Pathology and treatment of Eimeria zuernii coccidiosis in calves: investigations in an infection model

Two studies were conducted in the Eimeria zuernii infection model in order to investigate the pathology of E. zuernii coccidiosis and the efficacy of toltrazuril (Baycox 5% suspension) in this infection. For this purpose, a total of 30 calves were infected experimentally with E. zuernii oocysts and faecal samples taken regularly from the rectum and examined for faecal consistency and oocyst excretion. Six of the calves underwent pathological examination at various points in time after infection. Significant macroscopic and microscopic changes were demonstrated and parasitic stages were identified in the intestinal mucosa of infected calves during the late prepatent and patent period. Inflammatory reactions revealed by light microscopy were confirmed by electron microscopical investigations. Treatment of calves with toltrazuril during the late prepatent period resulted in significantly lower frequencies of diarrhoea and levels of oocyst excretion, and weight gain was significantly higher than in shamtreated animals.     

Efficacy of Monensin Against Bovine Coccidiosis in Young Holstein-Friesian Calves*

Monensin, an anticoccidial antibiotic, was incorporated into pelleted feed and given to 10-week-old Holstein-Friesian calves at 0.25 mg/kg, 1.0 mg/kg, or 2.0 mg/kg of body weight. Inoculated calves were inoculated by drenching with 500,000 sporulated oocysts of Eimeria bovis. Other calves served as inoculated or uninoculated controls. Observations were recorded on oocyst discharge in the feces, clinical signs, weight gain, food consumption, hemoglobin, packed cell volume, total serum protein, sodium and potassium content of the serum, and differential white cell counts. Calves in the inoculated control group developed severe infections, discharged large numbers of oocysts, developed clinical signs and 1 of 5 died. Uninoculated, untreated control calves were essentially free of coccidia. A few calves in the groups which received monensin developed light infections but none of them had clinical signs of coccidiosis. Calves which received the highest and the lowest dosages of monensin gained weight less rapidly than did the uninoculated controls or the animals which received monensin at 1.0 mg/kg of body weight. Inoculated control calves with severe infections had reduced food intake and a significant reduction in weight which was not regained during the experimental period. The only other significant change in any of the parameters measured was a reduction in the total serum protein of inoculated, nonmedicated control calves. The level returned to normal 5 weeks after clinical signs first appeared.     

Digestive and metabolic utilization of lauric,myristic and stearic acid in cows,and associated effects on milk fat quality

A study was carried out to examine the effect of dietary supplementation of oregano essential oil on performance of broiler chickens experimentally infected with Eimeria tenella at 14 days of age. A total of 120 day-old Cobb-500 chicks separated into 4 equal groups with three replicates each, were used in this study. Two groups, one infected with 5·10⁴ sporulated oocysts of E. tenella and the other not, were given a basal diet and served as controls. The other two groups also infected with E. tenella were administered diets supplemented with oregano essential oil at a level of 300 mg/kg, or with the anticoccidial lasalocid at 75 mg/kg. Following this infection, survival rate, bloody diarrhoea and oocysts excretion as well as lesion score were determined. Throughout the experimental period of 42 days, body weight gain and feed intake were recorded weekly, and feed conversion ratios were calculated. Two weeks after the infection with E. tenella supplementation with dietary oregano oil resulted in body weight gains and feed conversion ratios not differing from the non-infected group, but higher than those of the infected control group and lower than those of the lasalocid group. These parameters correspond with the extent of bloody diarrhoea, survival rate, lesion score and oocyst numbers and indicated that oregano essential oil exerted an anticoccidial effect against E. tenella, which was, however, lower than that exhibited by lasalocid.     

Maltose excretion by the symbiotic Chlorella of the heliozoan Acanthocystis turfacea

Chlorella sp. strain 3.83, a symbiotic Chlorella isolated from the heliozoan Acanthocystis turfacea, excreted between 8% and 16% of assimilated ¹⁴CO₂ as maltose in the light (15000 lx), with a pH optimum around 4.8. This percentage increased when the illuminance was lowered (36% at 1700 lx). Release of [¹⁴C]maltose continued in darkness and could be inhibited by the uncoupler carbonyl cyanide p-trifluoro-methoxyphenylhydrazone and by diethylstilbestrol. Net efflux of maltose was observed even at a concentration ratio of extracellular/intracellular maltose of 7.8. Exogenous [¹⁴C]maltose (5 mM) was taken up by the cells with a rate <2% of that of simultaneous maltose release, indicating a practically unidirectional transport. It is concluded that maltose excretion is an active-transport process.Abbreviations DES diethylstilbestrol - FCCP carbonyl cyanide p-trifluoromethoxyphenyl hydrazone - p.c. packed cells This work was supported by the Deutsche Forschungsgemeinschaft. Thanks are due to Doris Meindl for skillful experimental help.     

Genetic variability of resistance to Eimeria acervulina and E. tenella in chickens

Summary The genetic variability of 18 sire families of the Athens-Canadian randombred population infected with coccidiosis was assessed by examining the response variables of weight gain, packed red blood cell volume, mortality and coccidial lesions. A significant gain and PCV depression and high lesion scores for Eimeria tenella and E. acervulina were produced in the infected group compared to the noninfected group. Significant variation among the sire families was observed for all of the response variables except E. acervulina lesions and a significant sex x sire interaction was observed for weight gain. The heritability (h²) estimates for the response variables revealed that resistance to coccidiosis in chickens is moderately heritable. The h² estimates for gain and PCV increased with the coccidial infections indicating that maximum progress in selecting for resistance should be made when the population was exposed to coccidial infection. Gain was positively correlated to the other measures of resistance and thus selecting for coccidial resistance should not reduce growth rates. PCV was similarly correlated but had higher positive correlation with E. tenella lesion. Percent mortality which is the selection parameter in most coccidial selection programs was correlated with resistance to coccidiosis. The phenotypic and genotypic correlations demonstrated that chickens susceptible to E. tenella were also susceptible to E. acervulina. Total lesion scores were moderately to highly correlated with the other variables and would be a suitable variable to use in coccidiosis experimentation including a genetic selection program for resistance. This study shows that progress could be made in selecting for resistance to coccidiosis in chickens using one or a combination of these response variables.     

Immunotherapeutic effects of some sugar cane (Saccharum officinarum L.) extracts against coccidiosis in industrial broiler chickens

Present paper reports the effects of aqueous and ethanolic extracts of sugar cane (Saccharum officinarum L.) juice and bagasse, respectively on protective immune responses in industrial broiler chickens against coccidiosis. Immunotherapeutic efficacies of the extracts were measured by evaluating their effect on body weight gain, oocyst shedding, lesion score, anti-coccidial indices, per cent protection and elicited serum antibody responses against coccidiosis. Results revealed a significantly lower (P < 0.05) oocyst shedding and mortality in chickens administered with sugar cane extracts as compared to control. Further, significantly higher (P < 0.05) body weight gains and antibody responses were detected in chickens administered with sugar cane extracts as compared to chickens of control group. Moreover, ethanolic extract showed higher anti-coccidia index (227.61) as compared to aqueous extract (192.32). The organ body weight ratio of the lymphoid organs of experimental and control groups were statistically non-significant (P > 0.01). These results demonstrated that both ethanolic and aqueous extracts of sugar cane possess immune enhancing properties and their administration in chickens augments the protective immunity against coccidiosis.     

Toxoplasma gondii: Infection natural congenital in cattle and an experimental inoculation of gestating cows with oocysts

Two studies, of a natural infection and an experimental infection, were performed in order to study congenital transmission of Toxoplasma gondii in cattle. In the first study, 50 fetuses were harvested from gestating cows that were eutanasied at a municipal slaughterhouse in Jaboticabal, São Paulo state, Brazil. In the second study, 11 gestating cows were divided into four groups for inoculation with T. gondii: GI consisted of three cows inoculated with 1.0 × 10⁵ oocysts during their first trimester of gestation; GII consisted of three cows inoculated with 1.0 × 10⁵ oocysts during their second trimester of gestation; GIII consisted of three cows inoculated with 1.0 × 10⁵ oocysts during their last trimester of gestation; and GIV consisted of two control cows, one during its first and the other during its second trimester of gestation. In both studies, the presence of T. gondii was confirmed both indirectly by immunofluorescence assay (IFAT). In the natural infection experiment, 18% (9/50) of the gestating cows were confirmed to have specific antibodies (IFAT – 1:64) against T. gondii. The bioassay was able to diagnose the presence of T. gondii in the tissue samples from three calves. In the second experiment, the nine cows from groups I, II and III presented with specific antibodies (IFAT) against T. gondii. In contrast, T. gondii could not be detected by IFAT, histopathological examination or the bioassay in any of the nine calves born to cows experimentally infected with T. gondii oocysts. Based on the results from both studies, we conclude that congenital infection of T. gondii in cattle, while infrequent, does occur naturally. The pathogenicity of the strain of T. gondii may influence the likelihood of this route of transmission.     

Eimeria acervulina: The influence of inoculation dose on transmission between broiler chickens

The course and clinical appearance of an Eimeria species infection in chicken flocks depend on the response of an individual bird to infection and on population-dynamics of the infection in the flock. Differences in ingested numbers of oocysts may affect oocyst load in the flock and the subsequent infectious dose for not yet infected birds. To study the link between numbers of oocysts excreted by infected birds and transmission of Eimeria acervulina, experiments were carried out with 42 pairs of broiler chickens using inoculation doses with 5, 50, 500 or 50,000 sporulated oocysts. In each pair one bird was inoculated and the other bird was contact-exposed. All contact birds became infected, which occurred on average within 34 h after exposure to an inoculated bird. Although a higher inoculation dose resulted in higher oocyst excretion in inoculated and contact-infected birds, only small non-significant differences in transmission rates between groups were found.     

Clonal diversity of a malaria parasite, Plasmodium mexicanum, and its transmission success from its vertebrate-to-insect host

Infections of the lizard malaria parasite Plasmodium mexicanum are often genetically complex within their fence lizard host (Sceloporus occidentalis) harbouring two or more clones of parasite. The role of clonal diversity in transmission success was studied for P. mexicanum by feeding its sandfly vectors (Lutzomyia vexator and Lutzomyia stewarti) on experimentally infected lizards. Experimental infections consisted of one, two, three or more clones, assessed using three microsatellite markers. After 5 days, vectors were dissected to assess infection status, oocyst burden and genetic composition of the oocysts. A high proportion (92%) of sandflies became infected and carried high oocyst burdens (mean of 56 oocysts) with no influence of clonal diversity on these two measures of transmission success. Gametocytemia was positively correlated with transmission success and the more common vector (L. vexator) developed more oocysts on midguts. A high proportion (74%) of all alleles detected in the lizard blood was found in infected vectors. The relative proportion of clones within mixed infections, determined by peak heights on pherograms produced by the genetic analyser instrument, was very similar for the lizard’s blood and infections in the vectors. These results demonstrate that P. mexicanum achieves high transmission success, with most clones making the transition from vertebrate-to-insect host, and thus explains in part the high genetic diversity of the parasite among all hosts at the study site.     

Glucose excretion by the symbiotic Chlorella of Spongilla fluviatilis

Chlorella sorokiniana strain 211-40c, a symbiotic Chlorella isolated from a freshwater sponge, excreted between 3% and 5% of assimilated ¹⁴CO₂ as glucose in the light, with a pH optimum around 5. This percentage increased when the illuminance was lowered (to 15% at 20 lx). Release of [¹⁴C]glucose continued in the dark and could be inhibited by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Net efflux of glucose occurred even at a concentration ratio of extracellular/intracellular glucose of 4. This, together with the sensitivity to FCCP, is taken as evidence for active transport. Exogenous [¹⁴C]glucose was taken up by the cells under conditions of net glucose efflux, showing uptake and excretion to take place simultaneously.Abbreviations FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - p.c. packed cells     

Studies on construction of a recombinant Eimeria tenella SO7 gene expressing Escherichia coli and its protective efficacy against homologous infection

Eimeria spp. are the causative agents of coccidiosis, a major disease affecting the poultry industry. A recombinant non-antibiotic Escherichia coli that expresses the Eimeria tenella SO7 gene was constructed and its protective efficacy against homologous infection in chickens was determined. The three-day-old chickens were orally immunized with the recombinant non-antibiotic SO7 gene expressing E. coli and boosted two weeks later. Four weeks after the second immunization, the chickens were challenged with 5 × 10⁴ homologous sporulated oocysts. The protective effects of the recombinant non-antibiotic E. coli were determined by measuring body weight change, mortality, histopathology, lesion scores, oocyst counts, the specific antibody response and the frequency of CD4⁺ and CD8⁺ lymphocytes in peripheral blood. The results showed that immunization with SO7 expressing E. coli resulted in significantly improved body weight gain, reduced lesion scores and oocyst shedding in immunized chickens compared to controls. Furthermore, administration of recombinant SO7 expressing E. coli leads to a significant increase in serum antibody, CD4⁺ and CD8⁺ T cells in peripheral blood of chickens. These results, therefore, suggest that the recombinant non-antibiotic E. coli that expresses the SO7 gene is able to effectively stimulate host protective immunity as evidenced by the induction of development of both humoral and cell-mediated immune responses against homologous challenge in chickens.     

Methods to produce and safely work with large numbers of Toxoplasma gondii oocysts and bradyzoite cysts

Two major obstacles to conducting studies with Toxoplasma gondii oocysts are the difficulty in reliably producing large numbers of this life stage and safety concerns because the oocyst is the most environmentally resistant stage of this zoonotic organism. Oocyst production requires oral infection of the definitive feline host with adequate numbers of T. gondii organisms to obtain unsporulated oocysts that are shed in the feces for 3-10 days after infection. Since the most successful and common mode of experimental infection of kittens with T. gondii is by ingestion of bradyzoite tissue cysts, the first step in successful oocyst production is to ensure a high bradyzoite tissue cyst burden in the brains of mice that can be used for the oral inoculum. We compared two methods for producing bradyzoite brain cysts in mice, by infecting them either orally or subcutaneously with oocysts. In both cases, oocysts derived from a low passage T. gondii Type II strain (M4) were used to infect eight-ten week-old Swiss Webster mice. First the number of bradyzoite cysts that were purified from infected mouse brains was compared. Then to evaluate the effect of the route of oocyst inoculation on tissue cyst distribution in mice, a second group of mice was infected with oocysts by one of each route and tissues were examined by histology. In separate experiments, brains from infected mice were used to infect kittens for oocyst production. Greater than 1.3 billion oocysts were isolated from the feces of two infected kittens in the first production and greater than 1.8 billion oocysts from three kittens in the second production. Our results demonstrate that oral delivery of oocysts to mice results in both higher cyst loads in the brain and greater cyst burdens in other tissues examined as compared to those of mice that received the same number of oocysts subcutaneously. The ultimate goal in producing large numbers of oocysts in kittens is to generate adequate amounts of starting material for oocyst studies. Given the potential risks of working with live oocysts in the laboratory, we also tested a method of oocyst inactivation by freeze-thaw treatment. This procedure proved to completely inactivate oocysts without evidence of significant alteration of the oocyst molecular integrity.     

Eimeria stiedae: experimental infection in rabbits and the effect of treatment with toltrazuril and ivermectin

The aim of this study was to investigate the clinical, haematological, biochemical, lipid peroxidation, ultrasonographic and pathologic findings in hepatic coccidiosis induced by Eimeria stiedae in rabbits, and also to compare the treatment effects of both toltrazuril and ivermectin separately and in combination. In this study, 56 rabbits were divided into eight groups. The first group was designated as healthy control group. Rabbits were infected with 40.000 sporulated oocysts of E. stiedae. Groups 2, 3, 4, 5, 6, 7 and 8 were allocated as the infected control group, infected+toltrazuril-treated group, infected+ivermectin-treated group, infected+toltrazuril+ivermectin-treated group, non-infected+toltrazuril-treated group, non-infected+ivermectin-treated group, non-infected+toltrazuril+ivermectin-treated group, respectively. Haematocrit, Haemoglobin and MCV values as well as percentage of lymphocyte decreased in Groups 2 and 4 whereas leucocyte counts and percentage of granulocyte leucocyte increased. Serum GGT, ALT and AST activities increased but albumin value decreased. Plasma MDA concentrations increased whereas erythrocyte CAT, GSH-Px, and SOD activities decreased. Mean oocyst numbers in per gram faeces (epg values) increased in both groups during the study. Ultrasonographic examination revealed that the liver was enlarged and had hyperechogenic parenchyma. Bile ducts were dilated and hyperechogenic and the gall bladder was dilated. The livers of these animals were enlarged and typical macroscopic and microscopic findings of coccidiosis were present. Treatment with toltrazuril and toltrazuril+ivermectin combination were highly effective in reducing faecal oocyst output in infected rabbits. Haematological, biochemical and lipid peroxidation parameters and, ultrasonographic findings of the liver were close to control values for Groups 3 and 5. Necropsy of these animals showed no visible lesions related to hepatic coccidiosis although a few oocysts were detected in the bile duct epithelial cells.     

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with causing food- and water-borne disease outbreaks. The definitive host is the feline species where sexual replication occurs resulting in the development of the highly infectious and environmentally resistant oocyst. Infection occurs via ingestion of tissue cysts from contaminated meat or oocysts from soil or water. Infection is typically asymptomatic in healthy individuals, but results in a life-long latent infection that can reactivate causing toxoplasmic encephalitis and death if the individual becomes immunocompromised. Meat contaminated with T. gondii cysts have been the primary source of infection in Europe and the United States, but recent changes in animal management and husbandry practices and improved food handling and processing procedures have significantly reduced the prevalence of T. gondii cysts in meat^{1, 2}. Nonetheless, seroprevalence in humans remains relatively high suggesting that exposure from oocyst contaminated soil or water is likely. Indeed, waterborne outbreaks of toxoplasmosis have been reported worldwide supporting the theory exposure to the environmental oocyst form poses a significant health risk^3-5. To date, research on understanding the prevalence of T. gondii oocysts in the water and environment are limited due to the lack of tools to detect oocysts in the environment ^{5, 6}. This is primarily due to the lack of efficient purification protocols for obtaining large numbers of highly purified T gondii oocysts from infected cats for research purposes. This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation⁷.     

Survival of Oocysts of Eimeria alabamensis on Pastures under Different Climatic Conditions in Sweden

The extent to which oocysts of the coccidian parasite Eimeria alabamensis can survive the winter and cause clinical coccidiosis in different parts of Sweden was investigated. Fecal samples were collected between May and July 1993 from calves on 59 farms where calves had grazed the same pasture for at least 5 consecutive years. The farms were situated in 9 regions of Sweden with different climatic conditions in the winter. On each farm, 5 samples of feces were collected from the floor of the calf-house before the calves were turned out in the spring, and again from the pasture on days 4 or 5, 8 or 9 and 10 or 11 after they were turned out. Overwintering of oocysts of E. alabamensis was considered to have occurred if an increase in the excretion rate of oocysts of this species could be demonstrated 8 to 11 days after calves had been turned out to pastures that had not been grazed since the previous autumn. Oocysts were shown to have overwintered on 27 farms, representing all 9 regions. Samples from 20 (34%) of the farms representing all the climatic regions contained more than 850000 oocysts per g of feces. This was comparable with the numbers found in animals with clinical coccidiosis due to E. alabamensis. Delaying turnout until the beginning of July did not affect the infection rate of the calves. However, calves which were turned out to pastures that had been grazed by older cattle or horses, either earlier in the spring or in previous years, excreted significantly fewer oocysts than calves which were turned out to pastures that had been grazed only by calves. A questionnaire answered by 321 dairy farmers revealed that of the 298 farmers who turned their first-season grazing cattle out to traditional pastures, 179 (60%) had used the same pasture for at least 5 years. These 179 farmers had experienced a significantly higher incidence of diarrhoea in their calves during the first 2 weeks at pasture than those farmers who had used different pastures.     

Effects of ambient temperature and of temperature acclimation on nitrogen excretion and differential catabolism of protein and nonprotein resources in the intertidal snails,Littorina saxatilis (Olivi) and L. obtusata (L.)

Nitrogenous excretion in two snails, Littorina saxatilis (high intertidal) and L. obtusata (low intertidal) was studied in relation to temperature acclimation (at 4° and 21°C), including total N excretion rates, the fraction of urea in N excretion, corresponding O:N ratios and the partitioning of deaminated protein between catabolic and anabolic processes at 4°, 11° and 21°C. Aggregate N excretion rates in both species showed no significant compensatory adjustments following acclimation. Total weight specific N excretion rates at 21°C were higher in standard 3 mg L. saxatilis (739 ng N mg⁻¹ h⁻¹) than standard 5 mg L. obtusata (257 ng N mg⁻¹ h⁻¹) for snails acclimated to 21°C. Comparisons of Q₁₀ values of total weight specific N excretion to Q₁₀ values for weight specific oxygen consumption ({xxV}O₂) between 4° to 11 °C and 11° to 21°C indicated that, while total rates of catabolic metabolism ({xxV}O₂) and protein deamination in L. obtusata were essentially parallel, the relationship between N excretion and {xxV}O₂ in L. saxatilis revealed the partitioning of a larger share of deaminated protein carbon into anabolism at 4° and 21°C than at 11°C. Urea N accounted for a larger share of aggregate N excreted in L. saxatilis than in L. obtusata, but in both species urea N is a greater proportion of total N excreted when acclimated at 4°C (urea N: ammonia N ratio range: 1 to 2.15) than in snails acclimated to 21°C (urea N: ammonia N ratio range: 0.46 to 1.39). Molar O:N ratios indicate that the proportion of metabolism supported by protein catabolism is greater in L. saxatilis (O:N range: 2.5–8.4) than in L. obtusata (O:N range: 7.3–13.0). In both species, regardless of acclimation temperature, the O:N ratios are generally lowest (high protein catabolism) at 4°C and highest at 21°C.     

Glutamate excretion inEscherichia coli: dependency on thereIA andspoT genotype

Glutamate excretion due to amino acid starvation was investigated in “stringent” and “relaxed” strains ofEscherichia coli. The observed excretion process isrelA-dependent, carrier-mediated, and glutamate-specific. After induction, excretion was detected within less than 2 min and continued for more than 5h with a rate of 7–10 nmol (mg dry weight)⁻¹ min⁻¹. Using carbonyl cyanidem-chlorophenylhydrazone or polymyxin B nonapeptide, together with valinomycin, it was shown that glutamate excretion is driven by the membrane potential.     

Graft copolymerization of N-vinylformamide onto sodium carboxymethylcellulose and study of its swelling, metal ion sorption and flocculation behaviour

The present paper reports the graft copolymerization of N-vinylformamide onto sodium carboxymethylcellulose by free radical polymerization using potassium peroxymonosulphate/thiourea redox system in an inert atmosphere. The reaction conditions for maximum grafting have been optimized by varying the reaction variables, including the concentration of N-vinylformamide (12.0 × 10⁻²–28.0 × 10⁻² mol dm⁻³), potassium peroxymonosulphate (4.0 × 10⁻³–12.0 × 10⁻³ mol dm⁻³), thiourea (1.2 × 10⁻³–4.4 × 10⁻³ mol dm⁻³), sulphuric acid (2.0 × 10⁻³–10.0 × 10⁻³ mol dm⁻³), sodium carboxymethylcellulose (0.2–1.8 g dm⁻³) along with time duration (60–180 min) and temperature (25–45° C). Water swelling capacity, metal ion sorption and flocculation studies of synthesized graft copolymer have been performed with respect to the parent polymer. The graft copolymer has been characterized by FTIR spectroscopy and thermogravimetric analysis.     

Infection of Blissus antillus (Hemiptera: Lygaeidae) Eggs by the Entomopathogenic Fungi Metarhizium anisopliae and Beauveria bassiana

This study determined the pathogenicity and virulence of Beauveria bassiana and Metarhizium anisopliae to eggs of the chinch bug Blissus antillus (Hemiptera: Lygaeidae). Eggs were inoculated under laboratory conditions by immersion in concentrations of 1 × 10⁴ and 5 × 10⁶ conidia/ml. Inoculated eggs were kept under controlled conditions. Evaluations were carried out daily for 20 days. M. anisopliae isolates were highly virulent to eggs, even at 1 × 10⁴ conidia/ml. All B. bassiana isolates tested were considered to be of low virulence or avirulent. The most virulent isolate tested was ESALQ 818 (M. anisopliae), which caused 96.7% infection, when eggs were immersed in suspensions of 1 × 10⁴ conidia/ml. Conidial production on infected eggs was observed to be highest for M. anisopliae isolate CG144, with a mean value of 11.6 × 10⁵ conidia/ml/egg. Infection of Blissus eggs oviposited on plant stems was greater when M. anisopliae isolate CG144 was formulated in mineral oil (63.5% mortality) than when formulated in Tween 80 (27.1% mortality).     

Plasmodium vivax:A Monoclonal Antibody Recognizes a Circumsporozoite Protein Precursor on the Sporozoite Surface

Gonzalez-Ceron, L., Rodriguez, M. H., Wirtz, R. A., Sina, B. J., Palomeque, O. L., Nettel, J. A., and Tsutsumi, V. 1998.Plasmodium vivax:A monoclonal antibody recognizes a circumsporozoite protein precursor on the sporozoite surface.Experimental Parasitology90, 203–211. The major surface circumsporozoite (CS) proteins are known to play a role in malaria sporozoite development and invasion of invertebrate and vertebrate host cells.Plasmodium vivaxCS protein processing during mosquito midgut oocyst and salivary gland sporozoite development was studied using monoclonal antibodies which recognize different CS protein epitopes. Monoclonal antibodies which react with the CS amino acid repeat sequences by ELISA recognized a 50-kDa precursor protein in immature oocyst and additional 47- and 42-kDa proteins in older oocysts. A 42-kDa CS protein was detected after initial sporozoite invasion of mosquito salivary glands and an additional 50-kDa precursor CS protein observed later in infected salivary glands. These data confirm previous results with otherPlasmodiumspecies, in which more CS protein precursors were detected in oocysts than in salivary gland sporozoites. A monoclonal antibody (PvPCS) was characterized which reacts with an epitope found only in the 50-kDa precursor CS protein. PvPCS reacted with allP. vivaxsporozoite strains tested by indirect immunofluorescent assay, homogeneously staining the sporozoite periphery with much lower intensity than that produced by anti-CS repeat antibodies. Immunoelectron microscopy using PvPCS showed that the CS protein precursor was associated with peripheral cytoplasmic vacuoles and membranes of sporoblast and budding sporozoites in development oocysts. In salivary gland sporozoites, the CS protein precursor was primarily associated with micronemes and sporozoite membranes. Our results suggest that the 50-kDa CS protein precursor is synthesized intracellularly and secreted on the membrane surface, where it is proteolytically processed to form the 42-kDa mature CS protein. These data indicate that differences in CS protein processing in oocyst and salivary gland sporozoites development may occur.