Ultrastructure of schizonts in the liver of cats with experimentally induced cytauxzoonosis

Schizonts in the liver of 2 cats with cytauxzoonosis were studied by both light and electron microscopies. By light microscopy, the cytoplasm of macrophages in the sinusoids and small vascular channels contained schizonts with cytomeres or both cytomeres and mature merozoites. By electron microscopy, it was determined that schizogony occurred in 4 stages. The earliest stage was the presence of a multilobed structure containing finely granular protoplasm in the cytoplasm of the macrophage. The 2nd stage was an increase in height and number of the lobulations on the surface of the schizont. The 3rd stage involved the development of cytomeres and the appearance of a polar ring and rhoptries in everted sacculations on the cytomere membrane. Nuclei and mitochondria were incorporated into the sacculations before the release of mature merozoites into the host cell cytoplasm. In the last stage of schizogony, following massive merozoite formation and reduction in size of the schizont, residual nuclei divided by multiple fission. Each nuclear division became incorporated into a developing merozoite having preformed rhoptries, mitochondria, and a polar ring.     

Intraerythrocytic schizogony of Theileria sergenti in cattle

The characteristics of developing intraerythrocytic stages of T. sergenti were studied by light and transmission electron microscopy. The parasites with many ribosomes, acristate mitochondria, cytostome, and food vacuoles were morphologically regarded as the trophozoite stage. Although this type of parasites was frequently detected, intraerythrocytic merozoite stage with electron dense cisternae, rhoptries and small electron dense bodies was rarely observed in high parasitaemia. The intraerythrocytic stages of T. sergenti were divided mainly into four daughters by schizogony, and alternatively into two by binary fission. The daughter parasites in each division had the same ultrastructural features as of merozoites. As a result, it was suggested that T. sergenti trophozoites multiplied by schizogony to four organisms or by binary fission in the peripheral erythrocyte, and differentiated to the merozoites which acquired penetrating ability into the erythrocytes.     

Fine structure of Theileria parva in the bovine skin

Ultrastructural studies of Theileria parva in the bovine skin revealed ‘infective particles’ of the parasite. These parasite forms were pleomorphic and were found extracellular or within host lymphoid cells, neutrophils and erythrocytes. The parasites were a product of extracellular schizogony. They were phagocytosed by the host leucocytes but seemed actively to invade the erythrocytes. Several extracellular uninucleate schizonts were also observed. The presence of extracellular infective particles, uninucleate schizonts and multinucleate schizonts, some showing schizogony, suggests an extracellular life cycle of T. parva within bovine tissue.     

Sarcocystis neurona and Sarcocystis falcatula: monitoring of schizogony in cell culture using fluorescent nuclear labeling

The nuclei of merozoites of Sarcocystis neurona and Sarcocystis falcatula were labeled with the fluorescent marker Syto21. It was shown that the marker would label the parasites and that they would retain the marker throughout schizogony. Thus, there was sufficient marker in the daughter merozoites to make them easily visible with fluorescence microscopy. This technique will be helpful in studying the developmental biology of these parasites in vitro.     

Relative infectivity of Theileria annulata (Dchunkovsky and Luhs, 1904) stabilates derived from female and male Hyalomma ecavatum (Koch, 1844) ticks

Partially engorged female and male Hyalomma excavatum ticks infected with Theileria annulata were triturated separately in Eagle's minimum essential medium complemented with fetal calf serum. Glycerol was added to the supernatant obtained after centrifugation and the material was frozen at—79°C. Infectivity of the stabilate was titrated by inoculating susceptible calves with dilutions of the material.Stabilate prepared from female ticks was considerably more infective on a per tick basis than stabilate from males. All calves inoculated with an estimated equivalent of 0.1 or more female ticks, or 3.3 or more male ticks, became infected.Time to fever, prepatent period, and mortality did not appear to be dose-dependent in the range of tick equivalents employed.     

Transformation of Theileria parva derived from African buffalo (Syncerus caffer) by tick passage in cattle and its use in infection and treatment immunization.

A sporozoite stabilate (St. 199) of Theileria parva was obtained by feeding nymphal Rhipicephalus appendiculatus on an African buffalo (Syncerus caffer) and was used to immunize cattle by the infection and treatment method. Nymphal ticks were applied to one of the steers 90 days later and it was shown that the resultant adult tick had become infected. Using tick/cattle passage, two passage lines of T. parva were established. By the fifth tick/cattle passage, the parasite stocks had changed their behaviour to that of T. parva derived from cattle as the parasite produced relatively high schizont parasitosis and piroplasm parasitaemia in cattle, and had become highly infective to ticks. At various passage levels the parasite populations were characterized by behaviour and by monoclonal antibodies against T. parva schizonts using infected cell culture isolates from cattle during acute infections. The monoclonal antibody profile showed little evidence of antigen change of the parasite during passage through cattle, which was confirmed in a two-way cross-immunity experiment using sporozoite stabilate derived from ticks obtained from the buffalo and fourth passage in cattle. The implication of these results, particularly in relationship to immunization of cattle against T. parva derived from buffalo, is discussed.     

Encephalitic sarcocystosis in a newborn calf

Sporozoan schizonts were seen in histologic sections of cerebrum from a Hereford calf that died immediately after birth. Schizonts appeared in endothelial cells of small vessels in the gray and white matter. Rosette and palisade configurations of merozoites in schizonts, as well as the size of schizonts (15 to 40 X 21 microns) and merozoites (8 microns), resembled Sarcocystis stages described in cattle, and differentiated the organisms from Toxoplasma that infects nerve cells and undergoes endodyogeny. Specific identification of the infecting agent was not successful because tests of sera from the calf were not possible. Developmental, morphologic, and ultrastructural differences in schizogonic stages of the heteroxenous sporozoan species infecting cattle are poorly known and are presently unreliable criteria for species identification. Encephalitis, meningitis, and necrosis occurred in cerebral, cerebellar, and brain stem gray and white matter infiltrated with plasmacytes, lymphocytes, and macrophages. Microthrombi were often seen in small blood vessels within the reaction foci. Herd-mate sera tested against S cruzi were negative. The herd from which the calf was born was exposed to a variety of free-ranging domestic, feral, and wild scavengers with free access to dead range animals. Consequently, the cattle were undoubtedly exposed to infective cysts in feed or water contaminated by feces from carnivores.     

Experimental production of tick pyaemia

Lambs aged 2 weeks were inoculated with a tick-borne fever (TBF) stabilate on Day 0 and Staphylococcus aureus-contaminated ticks were applied on Day 5. Tick pyaemia was produced experimentally for the first time using Ixodes ricinus as a mechanical vector of S. aureus. Lambs aged 18 weeks were rechallenged with a homologous strain of TBF, and S. aureus-infected ticks applied 5 days later. No significant changes were noted at post-mortem examination.     

The effects of bovine tick resistance on the susceptibility of Hyalomma anatolicum anatolicum to infection with Theileria annulata (Ankara)

Two Bos taurus calves were made resistant to tick infestation by exposing them to approximately 500 rabbit-reared nymphs of Hyalomma anatolicum anatolicum twice at a 2-week interval. These two calves, together with a tick-susceptible control calf, were inoculated with a stabilate of Theileria annulata (Ankara). Patent infection resulted in all three calves. Seven-hundred and fifty gerbil-reared nymphs were then applied on each of these calves as well as another tick-susceptible calf that was Theileria free. This infestation was carried out on Day 8 post-inoculation. Ticks that dropped on Day 13 post-inoculation were examined to note the development of T. annulata in them and the histological changes that occurred in the gut and salivary glands. During the second phase of feeding, the gut epithelia of the ticks from the tick-resistant calves were less active. There were no notable differences in the characteristics of the developmental stages of T. annulata between the ticks from the tick-resistant calves and those from the susceptible calf. However, ticks from one calf that acquired a higher level of tick resistance were significantly less susceptible to infection by T. annulata. Bovine tick resistance therefore compromises the vector capacity of H. a. anatolicum and this may be of epidemiological significance in the endemic areas of tropical theileriosis.     

Babesia--a historical overview

The history of the genus Babesia is briefly outlined. The classical differences with the main other genus of non-pigment-forming hemoparasites, Theileria, are the absence of extra-erythrocytic multiplication (schizogony) in Babesia and the cycle in the vector tick, which includes transovarial transmission in Babesia but only transstadial transmission in Theileria. Also, the multiplication in the red cell of Babesia, by budding, most often results in two daughter cells (merozoites), while that of Theileria gives four merozoites, often as a Maltese cross. In particular this means that what is still commonly called Babesia microti is not a Babesia and that it would be just as logical to speak of human theileriosis as of babesiosis. The small piroplasm of horses, long known as Babesia equi, is already commonly designated as Theileria equi. However, on molecular grounds, it may be necessary to create a new genus for these parasites. The Babesia species of domestic animals are briefly discussed and presented in a table.     

In vivo comparison of susceptibility between Bos indicus and Bos taurus cattle types to Theileria parva infection

The objective of this study was to determine whether Bos taurus cattle differ form Bos indicus in their susceptibility to infection with the Muguga stabilate of Theileria parva and in their resistance to the resultant disease. Ten Friesians (B. taurus), ten improved Borans (B. indicus), ten unimproved Borans (B. indicus) and ten Zebus (B. indicus) born to dams from an East Coast fever (ECF) endemic area were inoculated with an infective dose50 dilution of T. parva Muguga stabilate 147. All the animals except one Friesian and one Zebu developed schizont parasitosis. All the improved Borans, nine of the Friesians, eight of the unimproved Borans and six of the Zebus developed a febrile response. Four of the improved Borans, four of the Friesians and three of the unimproved Borans died of theileriosis. No significant difference (P > 0.05) in the prepatent period occurred between the groups, but the Zebus had a significantly shorter duration of schizont parasitosis (P > 0.05) and took a significantly shorter time to recover (P > 0.05) than the other three groups. There was no significant difference in the two parameters between the other three groups. The study showed that three B. indicus breds and a B. taurus breed are equally susceptible to T. parva infection. However, Zebus born to dams from an ECF endemic area showed a better ability to control the course of disease than cattle from ECF free areas.     

Further evaluation of the use of buparvaquone in the infection and treatment method of immunizing cattle against Theileria parva derived from African buffalo (Syncerus caffer).

Three experiments were undertaken to determine the efficacy of different doses of buparvaquone in the infection and treatment immunization of cattle against Theileria parva derived from African buffalo (Syncerus caffer). Two of these experiments also compared buparvaquone with standard doses of long- and short-acting formulations of oxytetracycline. In addition, different dilutions of stabilates were used in the experiments. In the first experiment, a 10(-1.0) dilution of stabilate was used to infect groups of cattle treated with buparvaquone at doses of between 5 and 0.625 mg kg-1 body weight (bwt) on Day 0 after infection. All control cattle developed severe theileriosis and none of the treatment regimes (including those utilizing long-acting oxytetracycline) prevented the development of theileriosis. Treatment with buparvaquone at 2.5 mg kg-1 bwt or oxytetracycline gave the most satisfactory results. In the second experiment when the sporozoite dose was reduced to 10(-2.0) dilution, buparvaquone treatment at 5 and 2.5 mg kg-1 bwt and short- and long-acting formulations of oxytetracycline reduced reactions greatly. While all the oxytetracycline treated animals produced a serological response and were immune to a 50-fold higher challenge with the immunizing stabilate, several animals in the buparvaquone groups did not show a serological response and were not immune to challenge. In the third experiment, groups of cattle were infected with 10(-1.2), 10(-1.4) and 10(-1.6) dilutions of stabilate and were treated with 2.5 mg kg-1 bwt of buparvaquone. No animals developed severe theileriosis and all seroconverted. On homologous challenge, however, two out of 14 cattle showed severe reactions. It was concluded that further work on immunization using buparvaquone treatment at 2.5 mg kg-1 bwt and 10(-1.6) dilution of the stabilate would have to be carried out before such a system could be used in the field.     

Schizogony and gametogony of Eimeria zuernii (Rivolta, 1878) Martin, 1909

Calves were infected with Eimeria zuernii given by stomach tube and also placed directly into surgically prepared pouches of small intestine. The calves were killed at 2 day intervals or less from the 2nd to the 21st day after injection. First generation schizogony occurred in the lamina propria of the lower ileum and the first generation schizont was a giant schizont. Second generation schizogony and gametogony took place in the epithelial cells of the caecum and proximal colon. Measurements of the various stages of the life cycle of E. zuernii are given.     

Induction of protective immunity to Dictyocaulus viviparus in calves while under treatment with endectocides

Three groups of five parasite-naive calves were used. The treatments were: (a) Group 1 calves were weighed on Day 0 and injected with doramectin at 200 microg/kg. From Day 1 to 19 they were dosed orally with 2000 infective larvae of Dictyocaulus viviparus. On Day 28 they were again injected with doramectin, and infected with D. viviparus larvae from Days 33 to 41. They were then left untreated until Day 81 when they were infected with 20 infective larvae of D. viviparus per kg body weight. They were killed on Day 110 and lungworms were counted; (b) Group 2 calves were immunised with oral lungworm vaccine on Days 0 and 28, and infected and slaughtered as Group 1 on Days 81 and 110, respectively; (c) Group 3 calves acted as infection controls. Blood samples were taken at Days 0, 21, 49, 77 and 110 for antibody tests to D. viviparus. At autopsy there were no significant differences between the number of lungworms from Groups 1 and 2 (Means 17.4 and 31.3, respectively); Group 1 had significantly less value than Group 3 (Mean 228) (p < 0.05). Increased antibody titres to the larval sheath of the infective larvae were observed from Groups 1 and 2, showing that the larvae in Group 1 had penetrated the intestine before being killed by the circulating anthelmintic. This experiment shows that if calves are exposed to infective larvae while under systemic endectocide cover, an immune reaction is stimulated.     

The detection of IgM and IgG antibodies against Babesia bigemina in bovine sera using semi-defined antigens in enzyme immunoassays

Soluble extracts prepared from Babesia bigemina merozoites were tested for antigenicity in class-specific enzyme immunoassays currently being evaluated for the differential serodiagnosis of bovine babesiosis. Intact merozoites were harvested from erythrocytes from an experimentally-infected calf by controlled hypotonic lysis and differential ultra-centrifugation. The merozoites were disrupted by ultrasonication and a crude soluble extract obtained by ultracentrifugation. Fractionation of the crude extract on calibrated Sephadex G-200 columns consistently produced 4 fractions with molecular weights of 600, 40, 15 and 5 k (k = 10(3) daltons). Only the 600 and 15 k fractions proved to be antigenic when reacted against bovine immune sera. These fractions were incorporated into IgM- and IgG-specific enzyme immunoassays and used to determine the kinetics of the host-antibody responses to infection. The use of semi-defined antigens allowed assay standardization and good reproducibility of the results. A calf infected with a cryopreserved stabilate of B. bigemina originating from adult Boophilus microplus ticks developed a mild transient fever from 6-4 days post-infection (d.p.i.) and low parasitaemia levels from 7-16 d.p.i. IgG-antibodies first appeared at 7 d.p.i., peaked in intensity at 12 d.p.i. and then persisted at these levels until the end of the test period at 49 d.p.i. IgM-antibodies appeared at 7 d.p.i., peaked in intensity from 12-22 d.p.i., but then declined to low levels by 28 d.p.i. The importance of this transitory IgM-antibody response in the serodiagnosis of acute B. bigemina infections remains to be determined in clinical and field situations.     

Studies on Globidial Schizonts in the Abomasum of Norwegian Sheep: The Fine Structure of One of the Four Merozoite forms Investigated

Norwegian sheep were investigated for globidial schizont infection in the abomasum. The frequency of infection was found to be 78.2 %. Light microscope studies of the various mature schizonts revealed the existence of four morphologically different merozoites, small A, small B, intermediate and long forms. Each globidial schizont was found to contain only one form of these merozoites. However, these four schizont types occurred in the same abomasum.The intermediate form of globidial schizont merozoites was investigated by the aid of an electron microscope, with the aim of comparing its internal morphology with that previously published for Eimeria species. A striking resemblance was observed between the fine structures of the intermediate merozoite and that of Eimeria species, particularly the first generation merozoites described in giant schizonts of Eimeria bovis.The present status of globidial schizonts infecting the abomasum of sheep was discussed. It was concluded that these four forms of merozoites could represent different generations of one Eimeria species or different E. species producing giant schizonts in the abomasum.Due to the practical difficulties in studying the life histories of the different Eimeria species infecting sheep, it was proposed that the in vitro propagation of the individual species in cultured cells may shed some light on the corresponding asexual, as well as the sexual, stages. This would offer a new approach to the study of the ultra-structure of the developing parasite.     

Biological parameters of Trichostrongylus colubriformis in Meriones unguiculatus

In a series of three experiments, 64 jirds, Meriones unguiculatus, were infected with 700 infective larvae of Trichostrongylus colubriformis for the study of several biological parameters of this laboratory host-parasite model. In jirds the third stage larvae of T. colubriformis were shown to reach the fourth larval stage by the Day 6 post-infection (PI). By Day 10 PI, all the worms harvested had reached the immature adult stage. All immature adult stages of T. colubriformis developed to sexually mature adult stage by Day 12 PI when the female worms showed developing eggs in their uteri. Developed eggs were observed in the uteri of females on Days 13 and 14 PI. The first eggs of T. colubriformis appeared in the faeces of jirds on Day 13 PI. The peaks of egg production were recorded between Day 21 and Day 31 PI. Immunosuppression of jirds infected with 700 L3, by administration of dexamethasone from Day 57 to Day 94 PI led to increased faecal egg count when compared to untreated controls. All the worms were located in the small intestine. The jirds of dexamethasone treated group harboured higher number of adult worms than those of the untreated group. The number of adult worms was significantly higher in the first part of the small intestine than in the three other similar parts of the small intestine. The coefficient of correlation between the faecal egg count and worm number on the day of necropsy of jirds ranged between r = 0.58 and r = 0.89. Patent infections in jirds were maintained till the end of experiment on Day 100 PI, indicating that in this host and unlike other laboratory hosts, T. colubriformis is responsible for long lasting infections similar to what happens in domestic ruminants. The results of the present study suggest that the jird is a suitable laboratory model to study various aspects of this host-parasite relationship.     

Persistent efficacy of doramectin injectable against artificially induced infections with Cooperia punctata and Dictyocaulus viviparus in cattle.

Three studies were conducted to evaluate the persistent efficacy of doramectin injectable solution against experimental challenges with infective larvae of Cooperia punctata and Dictyocaulus viviparus. In each study, four groups of ten randomly-assigned calves, negative for trichostrongyle-type eggs on fecal examination, were treated subcutaneously in the midline of the neck with saline (1 ml/50 kg) on Day 0 or doramectin (200 microg/kg = 1 ml/50 kg) on Day 0, 7, or 14. Two additional calves from the same pool of animals were randomly assigned as larval-viability monitors and received no treatment. On Days 14-28, approximately 1000 and 50 infective larvae of Cooperia spp. and D. viviparus, respectively, were administered daily by gavage to each animal in Groups T1-T4. On Day 28, the two larval-viability monitor calves were inoculated in a similar manner with a single dose of approximately 30000 and 2000 larvae of Cooperia spp. and D. viviparus, respectively. Equal numbers of calves from each treatment group were killed on Days 42-45, as well as the two viability monitor animals to enumerate worm numbers. A 2% or 5% aliquot of small intestinal contents and washings were examined for worm quantification and identification, while 100% of the lung recoveries were quantified and identified. For each study and across the three studies, geometric mean worm recoveries for each treatment group were calculated from the natural log transformed data (worm count + 1) and were used to estimate percentage reduction. In the three studies, doramectin injectable solution was 97.5% efficacious against lungworms for up to 28 days and was 99.8% efficacious in reducing infection resulting from challenge with infective larvae of C. punctata for at least 28 days post-treatment.     

Cryptosporidium parvum: investigation of sporozoite excystation in vivo and the association of merozoites with intestinal mucus

Enteric cryptosporidiosis was studied in the small intestine of five-day-old sucking mice after infection with 10(6) Cryptosporidium parvum oocysts. It was shown that excystation and the majority of subsequent endogenous stages occurred predominantly in the ileum. During the first three days of infection the number of merozoites collected in ileal washings increased over 100-fold to approximately 10(6) merozoites per mouse on the third day. In contrast to control mice, wash fluid from infected mice contained numerous strands of dislodged mucus. Estimates of mucus in the ileal washings of infected mice were similar to those made in controls until day 4 after infection when they increased and remained high throughout the remainder of the experiment. This study describes a method whereby ileal mucus washings from C parvum infected infant mice could be used as a rich source of merozoites.