kidney, heart or lungs or peripheral blood vessels stained with Diff-Quick or 10 per cent Giemsa may be diagnostic¹²⁰. Regardless, great care should be taken to ensure clean slides are used and all particulates are filtered from the stain because debris can easily be mistaken for A. marginale infected erythrocytes. Smears should be visualized under oil immersion at 100x magnification. The A. marginale colonies are approximately 1-2 µm in diameter, deeply stained, uniformly round, and typically located at the periphery of the RBC.  Anaplasma centrale forms morphologically similar colonies that tend to be located in the centre of RBCs.

At the height of the anaemic crisis, A. marginale inclusion bodies may be difficult to find because of the removal of the infected erythrocytes from the circulation. In addition, the presence of a regenerative anaemia requires careful examination of blood smears to distinguish A. marginale inclusion bodies from Howell-Jolly bodies. PCR can be used in the place of, or in addition to, blood smears to identify A. marginale infected animals. In the absence of quantitation, a positive result with PCR indicates the presence of infection, not necessarily disease. Thus, in the absence of visualization of A. marginale-infected erythrocytes it is important to rule out other causes of anaemia such as bovine theileriosis, babesiosis, trypanosomosis before definitively diagnosing a case of bovine anaplasmosis.

PCR is generally a reliable method to identify A. marginale – infected animals. There are a number of platforms, and published targets for PCR-based assays to detect the pathogen in ruminant blood samples. Msp5, a highly conserved, single copy gene is the most commonly used target. The increased sensitivity of real-time PCR and nested PCR as compared to standard PCR is useful for identifying animals with low levels of circulating pathogen. Based on the specificity of the target gene, PCR is also well-suited for differentiating between A. marginale and other Anaplasma spp. or haemoparasites. Regardless of the platform and target gene, care should be taken to ensure the test has been validated for its intended purpose.

Serology is the most reliable method to detect persistently infected animals, which may be PCR negative due to low numbers of circulating organisms. However, serology should not be relied upon to diagnose clinical disease because seroconversion requires approximately 14-21 days and thus acutely infected animals may not yet have developed an antibody response. Additionally, for this same reason, identification of uninfected animals requires repeat testing to ensure recently infected animals that have not yet seroconverted are classified appropriately.

Historically, a number of serologic tests have been used to detect anti-A. marginale antibodies, including complement fixation, rapid card agglutination, and indirect immunofluorescent assays.  A commercially available competitive ELISA (cELISA) using recombinant Msp5 and the monoclonal antibody AnaF16C1 is widely used. When validated with a population of cattle from the US, it had 99.7 per cent diagnostic specificity and 100 per cent diagnostic sensitivity^{32, 38, 120}. However, this population of animals was unlikely to be infected with other Anaplasma spp. or other haemoparasites. Serologic cross-reaction among the Anaplasma spp. and Ehrlicha spp. is common. This cELISA will also detect antibodies directed against A. centrale, A. ovis, and possibly Ehrlichia spp.^{7, 102}, but does not detect antibodies from A. phagocytophilum infected dogs and humans¹⁶⁴. Serum from A. phagocytophilum infected animals will bind recombinant A. marginale Msp5 in other ELISA formats, such as the indirect ELISA¹⁶⁴. In short, validation of serologic tests for the detection of anti-A. marginale antibodies, if applied to non-cattle species, is necessary to ensure accurate results.

Apart from blood or tissue smears and determination of haematocrit to diagnose clinically affected animals, there remains a great need for commercially available, low input tests that can be readily used at the pen-side or in regions without access to diagnostic laboratories. Latex agglutination to detect anti-A. marginale antibodies using Msp5 as the antigen has been experimentally validated in cattle populations in Brazil¹³⁷. As low input thermocyclers become more available, reverse line blot, though potentially less sensitive than other PCR-based methods, may be useful³⁰.       

Differential diagnosis

The clinical signs of anaplasmosis should be differentiated from infectious and non-infectious conditions presenting with fever, anaemia, icterus, depression, inappetance, and reduced milk production. Similarly, on post-mortem examination, anaplasmosis should be differentiated from conditions which cause anaemia primarily due to erythrophagocytosis (extra vascular haemolysis).

In regions were both bovine babesiosis (redwater) caused by Babesia bigemina or B. bovis and bovine anaplasmosis are endemic, including Africa, Australia, and South and Central America, anaplasmosis can be confused clinically and pathologically with bovine babesiosis. The epidemiology of the two diseases can be quite similar as both diseases are tick-borne, have the same tick vectors, establish persistent infection in cattle, and young animals tend to be less susceptibility to severe disease. One of the main clinical features of babesiosis, but not bovine anaplasmosis, is haemoglobinuria. However, haemoglobinuria, is also not a constant feature of babesiosis, particularly during the early stage of B. bovis infection. Although diarrhoea, which commonly occurs in babesiosis, may be present in acute anaplasmosis, constipation and rumen stasis are more regular findings. The course of the clinical disease in anaplasmosis is often longer than that of babesiosis. Care should be taken as outbreaks of anaplasmosis and babesiosis may occur concurrently.

Some forms of theileriosis should be differentiated from bovine anaplasmosis. Theileria are apicomplexan, blood borne, intracellular tick transmitted parasites. Theileria orientalis, previously named T. buffeli in Australia, T. sargenti in Japan and East Asia, and T. orientalis in many other locations are now all classified as the T. orientalis complex and are separated into several genotypes⁵⁴. Most genotypes are non-pathogenic, however, genotype Ikeda and Chitose do cause disease. Since 2010, the Ikeda genotype has been responsible for a number of outbreaks of clinical disease in Australia, New Zealand, and more recently, the United States^{45, 94, 118}. Disease is due to the destruction of erythrocytes, and thus the clinical presentations are nearly identical to bovine anaplasmosis. In severe cases, intravascular haemolysis can occur leading to haemoglobinuria, which is not a clinical feature of bovine anaplasmosis. Detection of the pathogen is required to differentiate between these diseases in regions where they occur together. 

Theileria parva and T. annulata are transforming Theileria sp. because they cause lymphoproliferation and subsequent lymphadenomegaly. All aspects of the diseases caused by T.parva and T annulata are described in detail in the respective chapters(see-East coast fever and Theileria annulata theileriosis)Disease due to these two pathogens is predominantly due to the effects of lymphoproliferation and clinical signs typically include fever, weight loss, lymphadenopathy and often death¹³⁶. Animals infected with T. annulata can also develop progressive anaemia and icterus due to significant replication of the parasite in erythrocytes and thus may need to be differentiated from bovine anaplasmosis¹¹⁴. Additional clinical features of T. annulata which are not seen in bovine anaplasmosis include panleukopenia and marked thrombocytopenia. The later can lead to petechiae on the mucous membranes and serosal surfaces of many organs. 

Anaemia and progressive weight loss are also clinical signs often associated with Trypanosoma spp. infections. Bovine trypanosomosis (nagana), however, generally occurs within well-defined tsetse-infested areas.

Anaemia and icterus may also be encountered in cattle suffering from leptospirosis, bacilliary haemoglobinuria⁷², chronic copper poisoning and intoxications with Brassica and Allium spp^{71, 72}. In these conditions, haemoglobinaemia and haemoglobinuria are usually also present⁷².    

Control

Control strategies vary greatly in different countries and geographic regions. Generally, the effectiveness of a strategy in a specific region depends on a thorough knowledge of the epidemiology of the disease, but it is frequently the case that the scientifically-based measures are constrained by such factors as legislation, available infrastructure, funding, and the level of management. For these reasons no universal control programme can be recommended.

Following the introduction of anaplasmosis, whether by infected ticks, cattle or other reservoir hosts, into regions free of the disease and where an epidemic amongst the susceptible resident cattle populations could be expected, eradication of the disease is indicated. This is achieved by identifying infected cattle by PCR, microscopic examination of blood smears, and serology. Chemosterilization, the use of drugs to clear infected animals of the pathogen, require long-term doses of and require follow-up testing to ensure success in each animal, and thus may be difficult to achieve on a herd level (see section on chemosterilization). Concurrent stringent vector control measures to ensure eradication of infected ticks and to prevent mechanical spread of the infection must be implemented. In order to eradicate the disease successfully, a sophisticated veterinary infrastructure, the support of relevant legislation and the availability of ample funding are essential. Regulatory measures should also be implemented to prevent the subsequent reintroduction of the disease.

In southern Africa, where anaplasmosis is endemic in many of the important cattle farming areas, its eradication followed by the implementation and long-term maintenance of strict tick control programmes is not generally recommended, because stringent tick control could render the cattle population susceptible to several other tick-borne diseases including babesiosis and heartwater with potentially catastrophic consequences (see Vectors: Ticks), as has been experienced in Zimbabwe⁹³. Additionally, there are many financial and practical limitations regarding anaplasmosis eradication programmes. The development of acaricide resistance by ticks is common and the cost involved in high-frequency dipping programmes is too expensive for many farmers. Further complicating factors are that populations of wild ruminants sustain tick populations and some are reservoir hosts.

For these reasons, in endemic regions with high transmission rates, it is often advantageous to manage for a stable disease situation by allowing natural exposure of calves to tick-borne diseases, including anaplasmosis, during the period when they are naturally resistant to disease or protected by passively acquired maternal antibody. As far as anaplasmosis is concerned, the existence of epidemiological factors which inhibit the exposure of calves to infected ticks within the relevant period can be compensated for by the administration of the A. centrale live-blood vaccine. If this control strategy is followed, natural or artificial fluctuations in tick populations, or any other factor which may lead to episodic or low levels of pathogen transmission, are less likely to result in disease outbreaks.

Immunization

Anaplasma centrale, first identified in South Africa by Arnold Theiler, infects bovine erythrocytes, but is typically asymptomatic or causes milder disease than A. marginale.  Anaplasma centrale circulates in South. Africa in non-vaccinated animals and is likely maintained in wildlife^{65, 75}. Since its identification in 1912, intentional infection of animals with A. centrale is used as a means of vaccination to protect animals against bovine anaplasmosis^{150, 172, 183}. This live vaccine has been used extensively in Israel, Australia, southern Africa, and many parts of South America. However, A. centrale, although generally a mild pathogen, can cause severe clinical disease following vaccination^{16, 83, 133, 183}.           

Use of A. centrale as a vaccine requires amplification of the organisms in splenectomized cattle and cryopreservation of the infected erythrocytes. This procedure results in a number of difficulties including ensuring safety, viability and quality control. Additionally, it requires maintenance of the cold chain and carries the risk of the spread of additional blood-borne pathogens. Consequently, it is not practical in many circumstances and cannot be licensed for use in some parts of the world.

The surface proteins of A. centrale share moderate to high identity with the surface proteins of A. marginale.  Thus, it is likely that infection with A. centrale induces an immune response, including antibodies, that recognize A. marginale⁶. Anaplasma centrale generally provides protection against disease, but not infection. The degree of protection against disease is variable and likely depends on the challenge conditions and circulating strains of A. marginale. The highest recorded protection was in Kenya and Australia where parasitaemia upon challenge was 0.7 to 0.5 per cent in vaccinated animals and >7 to 10 per cent in nonvaccinated animals. In contrast, in studies conducted in Zimbabwe and South Africa, parasitaemia of 16 to 21 per cent developed in vaccinated animals following challenge, while, unvaccinated animals had parasitaemias of approximately 30 per cent. Intermediate, levels of protection have been recorded in Argentina and Paraguay with 2.3 to 5.7 per cent parasitaemia in vaccinated animals and 7.5 to 16 per cent parasitaemia in unvaccinated animals following challenge¹⁹.    

The use of A. centrale as a vaccine requires careful management and administration. As young calves tend not to develop severe disease, it is generally advised that they are vaccinated with A. centrale before or at approximately six months of age. In older animals more, severe disease can be expected and close supervision is indicated. Pregnant cows should not be vaccinated as they may abort. The incubation period following vaccination usually varies from five to seven weeks. Vaccinated animals should be closely observed for clinical signs of anaplasmosis during this period, and should, if necessary, be specifically and symptomatically treated. In view of the possible reaction to vaccination in older animals, only a limited number of animals should be immunized at any given time.

The use of inactivated A. marginale vaccines prevents serious clinical disease²⁰. However, little data exists concerning the efficacy of inactivated products. Repeated administration of products with residual proteins from bovine erythrocytes can result in fatal neonatal isoerythrolysis in calves born to vaccinated cows⁴¹. Additionally, cattle vaccinated with inactivated organisms are still highly susceptible to challenge with heterologous strains⁹².           

Improved methods to culture A. marginale could result in a safer, easier to produce live vaccine. Currently, A. marginale can be readily cultured in tick cell lines, but not bovine erythrocytes¹¹¹. However, protective immunity in cattle immunized with whole, killed A. marginale derived from cell culture is overall poor. In one study, protection was better with A. marginale derived from erythrocytes as compared to tick cells, though there were no statistically significant differences between any of the groups³⁹. In a second trial, A. marginale derived from tick cell culture provided protection from anaemia though not parasitaemia⁷⁹. There was no comparison of the protection provided with A. marginale from RBC in this study. While the major surface proteins of A. marginale are expressed during growth in cell culture, many other less immunogenic outer membrane proteins are greatly down-regulated in tick cells^{13, 116}. Thus the antigenic profile of A. marginale derived from tick cells is likely inadequate to provide protective immunity.

A recombinant vaccine offers a safe, efficacious, and low-cost method to protect cattle against bovine anaplasmosis. Purified outer membrane proteins of A. marginale consistently protect nearly all animals from high parasitaemia and disease, though protection from infection is inconsistent^{25, 115, 117}. The current model for vaccine-induced immunity against A. marginale proposes that clearance of the organism is affected by antibody against surface epitopes in combination with macrophage activation for enhanced phagocytosis and killing. Central to this model is the CD4+ T-cell expression of interferon gamma (IFN-γ), which enhances synthesis of the predominant opsonizing bovine IgG subclass, IgG2, concomitantly activating macrophages to increase receptor expression, phagocytosis, phagolysosomal fusion and release of rickettsiacidal nitric oxide.

The protective outer membrane proteins is a complex mixture, with over 25 immunogenic proteins⁹⁷. Within this mixture, a subset of outer membrane proteins have been identified as promising vaccine candidates¹¹⁵. A number of those candidates are highly conserved among widely geographically separated A. marginale strains. Thus, development of a single broadly protective vaccine, rather than a number of strain-specific vaccines, is possible^{51, 63}.

Several obstacles in the development of a recombinant vaccine remain, including: 1) Identification of a specific set of outer membrane proteins that provide protective immunity to homologous and heterologous challenge; 2) Development of an expression system that maintains the immunogenicity of the proteins, which is not the case for proteins expressed in E. coli; 3) Identification of an effective adjuvant.

Treatment

The treatment of clinical anaplasmosis in cattle requires, first, the suppression of the parasite (specific treatment) and, second, the alleviation or prevention of secondary complications and the hastening of recovery (symptomatic or supportive treatment)⁶⁸.             

Secondary complications, generally the result of hypoxia, are a frequent sequelae to anaemia. During acute anaplasmosis the severity of this anaemia is proportional to the extent of the parasitaemia^{23, 68, 169}. Specific treatment early in the course of infection while the PCV remains above 15 per cent is desirable^{24, 36, 48, 68, 89, 107, 108, 109, 145, 146}, and such treatment prior to the development of a high parasitaemia or the onset of severe anaemia is considered essential for a favourable outcome^{89, 182}. Often, however, by the time that acute anaplasmosis is diagnosed the level of parasitaemia is already declining as a result of the phagocytic removal of parasitized erythrocytes, and the animals are suffering from severe anaemia²³. Nevertheless, even when treatment is slightly delayed, treatment with tetracyclines or imidocarb is more beneficial than supportive treatment¹⁰⁸.

Specific treatment

Tetracyclines are the only effective chemotherapeutic agents approved for the treatment of anaplasmosis^{88, 89, 100, 145, 146}. Of these, chlortetracycline, tetracycline and oxytetracycline are the most commonly used drugs for the treatment of acute and chronic carrier infections^{87, 88, 89, 150}. Anaplasma marginale infections in cattle are not cleared at the usual recommended therapeutic doses of the tetracyclines^{90, 108, 161}.

There is some controversy about the desirability of treating acute anaplasmosis at the time of maximum anaemia²³. Much of this controversy is related to whether or not an animal will recover spontaneously, and the conflicting opinions which have been expressed range from those who consider treatment to be beneficial at this time to those who feel that the excitement and exertion incident to treatment are more likely to be detrimental to the animal. At the time when increased erythropoiesis becomes evident, specific treatment probably has little, if any, effect on the course of the disease, since at this stage the parasitaemia is rapidly decreasing as a result of the phagocytic removal of infected erythrocytes, and spontaneous recovery is frequent²³.

Tetracycline is the preferred treatment for clinical bovine anaplasmosis.  Tetracyclines are bacteriostatic, inhibit replication by reversible binding to the 30S ribosomal subunit, and have been widely used for the treatment of anaplasmosis^{21, 22, 23, 36, 48, 103, 108, 145, 146}.

Short acting oxytetracycline at 10mg/kg/day until 24 to 48 hours after symptoms subside for no more than 4 days will alleviate symptoms, if treatment is not initiated too late. Alternatively, a single intramuscular administrations of long-acting (LA) formulations of oxytetracycline at 20 mg/kg body weight  also effectively reduces disease severity^{90, 161, 173}. The use of a single treatment of long-acting oxytetracycline for acute anaplasmosis has an obvious practical advantage in that cattle receiving multiple injections of short acting oxytetracycline, or any of the other tetracycline drugs, need to be restrained repeatedly.

Imidocarb [3,3’bis-(2-imidazolin- 2-yl)-carbanilide dihydrochloride (or dipropionate)] has specific chemotherapeutic effects on A. marginale infections, and is approved for commercial sale in some countries^{14, 24, 40, 84, 85, 86, 87, 91, 105, 106, 148}. This drug is primarily used as an antibabesial^{3, 4, 89, 158, 174}. At normally recommended therapeutic dosage levels imidocarb does not clear A. marginale infections in cattle^{3, 4, 24, 40, 85, 87, 105}.           

Imidocarb hydrochloride⁵⁹ and its dipropionate salt¹⁰⁵both have a marked inhibitory effect on A. marginale. Therapeutic dosage levels of imidocarb for the treatment of acute anaplasmosis in cattle vary from 2 to 5 mg/kg body weight¹⁵⁰ administered subcutaneously or intramuscularly^{87, 88}. A single subcutaneous dose of imidocarb at 2,5 mg/kg body weight is as effective as higher rates varying from 5 to 10 mg/kg in the control of acute anaplasmosis,¹⁴⁸ and that a dosage rate of 2,5 mg/kg is the most efficacious under field conditions^{3, 106}.       

Imidocarb is carcinogenic, has a long withdrawal time and is not approved for use in the USA or Europe. Administration of imidocarb at recommended therapeutic dosage rates usually causes transient side-effects^{3, 4, 106, 148}. These include a localized swelling at the injection site (following subcutaneous administration) and systemic effects, characterized by increased salivation, lachrymation and nasal discharge^{3, 104, 148}. The severity of these signs is dose-related, and when doses are repeated or increased to eliminate infections (see Control: Chemosterilization), more severe clinical signs of toxicity including diarrhoea, dyspnoea and depression frequently occur². Since intravenous inoculation similarly results in more severe signs of toxicity^{87, 88, 106, 174} and even mortality at rates of 3 mg/kg or higher^{87, 174}, this route of administration is not recommended.

Following successful treatment of a primary A. marginale infection with any of the above-mentioned drugs, the parasitaemia decreases to 1 per cent or lower within four to seven days^{148, 182},  but there are frequent relapses (recrudescences of parasitaemia) of varying intensity at variable intervals thereafter^{3, 24, 40, 87, 106, 161}. Because treatment does not clear the bacteria, cyclical rises in parasitemia, as occurs in untreated animals is likely. Typically, these increases in parasitaemia are well controlled by the immune system and animals do not develop clinical disease.

Supportive treatment

Following treatment with an effective chemotherapeutic compound, parasitaemia declines over a period of several days, reaching levels of 1 per cent or lower only after approximately one week. The decline in parasitaemia is usually accompanied by a reduction in packed cell volume over a similar period of time. It is therefore important to bear in mind that the clinical condition of an animal may deteriorate considerably as a result of progressive anaemia notwithstanding successful specific chemotherapy, and that supportive symptomatic treatment may be required to prevent mortality²³.

Because of the risk of mechanical transmission of A. marginale, affected cattle (especially those with high parasitaemias) are a major source of infection to uninfected susceptible cattle in the herd and should preferably be separated from the healthy animals. When isolating diseased animals, consideration must be given to the provision of adequate protection against the elements, easy access to palatable feed and water, as well as facilities for continuing observation of the animals and for the administration of any additional chemotherapy, which would require the minimum amount of exertion on the part of the animals⁶¹.

Additional symptomatic treatment may be beneficial and is usually recommended. Furthermore, correction of any gastrointestinal disturbances such as ruminal stasis, constipation or lack of appetite by the oral or parenteral administration of rumenotorics, ruminal fluid, mild laxatives and appetite stimulants, may help prevent additional secondary complications and promote a more rapid recovery.

Control of vaccine reactions

Following the administration of live-blood vaccines, some animals may develop clinical anaplasmosis. For all practical purposes these cases should be appropriately treated specifically and symptomatically as though they were naturally acquired clinical infections.

Chemoprophylaxis

The chemoprophylaxis of anaplasmosis is largely based on the constant or repeated administration of tetracyclines to control A. marginale parasitaemias in naturally infected cattle. This practice, in the absence of clinical disease in a herd, is strongly discouraged and in some countries not allowed, as the judicious use of antibiotics is required to ameliorate the growing problem of antibiotic resistant bacterial populations in general.

Generally, chemotherapy during the prepatent period merely results in the prolongation of the prepatent period and delays the onset of clinical signs^{46, 48, 95}. With repeated administrations of long-acting oxytetracycline (at a rate of 20 mg/kg at seven-day intervals), commencing seven days after infection, the prepatent period is extended by approximately the same time-span as the duration of treatment^{46, 95}. Although all animals thus treated remain infected, fewer animals develop clinical signs of anaplasmosis as the number of treatments increases.

For animals exposed to natural infection, a treatment regimen consisting of intramuscular administrations of short-acting oxytetracycline (at a rate of 6,6 to 11 mg/kg or long-acting (LA), at a rate of 20 mg/kg at 21- to 28-day intervals) to all susceptible cattle throughout the season when vectors are most active and abundant, and continued for a further one or two months after its end, have been employed^{68, 88, 96, 140}. This treatment regimen should prevent clinical disease in most animals that become infected, but will not eliminate A. marginale from carrier animals.

When tetracyclines have been used to prevent clinical anaplasmosis, animals should continue to be closely observed for clinical signs of the disease after withdrawal of medication.

Chemosterilization

Anaplasma infections in cattle can be cleared, though typically long treatment regimens are required. The best method to determine if the pathogen was eliminated is via subinoculation of blood into a splenectomized animal. Additionally, a loss of the anti-Msp5 antibody response, as determined through cELISA can serve as a proxy. Real-time PCR should not be relied upon to confirm elimination of the pathogen.

Treatment to eliminate A. marginale infections in endemic areas is contraindicated because of the high risk of reinfection and the relative susceptibility of the cattle population in the absence of any long-term immunity which accompanies low-level persistent infections⁸⁸.

Sensitive serological tests such as the cELISA can be used to identify persistently infected cattle in a herd. Segregation or removal of these animals from the rest of the herd, or elimination of infection in these animals, if removal from the herd is undesirable, can be used to establish an A. marginale-free herd. This approach has successfully been used in parts of the USA and Canada, but is usually only feasible in areas with a low or moderate prevalence of anaplasmosis^{88, 145}.

Because of the possibility of transplacental infection, calves should also be screened serologically, and positive cases treated or eliminated in order to prevent them from becoming a future source of infection to adult cattle that have been cleared of infection. It must, nevertheless, be borne in mind that the serum of uninfected calves born of infected dams may also react positively in serological tests for periods of up to six months after birth, due to the presence of colostral antibodies¹³⁴.

Imidocarb has been used for chemosterilization, however there is considerable individual variation in tolerance to imidocarb⁸⁷ and severe toxicity and even mortality may result at the dosages recommended for chemosterilization^{85, 163}.

Oral administration of chlortetracycline for the elimination of A. marginale may have advantages over parenteral administration in that treatment on a herd basis in the form of feed premixes incorporating the drug is possible¹⁴⁰, but the consistent elimination of carrier infections by this method is often difficult to attain because of differences in amounts of medicated feed or salt-mineral mixes consumed by individual animals^{87, 145, 146}.            

Chemosterilization can be achieved in persistently infected animals using chlortetracycline as top dressing provided twice daily for 80 days. Animals receiving 4.4mg/kg/day, 11 mg/kg/day, and 22 mg/kg/day were cleared of A. marginale between 46-49 days of treatment based on real-time PCR, depending on the dose. Importantly, clearance of the bacteria was confirmed by subinoculation into a splenectomized animal. The animals became  seronegative approximately 110 days following a PCR negative results¹³⁸.

Alternatively, a single injection of long acting oxytetracycline at 20mg/kg followed by 30 days of treatment of chlortetracycline at 4.4mg/kg/day as top dressing on feed also resulted in chemosterilization¹³⁸. Following clearance of infection, animals can be re-infected, although these re-infected animals show resistance to clinical anaplasmosis for periods ranging from a few months up to 30 months following parasite elimination^{47, 99, 140, 141, 142, 149}.

Additionally, programmes aimed at the elimination of the carrier state are best conducted during the period of lowest vector activity and should be accompanied by the implementation or maintenance of vector control measures^{88, 89}.

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