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.

Bovine haemoparasites are cosmopolitan in distribution and are known to cause substantial losses to the cattle industry. In spite of their economic importance, there remains a dearth of information on their molecular epidemiology in many parts of the world including Malaysia. To ascertain the molecular prevalence and species co-infection of bovine haemoparasites in the country, blood samples were collected from 1,045 heads of beef and dairy cattle on 43 farms from six geographical zones throughout Peninsular Malaysia. Samples subjected to PCR amplification of parasite species-specific genetic fragments revealed that Anaplasma marginale was the most prevalent haemoparasite (72.6%),followed by Theileria orientalis(49.8%),Candidatus Mycoplasma haemobos ( 47. 0 % ),Babesia bovis(32. 5%), Babesia bigemina (30.5%) and Trypanosomaevansi(17.9%). A high percentage (92.1%) of cattle was infected with either one or more haemoparasites. Triple haemoparasite species co-infection was the most prevalent (25.6%), followed closely by double species co-infection (25.1%). The most common (8.8%) and significantly correlated(rs= 0.250; p<0.01) combination was A. marginale+ T.orientalis. The present study constitutes the first attempt in the country to document the molecular prevalence and species co-infection of bovine haemoparasites over a wide spatial distribution. The data obtained will facilitate treatment, control and prevention measures to improve the local cattle industry.

Development of the rickettsia, Anaplasma marginale, salivary glands of male Dermacentor andersoni exposed as nymphs or adult ticks, was studied indirectly by inoculation of susceptible calves with homogenates and directly by examination, using light microscopy and a DNA probe; some unfed ticks were incubated before tissues were collected. Salivary gland homogenates made from ticks in every treatment group caused anaplasmosis when injected into susceptible calves; prepatent periods decreased as the time that ticks had fed increased. Colonies of A marginale were seen only in salivary glands of ticks exposed as adults and not in those exposed as nymphs; the percentage of salivary gland acini infected in these ticks increased linearly with feeding time. However, the probe detected A marginale DNA in salivary glands of ticks from both groups; the amount of DNA detected increased as feeding time was extended. The amount of A marginale DNA appeared to remain constant in gut tissues, but to increase in salivary glands. Salivary glands of adult-infected male ticks that were incubated, but did not feed a second time, became infected with A marginale, and the pattern of infection of acini varied with incubation temperature. Development of A marginale in salivary glands appears to be coordinated with the tick feeding cycle; highest infection rate was observed in ticks exposed as adults.

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.

The infectivity and immunogenicity of Anaplasma marginale grown in a tick cell culture from embryonic Dermacentor variabilis ticks were assessed in splenectomized and intact calves, respectively. Culture 1 consisted of the cell line inoculated with midguts of adult ticks infected with the Mississippi isolate of A marginale and dissected 5 to 10 days after repletion and detachment from an experimentally infected calf. Cultures 2 and 3 consisted of the cell line inoculated with midguts of ticks infected with the Virginia isolate of the organism. Inoculum for culture 2 was derived from nymphal ticks dissected 5 to 10 days after repletion and detachment from the infected calf; inoculum for culture 3 was midguts from adult ticks that were fed as nymphs, allowed to molt in the laboratory and dissected 21 to 24 days after molting. In trial 1, cultures 1, 2, and 3 were maintained at pH 6.9 and incubated at 28 C; in trial 2, cultures 1 and 3 were maintained at pH 7.4 and incubated at either 28 C or 37 C. Cultures 1, 2, and 3 failed to induce infection when injected IV and SC into 6 calves in 2 separate trials. Prechallenge sera from these calves reacted with 2 purified Anaplasma antigens in the ELISA, but failed to react in the complement-fixation test. Results of a trial to use cultures 1 and 3 in combination with an oil-in-water adjuvant to immunize intact calves against A marginale were inconclusive. However, prechallenge sera from immunized calves reacted with the 2 purified Anaplasma initial body antigens in the ELISA but failed to react in the complement-fixation text. When reacted against electrophoretically separated A marginale initial body proteins disrupted by sodium dodecyl sulfate, prechallenge serums from calves used in infectivity and immunization trials reacted with a majority of the antigens precipitated by an animal experimentally infected by inoculation of infected blood. This offers additional evidence that A marginale was maintained in the tick culture for up to 11 months and that the organism in culture antigens similar, if not identical, to the erythrocytic stage of the rickettsial agent. The importance of the laboratory culture of A marginale is discussed.

On each day of feeding on susceptible calves, salivary glands obtained from groups of adult ticks that transmitted Anaplasma marginale were examined for A marginale colonies by use of light microscopy and transmission electron microscopy. On day 8 of feeding, salivary glands were examined, using fluorescein-labeled antibody and methyl green-pyronine stain. Use of fluorescein-labeled antibody consistently revealed small numbers of fluorescent foci in salivary gland acinar cells obtained from ticks that had fed for 8 days. Colonies of A marginale were seen by transmission electron microscopy only in salivary gland acini of male ticks; these colonies could not be identified, using light microscopy, in companion 1-micron plastic sections stained with Mallory stain. Methyl green-pyronine stain, used commonly to detect theilerial parasites in tick salivary glands, did not differentiate A marginale from cytoplasmic inclusions normally found in salivary gland acinar cells.

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.