(1) The central theme of this investigation was the part played by the chronically infected beast in the epidemiology of bovine besnoitiosis. The incentive was provided by the discovery that cyst organisms of B. besnoiti were transmissible to rabbits and cattle by inoculation. It brought the long-neglected, indomitable chronic case into the picture as a life-long reservoir.
(2) Transmission was observed to occur if susceptible cattle were allowed to cohabit with chronic cases in open paddocks. All the animals contracted a clinically in apparent form of the disease. About 50 per cent developed relatively small numbers of SC cysts, and in one-fifth of them cysts were detected in skin sections. In the other half immunity to challenge was the only evidence of infection. Transmission was almost exclusively confined to cattle that were in direct contact with the carriers, indicating a mode of transmission that operated over relatively short distances only. Venereal transmission could be excluded because infection occurred irrespective of whether or not bulls were present.
(3) These results, together with the well-known summer seasonal incidence, implied that mechanical transfer of the disease by a blood-sucking arthropod was a very likely mode of transmission. The feasibility of mechanical transmission was therefore investigated in rabbits using G. brevipalpis as tool and chronically infected cattle as donors. The majority became infected and developed typical reactions. The experiments were extended to cattle. They all contracted a rather mild form of the disease with no anasarca or scleroderma. Tsetse flies were no longer infective when tested three hours after potentially infective feeds, and there was no indication of cyclical development of the parasite within them. They also transmitted proliferative forms mechanically from rabbit to rabbit.
(4) The study was extended to flies that inhabit enzootic regions. Tabanid flies were also capable of transmitting cyst forms mechanically. Even a single fly could infect a rabbit. Cattle developed mild to fairly severe reactions but again no anasarca and scleroderma were evident. Tabanids remained infective by bite for 24 hours and were shown by rabbit inoculation with triturated flies to harbour viable organisms in their bodies for 29 hours after feeding; there was no sign of cyclical development in the three species tested. Gut smears of newly caught horse flies revealed the presence of flagellates, the "barleycorn" (choanomastigote) forms of which were remarkably similar to B. besnoiti at a cursory glance. Morphological, cultural and infectivity studies on rabbits and cattle indicated that they bore no relationship to B. besnoiti. The flagellates were apparently developmental stages of Crithidia and Blastocrithidia or Trypanosoma spp. Attempts to relate them to T. theileri were inconclusive.
(5) S. calcitrans also transmitted cyst organisms mechanically to rabbits and an ox. Subinoculation of small numbers of triturated flies into rabbits revealed that they had no difficulty in penetrating cysts and imbibing organisms, but large numbers of flies were required to transmit the disease mechanically. After ingestion organisms remained infective for an hour only and no biological development occurred. Three of the twelve attempts to isolate B. besnoiti from recently engorged stable flies that had been caught in an open paddock containing some chronically infected cattle were successful, which indicated that they also penetrated cysts when feeding naturally.
(6) Even C. simpsoni and unidentified Culex spp. penetrated cysts and ingested organisms, which retained their infectivity for up to 50 hours.
(7) Successful transmission in the experiment where cattle were exposed to conditions of cohabitation indicated that the transmissibility of B. besnoiti by various natural openings should be reinvestigated. Hamsters were susceptible to infection by ingestion of, as well as by inoculation with, tissues harbouring proliferative forms and cysts. Proliferation was enhanced, and symptoms of paralysis were seen in hamsters injected with cortisone. Rabbits could be infected quite readily by intranasal instillation of cyst organisms, but the conjunctival sac was a less reliable route. Cattle were susceptible to proliferative but not to cyst forms administered by mouth. Cyst organisms were, however, infective if dosed via the nostrils. Cattle developed fairly mild but quite distinct reactions with no anasarca or scleroderma. It is, however, difficult to envisage how cattle could be exposed to infection by these routes in nature.