Bovine Anaplasmosis

Kristin A. Clothier

Etiology

Molecular characterization and genetic analysis of Anaplasma and Ehrlichia have resulted in identification of six species in the Anaplasma genus: Anaplasma phagocytophilum (formerly called both Ehrlichia equi and Anaplasma equi), which infects granulocytes in a wide range of mammalian species including dogs, horses, and humans; A.

In livestock medicine, anaplasmosis traditionally refers to disease characterized by progressive anemia caused by intraerythrocytic infection with A. marginale in cattle and A. ovis in sheep and goats. In addition, A. centrale causes mild disease in cattle and has been used as a live vaccine to induce partial protection against A. marginale.ⁱ These organisms are obligate intracellular bacteria located either at the periphery (A. marginale and A. ovis) or center (A. centrale) of red blood cells (RBCs).^4,5 They reside and replicate inside parasitophorous vesicles and replicate by binary fission, with each vesicle contain­ing four to eight organisms.⁶

Pathogenesis

Anaplasma spp. are naturally transmitted by ixodid species ticks, most commonly Dermacentor in the U.S. mainland and Rhipicephalus spp. (including tick species previously classified in the genus Boophilus) in tropical and subtropical regions world- wide.^1,3 When ingested by feeding ticks, Anaplasma spp.

In the host, sequential rounds of bacterial invasion of mature erythrocytes, replication, and egress result in a progressively increasing cell-associated bacteremia, with a doubling time of roughly 24 hours. Depending on the strain of Anaplasma spp. and host susceptibility, 10% to 90% of erythrocytes may become infected; clinical signs appear when more than 15% of erythrocytes are infected.^7,8 Bacteremia results in RBC mem­brane peroxidation with infected cells showing a 10-fold increase in membrane leakage over uninfected RBC.^6,8 Anemia is due to splenic and hepatic macrophage-mediated phagocytosis of infected erythrocytes.

A. marginale expresses a variety of major surface proteins (MSPs), which facilitate interactions with vertebrate and invertebrate host cells.^7,9 This plasticity facilitates development of different bacterial surface structures leading to immune evasion and persistent infection in the animal. Phylogenetic analyses of a variety of strains have demonstrated the coevolution of A. marginale with tick vectors.^1,7 In addition, cases have been documented in which individual animals are infected by more than one genotype of A. marginale.⁶'^,'^v0

Protective immunity appears to require induction of both antibody against the outer membrane proteins and macrophage activation for enhanced phagocytosis and bacterial killing.⁷ The regenerative response to anemia can be vigorous and begins by 20 days post infection.

Epidemiology

A. marginale is the most prevalent of the tick-borne diseases of cattle worldwide and remains as a serious constraint to livestock production in tropical, subtropical, and even temperate regions.^11,12 In the United States, infection is endemic in much of the West, Midwest, and Southeast but occurs episodically in many states.⁷ As of April 1, 2014, the disease is no longer federally reportable in Canada, and testing for entry into the country is not required. The expansion of anaplasmosis into previously nonendemic regions has been attributed to wide­spread movement of cattle and to the effects of global warming 1712

on vector expansion.^1,7,12

Endemic regions are maintained by the prevalence of both competent tick vectors and persistently infected carrier cattle, which are typically asymptomatic but act as efficient reservoirs for tick-borne transmission to uninfected animals.^2,4 Vector activity varies by region, but outbreaks of anaplasmosis occur most frequently in the late spring and summer when arthropod activity is highest. Wild ruminants, including antelope, bighorn sheep, and elk have been experimentally infected with A. marginale, but the role they play in natural transmission is unknown. Male ticks can also remain persistently infected and a source for maintaining the bacteria in endemic regions.^4,11

While tickborne transmission is perhaps the most investi­gated route of spread, with at least 20 different species of ticks identified as sources of infection, other activities in which blood transfer occurs can result in infection.^3,4 Fomites (surgical instruments, dehorning equipment, ear tagging devices) are important sources of risk, particularly in nonendemic regions where tick vectors are not present.

Clinical Presentation

Clinical signs are highly variable, from acute severe disease to subclinical infection, and reflect variation in virulence among pathogen strains, breed resistance, and age-related host sus- ceptibility.^4,7 The incubation period is generally 10 to 30 days but can be as long as 60 days. Primary exposure in calf hood often produces asymptomatic or mild disease resulting in slight lethargy and anorexia for 24 to 48 hours.^6,7,10 Adult cattle are highly susceptible to acute disease characterized by a high fever ranging from 39.5° to 41° C (103° to 106° F), elevated heart rate, elevated respiratory rate, anorexia, lethargy, and in dairy cows, a dramatic decrease in milk production. Initial fever may be followed by subnormal temperatures, dry muzzle, and decreased rumination. Cattle may stagger or become aggressive as a result of cerebral hypoxia associated with anemia. Care must be taken not to stress severely anemic cattle because this may result in collapse and death. Mucous membranes are pallid or icteric if an animal has survived for 2 to 3 days past the acute crisis.^2,7 Additional clinical signs include constipation characterized by dark brown feces covered with mucus, pol- lakiuria with dark yellow urine, and abortion in late-gestation animals. Because hemolysis occurs extracellularly, hemoglo­binuria is not present.^3,5 Differential diagnoses include diseases that can produce anemia and/or icterus (babesiosis, bacillary hemoglobinuria, leptospirosis); hepatotoxic plant poisonings (Senecio), chemicals, and other causes of liver disease; and copper poisoning in sheep.

If an animal survives the acute crisis, the convalescent period is protracted (3 to 4 weeks) depending on the severity of the anemia, and icterus and weight loss are common.

Pathology

At necropsy, there are no pathognomonic lesions for the diagnosis of disease. In acute anaplasmosis the blood is thin, watery, and fails to clot readily. Mucous membranes, subcutane­ous tissues, liver, and skeletal musculature are pale; in later stages of acute disease, the same tissues may be icteric. Splenomegaly is a consistent finding; hepatomegaly and gallblad­der distention are commonly but less frequently observed.⁷ Urine is deep yellow, but neither hemoglobinuria nor hematuria occurs, which helps differentiate anaplasmosis from other hemolytic diseases. Occasionally petechiae may be found in the subepicardium, subendocardium, and other serous mem­branes. Detection of A. marginale-infected erythrocytes within capillaries of Giemsa-stained histologic sections can be used to confirm a diagnosis of anaplasmosis. ⁵

Diagnosis

Definitive diagnosis of acute anaplasmosis requires identification of A. marginale- or A. ovis-infected erythrocytes by microscopic examination or PCR in animals with severe anemia. A. marginale can best be detected in blood smears stained with Wright’s, new methylene blue, or Giemsa stain.^3,5,7 The inclusion is composed of a small morula of 2 to 8 individual organisms and as little as 5% or as many as 70% of erythrocytes may be infected. Creating a blood smear from freshly collected blood in the field may increase the likelihood of detecting parasites over blood smears made from blood exposed to anticoagulants. PCR provides increased sensitivity over direct exam and can differentiate animals infected with A. marginale, A.centrale, and A.

Following recovery, cattle and sheep remain persistently infected with 0.000001% to 0.1% of erythrocytes affected, and detection of organisms in erythrocytes is unreliable. Serol­ogy can be used to identify persistently infected animals.^3,7 The competitive enzyme-linked immunosorbent assay (cELISA [VMRD Inc., Pullman, Wash.]) provides high sensitivity and specificity and is used for official testing by many regulatory agencies (USDA, the Office of International Epizootics, most if not all state diagnostic laboratories.)^7,11

Treatment, Control, and Prevention

Therapy is aimed at minimizing or preventing the effects of A. marginale infection. Tetracyclines are the antibiotic of choice; however, once anemia is severe, treatment is often unsuccessful.¹² Short-acting oxytetracycline at 11 mg/kg IV q24h for 3 to 5 days or long-acting oxytetracycline at 20 mg/kg IM at 72-hour intervals may be effective.^2,7 Supportive therapy is important to maximize the chance of survival. If the PCV is 12% or below, whole blood transfusion (4 to 8 L) may be indicated to prevent death and shorten the convalescence period. A PCV of 8% or below indicates an unfavorable prognosis, and death often occurs despite appropriate antibiotic and supportive therapy. Antimicrobial therapy also neither eliminates persistent infection nor prevents disease, so alternative interventions are needed, especially in endemic areas.^7,11

Control measures used vary depending on the geographic region and type of livestock production system. In endemic regions with high transmission rates, beef cattle are often allowed to become naturally infected at a young age and remain asymptomatic carriers with minimal risk of later acute disease.² In regions with lower transmission rates, live blood­based vaccines may be used to ensure infection of cattle at a young age, such as the trivalent (A. marginale, Babesia bovis, Babesia bigemina) live vaccine used in Australia. Similarly, live vaccines based on A. centrale or weakly virulent strains of A. marginale are commonly used in Africa, Asia, and Central and South America.^3,9 These vaccines are not licensed for use in the United States, largely because of the risk of transmit­ting known or newly emergent pathogens contaminating the blood-based vaccine. The exception to this is the licensing of a live vaccine for use in California (AnaVac [PHL Associates, Davis, Calif.]). Importantly, live vaccines should only be used in young animals and are contraindicated for pregnant and older animals. Killed vaccines are less efficacious and require multiple immunizations but can induce at least partial protection against severe morbidity and mortality. An experimental killed vaccine (Experimental Anaplasmosis Vaccine [University Products LLC, Baton Rouge, La.) has been licensed for use in 14 states and Puerto Rico.¹¹

In the absence of immunoprophylaxis, anaplasmosis is usually controlled by preventing transmission. Advances in molecular characterization may provide additional targets for successful vaccine development.^12,15 Maintenance of an A. marginale-free herd in nonendemic areas can be accomplished by quarantine and serologic screening of all additions using the cELISA. In endemic regions, minimizing the exposure to vectors through strategic use of pastures, insecticides, and management of herd additions, as well as preventing iatrogenic transmission, can reduce the impacts of anaplasmosis.^2,7 Restricted use of antibiot­ics can also prevent elimination of the carrier status, which can protect from acute disease.¹¹ The risk of maintaining a fully susceptible herd within an endemic region should not be taken lightly.