BACKGROUND: There is paucity of information about Anaplasma marginale (A. marginale) infection in water buffaloes and there have not been any reports of clinical anaplasmosis in the buffaloes in Iran. OBJECTIVES: Molecular and hematologic survey on A. marginale infection in apparently healthy buffaloes referring to Ahvaz abattoir. METHODS: Samples of blood and spleen tissue were obtained from 103 healthy buffaloes referring to the slaughterhouse. Blood samples were subjected to microscopic examination and PCR assay while spleen specimens were only analyzed by PCR. In this study, a nested-PCR method was used to amplify a fragment of the groEL gene of the bacterium. RESULTS: According to PCR, 31.1% and 1.9% of examined blood and spleen samples were found positive for A. marginale, respectively. The buffaloes which were positive in spleen tissue PCR test were positive in blood PCR, as well. Microscopically, Anaplasma-like organisms were found in 15.5% of stained blood smears. There was a slight Kappa agreement between stained blood smears and PCR. No significant difference was found in hematologic values between the infected and non-infected buffaloes based on PCR results. CONCLUSIONS: Significant occurrence of infection with A. marginale in the studied buffaloes can indicate the probable role of buffalo as a reservoir of the disease agent and its transmission to the cattle.

Anaplasmosis is a tick-borne disease of ruminants and wild animals that caused by an intra erythrocytic bacterium, Anaplasma marginale. Under natural conditions, camels become infected in areas where the disease is endemic. Camels that survive from acute infection become carriers because of the capability of these bacteria to deception the immune system using antigenic variations. Although, several serological methods were concerned for Anaplasma marginale IgG antibodies detection, but the competitive indirect ELISA test was more sensitivity and specificity. The present study was conducted at Al-Najaf and Wasit provinces on 120 camels, selected randomly from both sexes and divided into two aged groups. The total sero positivity prevalence was (10.83%); and depending on provincial basis was (8.57%) in Al-Najaf and (14%) in Wasit provinces. Clinically, the sero positive prevalence two age groups (5 years old) had (6.67%) and (15%), respectively. No significant differences (P

Three available differential stains, Camco-Quik, Diff-Quik, and Wright-Giesma were compared for detection of intraerythrocytic Anaplasma marginale in bovine blood smears. In samples where < 1% to more than 51% of the RBC were infected, statistical analysis of the data indicated no significant difference in the detection of A marginale with Camco-Quik or Diff-quik stains. However, a significantly lower percentage of infected RBC were detected when blood smears were stained with the Wright-Giemsa stain, compared with the other 2 methods.

Transstadial and transovarial transmission of Anaplasma marginale by Dermacentor variabilis were attempted with ticks exposed to the organism once by feeding as larvae or nymphs, and twice by feeding as larvae and nymphs. Typical colonies of A marginale were in gut tissues of adults that were infected as larvae, larvae and nymphs, and as nymphs; repeated exposure of ticks did not appear to result in an increase in the number of colonies in the gut of subsequently molted adults nor did it affect severity of the clinical disease that developed in cattle they fed on. In contrast, colonies of A marginale were not found in the midgut epithelium of unfed nymphs exposed as larvae, even though companion nymphs transmitted the parasite, causing severe clinical anaplasmosis in susceptible calves. The organism was not transmitted transovarially by F1 larvae or nymphs from the groups exposed as parent larvae, nymphs, larvae and nymphs, and as adults. Some of the calves fed on by F1 progeny had a few erythrocytic marginale bodies that looked suspiciously like A marginale, as well as postchallenge exposure prepatent periods that were longer than other calves in the transovarial transmission study. Sera from these calves were tested for antibody to A marginale, using a highly sensitive immunoblot technique. Antibodies were not detected in any of the sera.

Anaplasma marginale was propagated in a tick cell line derived from Dermacentor variabilis embryos. The rickettsial organism was identified and monitored in culture by transmission electron microscopy and the indirect immunofluorescence technique, using specific monoclonal antibodies. Inoculation of the embryonic tick cell line with midguts of infected adult ticks (culture 1), nymphal ticks (culture 2) and adult ticks that were infected as nymphs and dissected as adults (culture 3) resulted in 3 continuous cultures of A marginale. Culture 1 had been maintained through 22 passages over a 11-month period; cultures 2 and 3 had been maintained for 18 passages over a 9-month period. Growth of A marginale in the cell line began in the area of the nuclear membrane at approximately 4 days after inoculation or transfer. Thereafter, the organisms were observed in inclusions scattered throughout the cytoplasm of the host cells. Maximal growth of the organism occurred at 7 to 14 days, after which numbers of inclusions rapidly decreased to minimal or undetectable levels. The organism began new cycles of growth with each 1:5 to 1:10 split and transfer of the host cells. Electron microscopy of recently infected cells revealed a morphology of the organism that closely resembled that observed in marginal bodies of infected erythrocytes. After several passages, A marginale organisms had a varied morphology and resembled the organism described in midgut cells of naturally infected ticks. Substitution of adult bovine serum for fetal bovine serum and adjustment of the pH of the medium from 6.9 to 7.4 resulted in several-fold increases in amount of growth and reduced the period required to reach maximal growth to a predictable time of 5 to 7 days. The importance and potential of this method of continuous laboratory propagation of A marginale are discussed.

Bovine anaplasmosis is the disease caused by the bacterium Anaplasma marginale. It can cause production loss and death in cattle and bison. This was a reportable disease in Canada until April 2014. Before then, infected herds were quarantined and culled, removing infected animals. In North America, A. marginale is biologically vectored by hard ticks (Acari: Ixodidae), Dermacentor variabilis and D. andersoni. Biting flies, particularly horse flies (Diptera: Tabanidae), can also act as mechanical vectors. An outbreak of bovine anaplasmosis, consisting of 14 herds, was detected in southern Manitoba in 2008. This outbreak lasted multiple rounds of testing and culling before eradication in 2011, suggesting local maintenance of the pathogen was occurring. We applied novel approaches to examine the vector ecology of this disease in this region. We did not detect A. marginale by screening of 2056 D. variabilis (2011 and 2012) and 520 horse flies (2011) using polymerase chain reaction (PCR).

The ionophore A23187 was used to facilitate release and continued development of Anaplasma marginale in short-term erythrocyte cultures. Addition of 10 micromolar 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 micromolar 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 micromolar 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 micromolar 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.

The 1-host tick Boophilus annulatus was found to transmit anaplasmosis in cattle transstadially. Anaplasma marginale was invariably transmitted when ticks that had been pulled off Anaplasma-infected calves either after 7 days (as fully engorged larvae) or after 14 to 15 days (as fully engorged nymphs) were transferred within 4 days to susceptible calves. Three morphologically different A marginale isolates, 1 round (tailless) and 2 with different types of appendages (tailed) were transmitted by the ticks. These findings might explain the overlap of the geographic distribution of the disease and that of Boophilus spp in some areas of the world.

The development of Anaplasma marginale was studied in Dermacentor andersoni nymphs after they had fed on a calf with ascending Anaplasma infection. Gut tissues were collected on day 4 of tick feeding, from newly replete (fed) nymphs and on postfeeding days (PFD) 5, 10, 15, 20, and were processed for light and electron microscopy to determine density of A marginale colonies. Homogenates of gut tissues were prepared from nymphs collected on the same days and inoculated into susceptible, splenectomized calves to test for infectivity. Anaplasma colonies were detected in gut cells on PFD 5, 10, 15, and 20. Although colony density appeared to be higher on PFD 10 and 15, differences were not significant. Nymphal type-1 colonies were detected in highest numbers on PFD 5 and 10, transitional colonies were seen in highest numbers at PFD 10 and 15, and nymphal type-2 colonies were observed only on PFD 20. Gut homogenates that were collected from ticks at 4 days of feeding, when newly replete, and on PFD 20 caused anaplasmosis when injected into susceptible calves, but homogenates made from ticks collected on PFD 5, 10, and 15 were not infective. The data indicate that of the colony types of A marginale that develop in replete nymphs, nymphal type-1 and transitional colonies may contain organisms that are not infective for cattle.