Anaplasmosis and taxonomic issues Paul R Earl Facultad de Ciencias Biológicas Universidad Autónoma de Nuevo León San Nicolás, NL 66451, Mexico Paul R Earl

Tick-borne, anaplasmosis is a worldwide disease of cattle and other ruminants like deer and elk caused by a nonmotile gramnegative blood-inhabiting coccus: Anaplasma marginale Theiler, Theiler found A. centrale in the same South African cattle in While A. marginale found mostly at the edge of the red blood cell (RBC) causes anemia running to half the RBCs, A. centrale found often in the center of the RBCs usually has mild infections. Thus, A. centrale is long used universally as a live vaccine against A. marginale. However, this successful vaccine is not licenced in the US, nor does A. centrale occur in the US. More, A. marginale has been grown in tick cell cultures, and A. centrale has not been—a bit strange.

The acute phase of the disease is characterized by severe anemia, weight loss, fever, abortion, lower milk production and often death when cows are infected at 2 years or over. The only known site of development of A. marginale in cattle is within the erythrocytes in vacuoles. The number of infected erythrocytes increases logarithmically and removal of these infected cells by phagocytosis results in development of anemia and icterus. Cattle that recover from acute infection remain persistently infected and are protected from clinical disease, serving as reservoirs for mechanical transmission and biological transmission by ticks.

Human anaplasmosis (HA) This disease used to be called erhlichiosis or human granulocytic ehrlichiosis (HGE). This anemia is caused by Anaplasma phagocytophila, Ehrlichia equi and E. chaffeensis. Is E. canis involved ? HGE seems to be caused by one complex agent that is very similar to being just one species, even when combined with 2 companion strains. If so, then Ehrlichia is a synonym of Anaplasma.

Onset of illness occurs 1-3 weeks after exposure to an infected tick. Common signs and symptoms include fever, chills, headache and myalgias. Nausea, vomiting, anorexia, acute weight loss, abdominal pain, cough, diarrhea and change in mental status are reported infrequently. Highly suggestive laboratory findings include leukopenia (white blood cells, WBC< 4,500/mm³), thrombocytopenia (platelets <150,000/mm³) and increased aminotransferase levels. Unusual presentations may be the result of coinfections with Borrelia burgdorferi (Lyme disease agent) or Babesia microti (babesiosis agent) as a single feeding tick may transmit multiple disease agents.

Diagnostic tests and case definitions for HA An indirect immunofluorescence assay (IFA) is the principal test used to detect HA. Acute and convalescent sera can be evaluated to look for a 4-fold change in antibody titer to HA. Intracellular inclusions (morulas) also may be visualized in granulocytes (monocytes?) of Giemsa or Wright-stained blood. Also, polymerase chain reaction (PCR) assays are being used increasingly to detect and sequence HA DNA.

Treatment HA patients typically respond dramatically to doxycycline therapy (100 mg twice daily until the patient is afebrile for at least 3 days). Other tetracycline drugs also are also effective. Even if the diagnosis of HA is not confirmed, patients with unexplained fever after a tick exposure should receive empiric doxycycline therapy, particularly if they experience leukopenia or thrombocytopenia.

The new phylogeny At this point, the practical story has been told, at least for HA. Related bacteria are the genera “Ehrlichia,” “Cowdria,” Neorickettsia and Wolbachia, and also Aegyptianiella of chickens, ducks, etc., all obligate intracellular bacteria that live in vacuoles of eukaryotic cells. Recent genetic nucleotide sequencing of 16S rRNA genes, groESL and sequencing of surface protein genes indicate that the family of Anaplasmataceae was incorrectly ordered until corrected by Dumler and others (Internatl J Sys Evol Microbiol, 2001, 51: 2145–2165).

They found 4 clades: 1/ Anaplasma, including the Ehrlichia phagocytophila group, Ehrlichia platys and Ehrlichia bovis), 2/ Ehrlichia, including E. ruminantium, 3/ Wolbachia and 4/ Neorickettsia, including N. sennetsu and N. risticii ) Maximum similarity between clades ranged from 87<1 to 94<9 %.

The Anaplasma tree The top cluster of the tree of 7 species (5 + 2) belongs to Anaplasma, or is 2 species neither belonging to Ehrlichia. The middle cluster is possibly Ehrlichia. Wolbachia pipientis has no convincing taxonomic characters. Neorickettsia has no convincing characters that demonstrate its relationships.

Ehrlichia appears as 2 distinct groups which is quite wierd and unacceptable. This means that some organisms ORIGINALLY assigned to Ehrlichia do not belong to it. More, some strains that are very close indeed are split into species in a most arbitrary way. Some of these taxonomists may have rankraising disease. A strain becomes a species, a species a genus and so on. Anaplasma phagocytophila, A. equi & HGE equals Group A that is close to A. bovis & A. platys, and not far from Anaplasma marginale that has A. centrale as a likely synonym. All of Group A can be named Anaplasma spp.

Bovine anaplasmosis revisited Infection immunity Anaplasmosis occurs in most parts of the world. It is recognized in 40/50 United States and is particularly important in the gulf coast and southwestern states. See Richey, EJ: Bovine Anaplasmosis,1999, College of Vet Med, U of Florida, Gainsville.

Cattle that recover from acute infection remain persistently infected and are protected from clinical disease, serving as reservoirs for mechanical transmission and biological transmission by ticks. The incubation time for the disease to develop varies from 2 weeks to over 3 months, but averages 3-4 weeks. Calves, less than 1 year of age when infected by Anaplasma marginale do not show any signs of disease and become immune carriers. They are protected against the anemia and clinical disease as long as they are infected (infection immunity).

Transmission Ticks like Dermacentor andersoni and Boophilus microphilus are unique biological vectors, in that they carry A. marginale in their tissues and can infect cattle when feeding at subsequent molts or stages of their life cycles. Some species of ticks can transmit the organism from one tick generation to the next via their eggs.

The male and female of the tick Boophilus microphilus

Male of the tick Dermocentor andersoni

Diagnostic Tests Three tests are routinely used to detect the presence of anaplasma antibodies in blood. They are the complement fixation (CF), fluorescent antibody (FA), and the rapid card agglutination (RCA) tests. Acute cases seen by Giemsa stain at 1000 power have many parasirized RBCs. Acridine orange with a fluorescence microscope can be most helpful.

Treatment Once an infection has been diagnosed, the most effective treatment is the administration of oxytetracycline. One intramuscular injection (IM)of 9 mg/lb or 20 mg/kg of body weight of long-acting oxytetracycline during acute infection will usually prevent death if the disease is not too far advanced. Death is most often by hypoxia caused by anemia. In severe cases, however, the stress of capture and treatment may kill the animal. These animals may survive by just being provided with easy access to food and water. Nevertheless, blood transfusions must help, but—oddly enough—it is not used as a general treatment.

Vaccines Australia wins. This is an attenuated live vaccine based on mild Anaplasma centrale PLUS Babesia bigemina and B. bovis. Are those vaccinated cattle then carriers of these 3 infections for life ? Note that tick cells grow in several cell lines so that anaplasmas can be grown in vitro. Then the mix is tick and bacterial cells, not host cells. Does this make a difference? How? What is the objective ? The goal is to protect cattle against anemia leading to severe hypoxia. Why hasn’t A. centrale been grown in tick cultures ?

There are 2 types of vaccines commonly used to prevent clinical disease due to A. marginale. One type is a killed vaccine which contains killed organisms that stimulate an immune response that is adequate to protect against anemia and illness. The other type of vaccine is a modified live A. marginale that produces a "controlled infection". The live vaccine uses a modified live organism to infect the animal and the animal becomes immune to disease and also becomes a carrier of the vaccine organism. As calves are not susceptible to the anemic disease that affects adult cattle, they can safely be given modified live vaccines. Obviously, the modified live vaccine should not be given to adult cattle as it can even kill them.

Cattle traffic If unprotected adult cattle are taken to a locale where A. marginale is common they can become infected and can experience heavy losses due to anaplasmosis. Cattle such as these should be vaccinated with a killed vaccine a month or more before being taken into high risk areas. Carrier cattle may be cleared of infection by antibiotics. Such animals revert to a negative status when tested with CF, FA or RCA tests. Unlike those few animals that naturally and spontaneously clear themselves, those cleared via antibiotics remain resistant to severe illness when re-exposed. Methods employed to clear anaplasma-positive animals include feeding tetracycline or chlortetracycline at 5 mg/lb or 10 mg/kg of body weight for days.

What do we have left? 1/ Can some vaccine drive out (eradicate) anaplasmosis? 2/ Is vector control really possible? 3/ By insecticide dipping? 4/ Per district, does the RISK call for vaccination? 5/ What is the story of insecticide- impregnated ear tags? 6/ What about babesiosis? 7/ Are sudden outbreaks of cattle diseases properly recorded and analyzed? 8/ Is there a profound epidemiology of anaplasmosis beyond personal field experience? 9/ Is there a system for farmers and others to report morbidity & mortality? 10/ Can you have disease-free, or must you have infected?