First Survey on the Presence and Distribution of Oxytetracycline-Resistance Genes in Anaplasma Species.

INTRODUCTION: Anaplasma sp. is an obligatory intracellular Gram-negative tick-transmitted bacterial pathogen of humans and animals. Oxytetracycline and chlortetracycline are the drugs of choice for treating domestic animals with acute anaplasmosis. Lack of documented information about oxytetracycline resistance in Anaplasma species in the world was the scope of this study to screen by PCR for the detection of the oxytetracycline-resistance genes in Anaplasma species from infected cattle and sheep in the Northwest and Southwest of Iran. MATERIALS AND METHODS: Total of 100 cattle and sheep blood samples collected from 2 provinces in the Northwest and 1 province in the Southwest of Iran were tested microscopically by the Giemsa staining examination and confirmed by PCR. Then the presence of two different oxytetracycline-resistance genes (otrA, and otrB) was detected by PCR in positive samples. RESULTS: The results showed that 60% of Anaplasma-infected samples were identified to have an otrA-resistance gene, and 26.67% had an otrB-resistance gene. The coexistence of two oxytetracycline-resistance determinants was encountered in 13.33% of the isolates. The significant difference in the frequency of otr genes was found among three Anaplasma species (A. marginale, A. centrale and A. ovis), and among three studied regions in Iran (p < 0.05). The identified sequences were submitted to the GenBank and deposited under accession numbers MN880729 and MN895439 for otrB and otrA genes. CONCLUSION: This study, for the first time, indicated the oxytetracycline-resistance genes in the three most prevalent Anaplasma species in ruminants. This finding helps to select an appropriate treatment strategy for eradication of anaplasmosis.

Ehrlichia canis in dogs experimentally infected, treated, and then immune suppressed during the acute or subclinical phases.

BACKGROUND: Concerns for recrudescence of Ehrlichia canis infection arise when immunosuppressive drugs are used to treat immune-mediated diseases in dogs previously infected with E. canis. OBJECTIVES: Determine whether administration of prednisolone and cyclosporine would reactivate E. canis infection in dogs previously treated with doxycycline during the acute or subclinical phases. ANIMALS: Seven beagles previously experimentally infected with E. canis and administered doxycycline for 4 weeks were included. Three of the 7 dogs were incidentally concurrently infected with Anaplasma platys and Babesia vogeli and were administered 2 doses of imidocarb 2 weeks apart before enrollment in the current study. METHODS: Experimental study. Each dog was administered prednisolone and cyclosporine for 6 weeks. Clinical signs, complete blood cell count (CBC), polymerase chain reaction (PCR) assays for E. canis, A. platys, and B. vogeli DNA in blood, E. canis indirect fluorescent antibodies (IFA) titers, and flow cytometry for antiplatelet antibodies were monitored. RESULTS: All dogs completed the immunosuppressive protocol. No evidence for recrudescence of E. canis, A. platys, or B. vogeli were detected based on clinical signs or results of CBC, PCR, IFA, and flow cytometry for antiplatelet antibodies. E. canis IFA titers were negative in 5/7 dogs at the end of immunosuppressive protocol and were negative 6 months after the protocol in 5/5 dogs available for testing. CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs administered with a 4-week course of doxycycline with or without imidocarb failed to show evidence of activation of E. canis infection after administration of a commonly used immune suppressive protocol.

Effect of tetracycline on development of Anaplasma marginale in cultured Ixodes scapularis cells.

Infections of the tick-borne ehrlichial pathogen, Anaplasma marginale, in cattle have been controlled, in part, by administration of low doses of tetracycline. Recently, a cell culture system was developed for A. marginale using a tick cell line derived from embryonic Ixodes scapularis. This study was designed to determine the effect of tetracycline on A. marginale propagated in a tick cell culture assay. Various concentrations of tetracycline (0, 0.01, 0.10, 1.0, 5, 10, 20 or 100 microg/ml) were added in medium to cultures 48h after cell monolayers were inoculated with A. marginale. A. marginale growth in the drug treated and control cultures was subsequently evaluated by indirect ELISA at 7 days post-infection (PI) and daily by light and electron microscopy (LM and EM). Infectivity of the culture-derived A. marginale was determined by inoculation of susceptible cattle with treated and untreated control cultures. Tetracycline doses of 5, 10, 20 and 100 microg/ml resulted in significant inhibition of A. marginale growth as determined by ELISA. Morphologic deterioration of Anaplasma, as determined by LM and EM, occurred in cultures treated with the same drug concentrations. A. marginale replication, inhibited in cultures treated on days 2-6 PI with 20 microg/ml tetracycline, was not apparent 96 days after antibiotic removal. Infected cell cultures treated with medium containing 20 microg/ml tetracycline proved to be non-infective when inoculated into susceptible splenectomized calves. All parameters studied herein demonstrated that tetracycline killed A. marginale in cultured tick cells. The Anaplasma-tick cell culture drug assay therefore, would be useful for screening and evaluating novel antibiotics for control of anaplasmosis.

Effect of 4-bromo-calcium ionophore A23187 on release of Anaplasma marginale from bovine erythrocytes in vitro.

The ionophore A23187 was used to facilitate release and continued development of Anaplasma marginale in short-term erythrocyte cultures. Addition of 10 microM A23187 to the cultures resulted in significant decrease in percentage of parasitized erythrocytes (PPE) by 24 hours after treatment; further development and increase in PPE was not observed. In contrast, the PPE of untreated cultures, those treated with dimethyl sulfoxide (DMSO) only and with 1 microM A23187 increased slightly during that time. Total erythrocyte count decreased in treated cultures in excess of that expected after samples of the medium were taken for analysis. The greatest cell loss and increased hemoglobin concentration in culture medium was observed in cultures treated with 10 microM A23187 and with an equivalent volume of DMSO. The DMSO appeared to cause hemolysis of some erythrocytes, but not of infected cells selectively. Release of A marginale inclusion bodies was seen by electron microscopy in samples from the 10 microM A23187-exposed cultures. At 30 minutes after treatment, free initial bodies were frequently seen. Inclusion body membranes and individual A marginale were associated with membranes of adjacent erythrocytes. Individual rickettsiae were seen in cell depressions and appeared to be entering erythrocytes. However, neither further invasion nor development of the parasite in erythrocytes was observed. Ionophore A23187 appeared to promote release of A marginale from erythrocytes, but did not enhance infection of erythrocytes or development of organisms in vitro.

Influence of a second Anaplasma exposure on the success of treatment to eliminate Anaplasma carrier infections in cattle.

Treatment of adult Anaplasma carrier cows, with long-acting oxytetracycline at dosage levels generally successful in eliminating infection, was unsuccessful when the treatment was preceded or accompanied by a 2nd exposure to A marginale on days 0, 7, or 14 before treatment. Noninfected calves exposed to A marginale 7 days before a similar treatment developed anaplasmosis and became carriers of infection.

Morphologic alteration of Anaplasma marginale in calves treated with a dithiosemicarbazone.

Blood and clotted blood samples from 3 calves with anaplasmosis were examined by light and electron microscopies before and after the calves were treated twice with alpha-ethoxyethylglyoxal dithiosemicarbazone (given IV). In the treated calves anaplasma bodies were swollen and irregularly shaped and had rough surfaces when viewed by light microscopy. Posttreatment electron microscopic examinations revealed swelling and vacuolation of initial bodies of anaplasma bodies, and ballooning of the vesicular membrane surrounding the anaplasma body. Other initial bodies were ameboid in shape this was or was not accompanied by rupture of the vesicular membrane of the anaplasma body. Necrotic, ameboid-shaped initial bodies also were seen. Some necrotic initial bodies were surrounded by complete vesicular membrane, and others were close to a broken, curled vesicular membrane.

Morphologic alterations of Anaplasma marginale in calves after treatment with oxytetracycline.

The morphologic features of Anaplasma marginale were determined after treatment of infected calves with oxytetracycline. By light microscopy, anaplasma bodies in erythrocytes of treated calves were enlarged and vacuolated, small and dense, or comma shaped. Several degenerated forms of anaplasma bodies were observed by electron microscopy. There was a blending of the 2 membranes surrounding initial bodies, aggregation of nucleoprotein at the periphery of the subunit, and vacuolation. A 2nd form of degeneration was coalescence of subunits in marginal bodies. A 3rd form of degeneration was unification of subunits of anaplasma bodies and clumping of nucleoprotein. A 4th form of degeneration was the persistence of an anaplasma body as a solitary, irregularly shaped mass containing electron-opaque clumps.

Propagation of Anaplasma marginale in bovine lymph node cell culture.

Anaplasma marginale was propagated in cell cultures derived from bovine lymph node (LN). Treatment of host cells with diethylaminoethyl dextran (DEAE-D) before inoculation and centrifugation of inoculum onto the monolayers resulted in significant numerical increases of A marginale. The direct fluorescent antibody technique (FAT) was used for detection of the organism in culture. The rat was combined with the standard microscopic count procedure to obtain numerical estimates of the organism in cell culture. Infection of LN cells was irregular, with some cells containing many organisms and others containing none. The organisms were dispersed or in inclusions in the cytoplasm of LN cells. Numerical increases of organisms occurred within 6 hours and these were greatest at 12 to 24 hours. After 24 hours, the organisms decreased rapidly, but small numbers of them were observed for at least 7 days. The average generation time in culture was approximately 17.1 hours.