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 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.

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.

Salivary glands from males of 3 Dermacentor species (D andersoni, D variabilis and D occidentalis) that were infected with either the Virginia or Idaho isolate of Anaplasma marginale as nymphs or adults were examined for colonies of A marginale by use of light and electron microscopy. Prior to dissection of salivary glands, exposed ticks were held at 25 C for 15 to 18 days, followed by a 3-day incubation at 37 C. Ticks of 2 species transmitted A marginale to calves; the third tick species was confirmed infected by demonstration of typical colonies in tick gut cells, but transmission was not attempted; Colonies of A marginale were seen with light microscopy in salivary glands of all 3 species of ticks; they were located in acinar cells that contained simple granules. Colonies varied morphologically from small, compact ones to larger structures that contained distinct organisms and often were adjacent to the host cell nucleus. Electron microscopy confirmed that the colonies were rickettsial organisms. Morphologic features of A marginale varied and included reticulated forms, forms with electron-dense centers, and small particles; these various forms were similar to those described previously in midgut epithelial cells of ticks. We believe that the organism seen within tick salivary glands may replicate in the glands before its transmission to the vertebrate host.

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.