An immunocytochemical marker for the complex granules of tick salivary glands which traces e-granule shedding to interstitial labyrinthine spaces.

An antigenic protein (12C), previously isolated from salivary glands of Rhipicephalus appendiculatus, as a possible factor in host resistance to repeated tick infestation, is labeled by protein A-gold immunocytochemistry in adult feeding ticks. It is a component of the gland's complex a-, d- and e-granules and also appears within the chitinous walls of intercalated ducts. In females, between days 4 and 7 of feeding, labeled e-granules appear also within the labyrinthine spaces of acinus type III, apparently released from e-cells as the abluminal interstitial cells initiate formation of a basolateral labyrinth. Granules thus shed are fragmented by interstitial cell processes, some fragments being phagocytized, others disintegrating to single point label scattered throughout the labyrinth. The latter possibly may pass into the acinar lumen. By the eighth day the label is gone from the labyrinth.

Ultrastructure of anaplasmal inclusions (Pawhuska isolate) and their appendages in intact and hemolyzed erythrocytes and in complement-fixation antigen.

Hemolysis of parasitized erythrocytes augmented visualization of the anaplasmal inclusion, including its initial bodies, inclusion membrane, and inclusion appendage ("tail" or "band"). A dense attachment complex joined the appendage to the inclusion membrane (wall of the inclusion vacuole). The inclusion appendage consisted of tightly packed, interconnected laminae and assumed loop, dumbbell, and comet configurations described by other workers. The erythrocytic plasmalemma and the inclusion membrane had a thickness of 9.0 +/- 0.8 nm and similar structures. The initial bodies were covered by a thin inner organismic membrane (7.0 +/- 0.7 nm thick) attached to the organismic chromatin, an intermembranous matrix, and by an outer membranous sheath or pellicle (12.5 +/- 1.2 nm thick). Dense granular aggregates (24 to 40 nm in diameter) within chromatin clumps were the only structures in the initial body remotely similar to ribosomes, yet they were too large, were never free of chromatin, and appeared to disappear upon hemolysis. Complement-fixation antigen prepared by fractionation contained initial bodies, inclusion appendages, a few mitochondria, vesicularized membranes, and stromal debris. The preparatory treatment caused segregation of the organismic chromatin into independent dense particles 103 +/- 12 nm in diameter still bound by inner organismic membrane. Similar particles were seen also in the plasma and inclusion vacuoles of hemolyzed erythrocytes.

Ultrastructural localization of anaplasmal antigens (Pawhuska isolate) with ferritin-conjugated antibody.

Antibodies from a cow with an experimentally induced infection of the Pawhuska isolate of bovine anaplasmosis were conjugated with ferritin and used to label antigenic sites in preparations of parasitized erythrocytes. Intact erythrocytes did not label on the extracellular surface. Ferritin-conjugated antibody did not pass through the intact erythrocyte to label the parasite, probably due to the large molecular size of the antibody. Damage to erythrocytic plasmalemma and inclusion body in the hemolyzed erythrocytes and complement-fixation antigen allowed labeling of anaplasmal inclusion structures. The positively labeled structures were outer surface of the pellicle, chromatin of the initial body, and inclusion appendage. Unlabeled structures included inner organismic membrane of the initial body, inclusion membrane, fibrillar protoplasmic network of the initial body, and small electron-dense bodies derived from the initial body.