Browsing by Author "McKenna, Philip Bernard"

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    Coccidia (Protozoa sporozoasida) of cats and dogs : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philsophy [sic] in Veterinary Science at Massey University
    (Massey University, 1978) McKenna, Philip Bernard
    Studies were undertaken to establish for the first time the identity and prevalence of coccidian parasites of New Zealand cats and dogs and to determine the effect of various factors on the activation of Isospora felis and Isospora rivolta sporozoites. The associations between these protozoa and such organisms as Toxoplasma, Sarcocystis, Besnoitia and other related genera are examined and the literature concerning their life cycles, nomenclature and general biology reviewed. Examination of faecal samples from 508 cats and 481 dogs from North Island localities revealed that 155 (30.5%) and 307 (63.8%) respectively, contained coccidia. The majority of infected samples were found to contain a single coccidian but in total four valid coccidians from cats and four from dogs, as well as several pseudoparasitic coccidia, were recorded and described. The identities and prevalences of these valid coccidians were: (a) Cats : Isospora felis (17.5%) Isospora rivolta (2.2.%) Toxoplasma gondii (0.98%) Sarcocystis sp. (16.9%) (b) Dogs : Isospora canis (4.0%) Isospora ohioensis (9.2%) Hammondia heydorni (2.7%) Sarcocystis sp. (58.8%) The sex of the host had no significant effect on the prevalence of infection. The effect of other factors, such as season, host age and host origin, however, was found to vary from coccidian to coccidian and appeared to be explicable in terms of differences in routes of transmission, host immunity and intermediate host specificity. Levels of sporocyst shedding in cats and dogs naturally infected with Sarcocystis sp. tended to be low with the majority excreting 200 sporocysts per gram of faeces or less. The specific identities of such sporocysts are unknown but at least some from cats were demonstrated, by mouse infection, to be those of S. muris. Attempts to induce similar Sarcocystis infection in mice, using isolates of I. felis recovered from the faeces of naturally infected cats were unsuccessful. After completion of the main survey, a further coccidian showing similarities to Besnoitia wallacci was recovered from the faeces of one of five feral cats. The feeding of sporulated oocysts of this coccidian to mice, rats, rabbits and guinea pigs resulted in the formation of typical Besnoitia cysts in all hosts except the last. Studies on the activation of I. felis and I. rivolta sporozoites revealed that, although some differences were apparent between the two, both were capable of activation over a wide range of conditions. Activation of both species was observed to take place in trypsin and bile between temperatures of 21° and 43°C (the range tested) and to occur rapidly at 39°C. While the presence of bile appeared to be essential for this process that of trypsin did not. In general, neither the concentration of bile (above 5%) nor the type of bile was found to have any marked effect on the level of activation attained while hydrogen ion concentration (pH range 5.0 to 10.0) also appeared to have little influence. Unlike many species of coccidia which have been studied, pretreatment of oocysts before exposure to trypsin and bile was found not to be an essential prerequisite for the activation of I. felis and I. rivolta. However, higher levels of activation were attained when pretreatment was used than when it was not although for I. rivolta at least, the level of activation appeared to be less dependent on pretreatment for oocysts stored in sulphuric acid than for those stored in potassium dichromate. The process of activation and excystation of both species was observed to be essentially similar to that described for other species of coccidia which also lack either sporocyst stieda bodies or oocyst micropyles. Sporozoites escaped following the collapse of the sporocyst wall and were observed to complete excystation through identations and fractures at one or both ends of the oocyst.
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    Sarcocystis gigantea : studies on sporocyst production, excystation and viability : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science
    (Massey University, 1984) McKenna, Philip Bernard
    Recent advances in knowledge about the sporozoan genus Sacocystis (Protozoa: Apicomplexa) are reviewed and studies on the production, excystation and viability of sporocysts of Sarcocystis gigantea, undertaken. Investigation of a sedimentation/floatation procedure for the mass recovery of S. gigantea sporocysts from cat faeces showed that the greatest yields were obtained when a proportion of faeces to floatation medium of 5% and a centrifugal force of 6000 x g for at least 5 min were used. Ninety-six percent of the sporocysts recovered were obtained from the first centrifugation in aqueous NaCl solution, specific gravity 1.2. Although neither sieving nor additional washing of homogenised samples prior to floatation significantly affected sporocyst recovery both reduced the amount of debris present. A considerable reduction in the amount of debris resulted from feeding infected cats on tinned fish rather than tinned meat. The addition of CCl4 to the NaCl solution also improved sporocyst purity but with a marked reduction in the numbers recovered. A technique for determining the concentration of sporocysts in faeces, using a modification of the mass recovery procedure and a haemocytometer, was developed. This was shown to be more accurate and reliable than the McMaster method for performing faecal sporocyst counts. It resulted in a mean sporocyst recovery of 75.5% and was used to obtain information about patterns of sporocyst excretion and numbers of S. gigantea sporocysts shed by 28 experimentally infected cats. In all cats, sporocyst excretion commenced 10 or 11 days post-infection(PI). Peak production occurred between 13 and 22 days PI, in most instances on days 17 and 18. Peak numbers (rounded) ranged from 550 to 260,000 (mean = 53,000) sporocysts per gram of faeces or from 38,000 to 6.6. million (mean = 1.7 million) sporocysts per day. The number of days sporocysts were shed ranged from 26 to at least 60 days PI but in 26 of the 28 infections examined, more than 80% of the total sporocyst yield was produced within 30 days of infection. The total numbers of sporocysts produced by individual cats over the patent period ranged from 164,000 to 56.6 million (mean = 12.7 million). These numbers tended to increase with increasing infective dose and to be greater in those cats receiving multiple rather than equivalent single doses. Neither the sex of the cat, nor experience of one or two previous infections, had any significant effect on the numbers of sporocysts shed. Studies on the in vitro excystation of S.gigantea sporocysts revealed that pretreatment before exposure to trypsin and bile was an essential pre-requisite. However, in contrast to S. tenella and S. capracanis, incubation in cysteine hydrochloride under CO2 was largely unsuccessful for excysting S. gigantea: of the pretreatments tested only exposure to sodium hypochlorite proved effective. Excystation from sodium hypochlorite-pretreated S. gigantea sporocysts took place in trypsin and bile between temperatures of 30° and 43°C and occurred rapidly at 39°C. While the presence of bile or bile salts was essential for this process, that of trypsin was not although more sporocysts excysted in its presence than in its absence. Excystation occurred in the presence of all bile types tested but not when 'Tween 80' was substituted for bile. The highest levels of excystation were recorded when cattle or sheep bile or sodium taurocholate were used and the lowest when chicken or pig bile were employed. Neither the concentration of sheep bile above 2.5%, nor hydrogen ion concentration (pH range 5.0 to 10.0) appeared to have any marked effect on the level of excystation obtained. The excystation process for S. gigantea was similar to that described for other Sarcocystis species and for other coccidian genera that lack sporocyst Stieda bodies. Sporozoites escaped following the collapse of the sporocyst wall and its eventual separation into four elongated pieces. In vivo studies on excystation of S. gigantea indicated that this process was, as in vitro, diphasic involving pretreatment and treatment phases. They also tended to support in vitro observations that the requirements for the excystation of S. gigantea and S. tenella sporocysts were quite different. Although the results suggested that for neither species was the pretreatment stimulus likely to be provided by conditions in the rumen alone, exposure to abomasal conditions only, induced moderate levels of excystation in both when they were subsequently treated with trypsin and bile. For S. gigantea, 0.25 to 4 hr abomasal exposure was most effective,for S. tenella 24 hours. The stimuli necessary to complete the excystation process could, apparently, be provided by 1 hr placement in the duodenum for S. gigantea but not for S. tenella. Using in vitro excystation as a measure of viability, it was found that at 4°C, S. gigantea sporocysts survived considerably better in tap water (85% excystation after 174 days) than in either 2.5% potassium dichromate (15% excystation after 174 days) or 2% sulphuric acid (0% excystation after 5 days). Although they were able to resist 48 hr suspension at room temperature in most laboratory reagents, disinfectants and anti-coccidial drugs tested, six (sulphuric acid, ammonia, methanol, ethanol, potassium hydroxide, sodium hydroxide, 16% synergistic mixture of five chlorinated phenols.) had major sporocysticidal properties. Further investigation with three of these, showed that sporocyst excystation was reduced from 65% to less than 10% following contact with 2.5% sulphuric acid for 1 hr or with 2% ammonia or 4% Medol for 4 hours. Sporocysts were either killed, or their ability to excyst severely impaired, by heating to 60° and 55°C for 5 and 60 min, respectively, by exposure to ultraviolet radiation at a dose of 4000 ET, or by prolonged storage in water at 24°C. Sporocysts exposed to either constant or intermittent freezing at -18°C suffered a comparatively slow decline in excystation rate with time as did those subjected to desiccation. The duration of survival of desiccated sporocysts was inversely related to relative humidity and after 245 days at 33% RH and temperatures of 15 or 24°C, 60% of such sporocysts excysted. Studies on the survival of S. gigantea sporocysts in faeces outdoors showed that, viability declined most rapidly over the summer months and suggested that they were unlikely to remain infective for more than one year. Possible associations between the reported findings and both the epidemiology of S. gigantea infection and some of the previous unsuccessful or equivocal attempts to experimentally infect sheep with this species, are discussed.