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.

Rhipicephalus bursa is a common tick parasite of small-to-medium size ungulates, principally in warm, temperate, and subtropical areas. Although common in livestock and showing a wide geographic distribution, its epidemiological role in tick-borne bacterial disease is barely known. This study addressed the knowledge gap and aimed to screen for the presence of Anaplasmataceae and spotted fever group (SFG) Rickettsia species in R. bursa ticks collected from domestic animals in Romania, Eastern Europe. A total of 64 pools of R. bursa ticks collected from small ungulates were tested by PCR for Anaplasmataceae DNA presence using group-specific primers. Specific testing was performed for Anaplasma marginale/A. centrale/A. ovis, A. platys, A. phagocytophilum, Ehrlichia canis, and SFG Rickettsia. The positive samples were purified and sequenced, and sequences analysis was used to identify the species and to confirm the PCR results. The only pathogen identified in this study was E. canis. The obtained sequences confirmed the PCR results. The presence of E. canis in R. bursa in Romania and in ticks from sheep was shown for the first time in this study. Based on these findings, it may be presumed that the E. canis DNA originated from ticks; however, the vectorial role of R. bursa (and other arthropod species) in the transmission of E. canis should be proved experimentally.

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.

Isolated Anaplasma marginale initial bodies were successfully used in a dot ELISA for rapid detection of antibodies to Anaplasma organisms. The enzyme immunoassay used only 25 ng of antigen dotted onto nitrocellulose disks. Antigen-antibody complexes were detected by use of alkaline phosphatase-conjugated protein A, and reactions were read visually after addition of a precipitable, chromogenic substrate. The test allowed the processing of multiple sera, either for screening or for titer determination, in < 3 hours and was found to be as sensitive as the indirect fluorescent antibody test. The overall performance of the dot ELISA, using isolated A marginale initial bodies, for 580 bovine serum samples was as follows: sensitivity, 93%; specificity, 96%; and predictive value, 95%. Cross-reactivity was not observed with sera positive to Babesia bovis and B bigemina, Trypanosoma vivax, or common bacteria or viruses infecting cattle. The antigen dotted onto nitrocellulose disks was stable when stored at -20, 4, or 25 C. Compared with the indirect fluorescent antibody test, the dot ELISA allowed easier, faster, and more objective interpretation of results. Its simplicity and low cost combined with high sensitivity and specificity indicate that this assay could effectively replace serologic assays currently used for diagnosis of anaplasmosis in cattle.

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 one of the most important diseases that threaten livestock production worldwide especially in developing countries, in cattle mainly caused by obligate intra-erythrocytic Anaplasma marginale. A. marginale is transmitted biologically by ticks (Hyalomma, Rhipicephalus). Bovine anaplasmosis causes mild to severe clinical signs ranging from anorexia, fever, anemia, and respiratory manifestations to icterus and death. Molecular detection is the best method for Anaplasma diagnosis because of its ability to detect sub-clinical and carrier hosts. This study investigated the occurrence of A. marginale infection among cattle in the Qena governorate utilizing a molecular assay based the msp5 gene. A total of 100 whole blood samples were collected randomly from apparently healthy and diseased cattle. Such cattle were examined clinically, and their samples were included for microscopic examination. PCR screening of the tested cattle showed 23% (23/100) as a positive rate. While 6 samples from 100 (6%) showed A. marginale parasite in the microscopic examination. Several risk factors were analyzed in the current study, higher incidence rates were detected in animals less than 2 years than older than 2 years, Holstein-Friesian breeds than crossbreeds and in animals kept in small farms than in the mass farming system. Clinical and hematological variables were also investigated in several infected and non-infected cattle based on PCR reactivity. Fever, anorexia, respiratory manifestations, enlarged lymph nodes, pale or icteric mucous membranes and digestive disorders were reported in infected cattle (n= 23) but not in non-infected animals (n=77). Consistently, hematological variables in infected cattle (n = 10) revealed significantly lower RBCs count and hemoglobin content than those in the non-infected group (n=20) indicating hemolytic anemia. This study shows the high prevalence of A. marginale in cattle in Qena governorate associated with health hazards and multi-risk factors, so frequent usage of acaricides, regular examination of cattle, and successful chemoprophylaxis are recommended.

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 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 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.

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.