Development of the rickettsia, Anaplasma marginale, in 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.

The persistence of Anaplasma marginale Theiler in male Dermacentor andersoni Stiles ticks exposed to the organism as adults was studied as the ticks were successively transferred to five susceptible calves. All calves fed upon by these ticks rapidly developed clinical anaplasmosis; incubation periods of infection ranged from 19 to 26 d and did not change significantly with successive feedings. Development of A. marginale in tick midgut and salivary glands was followed daily during tick feeding (total, 35 d) with light microscopy and DNA hybridization. With microscopy, A. marginale colonies persisted in midgut cells throughout the experiment. Large colonies were observed in gut muscle cells on days 8 through 35 and were the predominant infected cell type during this part of feeding. Colonies were seen in salivary gland acini from day 2 throughout the 35-d experiment. The DNA probe confirmed the presence of Anaplasma DNA in midgut and salivary glands throughout the experiment. Quantitative estimates of infection intensity in tissues of individual ticks approximated 10(7) initial body equivalents, confirming heavy infections. A marginale in midgut tissues decreased with feeding time, whereas the estimated number of organisms in salivary glands remained constant. These data demonstrate that D. andersoni males are efficient vectors of A. marginale and may be potential reservoirs of infection for ruminants for extended periods.

The development and transmission of Anaplasma marginale was studied in Dermacentor andersoni males. Laboratory-reared male D andersoni were allowed to feed for 7 days on a calf with ascending A marginale parasitemia. The ticks were then held in a humidity chamber for 7 days before being placed on 2 susceptible calves. Anaplasmosis developed in the calves after incubation periods of 24 and 26 days. Gut and salivary glands were collected from ticks on each day of the 23-day experiment and examined with light and electron microscopy. Colonies of A marginale were first observed in midgut epithelial cells on the sixth day of feeding on infected calves, with the highest density of colonies found in gut cells while ticks were between feeding periods. The first colonies contained 1 large dense organism that subsequently gave rise to many reticulated organisms. Initially, these smaller organisms were electron-lucent and then became electron-dense. On the fifth day after ticks were transferred to susceptible calves for feeding, A marginale colonies were found in muscle cells on the hemocoel side of the gut basement membrane. A final site for development of A marginale was the salivary glands. Colonies were first seen in acinar cells on the first day that ticks fed on susceptible calves, with the highest percentage of infected host cells observed on days 7 to 9 of that feeding. Organisms within these colonies were initially electron-lucent, but became electron-dense.

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.

Dermacentor variabilis were infected as nymphs with Anaplasma marginale by allowing the ticks to feed on a single infected donor calf. Two weeks after molting to the adult stage, the ticks were allotted into 1 of 3 groups and were allowed to overwinter at room temperature (25 C) in the laboratory (group 1), cold storage (4.5 C) in the laboratory (group 2), or outdoors in leaf litter (group 3). Persistence of A marginale was assessed by determining density of colonies (number of colonies/0.1 mm2 of gut tissue examined) in tick gut specimens at 3, 5, 7, 9, and 12 months after molting to the adult stage. Colonies of A marginale were found in all groups at every density evaluation period. Highest colony densities were observed uniformly in specimens collected at month 7 (May); densities decreased at month 9 and were lowest at month 12. Statistical analysis indicated that ticks subjected to cold storage and to outdoor conditions had similar colony densities of A marginale; the density curve in these 2 groups indicated significant quadratic effects over time, with peak densities in May. Mean colony density in ticks kept at room temperature fit a different quadratic equation. The morphologic data indicated that A marginale overwinters in Dermacentor variabilis, and that increasing numbers of organisms are found from January to May.

Colonies of Anaplasma marginale in midgut epithelial cells of experimentally infected Dermacentor andersoni were studied in adult ticks 1, 3, and 6 months old. Longevity of the parasite in ticks was assessed by evaluating its infectivity for splenectomized calves; calves were exposed by feeding ticks and by inoculation of tick gut homogenates. Longevity was also evaluated by determining size, type, and density of colonies in male and female ticks. The effect of incubation (2.5 days at 37 C) on colony density was also examined for ticks at each age period. All methods used to assess longevity of A marginale in ticks (tick transmission, calf inoculation, and histologic studies) indicated a decrease of the numbers of organisms in 6-month-old ticks. Furthermore, when tick gut homogenates from 6-month-old nonincubated ticks were not infectious for susceptible calves, incubation of ticks before dissection restored infectivity of homogenates. Colonies of A marginale were detected in gut tissues of 6-month-old ticks that were not infective; therefore, infectivity of ticks could not be confirmed merely by presence of A marginale colonies.