Coproantigen detection in dogs experimentally and naturally infected with Echinococcus granulosus by a monoclonal antibody-based enzyme-linked immunosorbent assay

A sandwich ELISA for the detection of Echinococcus granulosus coproantigen in formalin and heat-treated faecal supernatants of dogs was developed. The assay used affinity-purified polyclonal antibodies obtained from rabbits hyperimmunised with E. granulosus excretory/secretory antigens and biotiaylated monoclonal antibody EmA9 produced against adult E. multilocularis somatic extract. The test was sensitive to 7 ng and 2.3 ng of E. granulosus protein and carbohydrate/ml of faecal supernatant, respectively. Thirteen helminth-free dogs were infected with different amounts of E. granulosus protoscoleces and the presence of coproantigen was monitored during the prepatent period until day 35 post-infection, when they were necropsied. Faecal antigen levels started to rise above the normal range between days 10 and 20 post-infection, and typically peaked at the end of the experiment. All the dogs, bearing from 3 to 67 700 worms, showed positive values in the ELISA during the prepatent period. One dog experimentally infected with Taenia hydatigena metacestode and harbouring three worms, tested positive only after the prepatent period at day 52. The test was applied to 98 stray dogs. The ELISA detected all of four dogs naturally infected with E. granulosus, two dogs with patent infections of T. hydatigena and two dogs with no cestode infections, sbowing a sensitivity of 100% and a specificity of 96%.     

Effects of experimental infection with Eimeria bovis on the balance of sodium, potassium and water in calves

Balance trials were performed to investigate the effects of experimental Eimeria bovis coccidiosis on the metabolism of water, sodium and potassium in calves. Non-infected pair-fed controls and controls fed according to plan were included in the study to allow differentiation between the effects due to infection and due to changes in feed intake. Primary infection with 5 × 10⁴ (group A) or 1 × 10⁵ (group B) oocysts caused mild diarrhoea in three out of four group A calves and mild to severe haemorrhagic diarrhoea in all five group B calves. Losses of sodium and potassium via faeces tended to increase in the infected calves during patency and apparent digestibility (AD) of these minerals was comparably low. In the urine of the infected calves the Na/K-ratio decreased due to a reduced urinary excretion of sodium. The retention (RT) of sodium was particularly high in the calves that had received the higher oocyst dose. Potassium RT did not underlie significant changes during the course of coccidiosis. In the infected calves the plasma level of sodium was reduced transiently while the level of potassium remained fairly stable. Infections with the higher oocyst dose caused a distinct reduction of fluid excretion via urine which compensated for the increased faecal water losses during severe diarrhoea. Reinfection of the group A calves with 1 × 10⁵ oocysts did not cause any significant metabolic impairment. The results of this study indicate that although acute sublethal bovine coccidiosis alters electrolyte and water metabolism the overall balance of electrolytes and water is largely maintained by physiologic adaptation.     

Neospora caninum in cattle: experimental infection with oocysts can result in exogenous transplacental infection, but not endogenous transplacental infection in the subsequent pregnancy

Whilst it is presumed that infection of pregnant cattle with Neospora caninum oocysts can provoke abortion and is the likely cause of epidemic abortion outbreaks, only two previous experiments have involved inoculation of pregnant cows with oocysts (and only one abortion was provoked in 22 pregnancies). Here, we describe the oral oocyst challenge of 18 cows synchronously bred and inoculated precisely at 70 (n = 6), 120 (n = 6) and 210 (n = 6) days in pregnancy with a nominal dose of 40,000 oocysts. Only one abortion occurred (at the 120 days challenge) which could be definitively ascribed to N. caninum and no transplacental infection (TPI) was detected in any of the other 11 calves born in the 70 and 120 day challenge groups. In contrast, 4/5 live calves born to cattle challenged at 210 days were transplacentally infected. When cows which had transplacentally infected their calves in the first pregnancy were rebred, no TPI occurred. The results show that the timing of challenge influences clinical and parasitological outcomes and that cattle in late pregnancy are exquisitely sensitive to oocyst challenge leading to exogenous TPI and congenitally infected calves. However, cattle which were indisputably systemically infected in their first pregnancy did not induce endogenous TPI in their subsequent pregnancy. This confirms previous results with experimental tachyzoite challenge and suggests that post-natal infection does not lead to persisting infections which can recrudesce in pregnancy.     

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.     

Induction of protective immunity to Brugia pahangi in jirds by drug-abbreviated infection.

Protective immunity of homologous challenge infection was examined in jirds after drug-abbreviated infection with Brugia pahangi. Mebendazole (MBZ) treatment at the early prepatent (5-7 weeks of post infection) or the late prepatent (7-9 weeks of post infection) period was highly effective in causing almost complete eradication of the primary infection. After challenge infection, the worm burden was significantly reduced 19% (31.1 in average) and 77% (9.5) to that of the controls (38.8 and 41.7), respectively. The magnitude of eosinophil response paralleled the degree of protection. No or only a few microfilariae were seen after challenge infection in jirds treated during the prepatent periods. They were also resistant to intravenous challenge with the microfilariae of B. pahangi. MBZ treatment at the patent period was, on the contrary, incomplete against primarily infected adult worms, and was not able to induce either significant protection (30.1 vs 33.1 in control) or eosinophil response to the challenge infection.     

Treatment with beta-cyclodextrin of natural Cryptosporidium parvum infections in lambs under field conditions.

Following the unexpected activity of the excipient beta-cyclodextrin against experimental infection by Cryptosporidium parvum in suckling mice, its efficacy in the prevention and treatment of natural infections in lambs was evaluated under field conditions. Fifty-three crossbred neonatal lambs were randomly selected for the study. Treatment consisted of oral administration of an aqueous suspension of beta-cyclodextrin at a dose of 500 mg/kg of body weight. To test prophylactic efficacy, the suspension was administered at 1, 2 and 3 days of age. To evaluate therapeutic efficacy, the suspension was administered on each of the 3 days following onset of diarrhoea. Infection was monitored by daily examination of faecal samples, from birth to 30 days. The criteria studied in evaluating efficacy were: oocyst shedding, the presence of diarrhoea, and weight gain at 15 and 30 days. In the group that received prophylactic treatment with beta-cyclodextrin, there were no mortalities and, compared with control lambs, there was a decrease in the number of animals infected, a longer prepatent period and notable reduction in the patent period and the duration of diarrhoea. Therapeutic treatment also reduced the patent period and the severity of diarrhoea. beta-cyclodextrin was well tolerated by all of the treated animals.     

Influence of hydrogen peroxide on acid-fast staining of Cryptosporidium parvum oocysts

Infections by the protozoan parasite Cryptosporidium parvum are routinely diagnosed by modified Ziehl-Neelsen (acid-fast) staining of faecal preparations despite the counterstaining and ghost-like appearance of some oocysts. Quantitative studies demonstrated that only a small percentage of oocysts excreted by naturally infected newborn calves displayed acid-fast characteristics, but that percentage increased when the time between excretion and sample staining was increased. The treatment of faecal samples with hydrogen peroxide (10 min, 5 vol. final concentration) caused all oocysts to become acid-fast, with up to 40-fold increases in test sensitivity in samples treated and stained within 3 h of excretion. Flow-cytometry analysis of hydrogen peroxide-treated oocysts also demonstrated increased labelling of oocysts by a commercial monoclonal antibody preparation commonly used for diagnosis.     

Biology and pathogenicity of Eimeria neodebliecki Vetterling, 1965 in experimentally infected pigs

The endogenous development and pathogenicity of Eimeria neodebliecki Vetterling, 1965 are described in weaned pigs inoculated with 250 000 oocysts. The endogenous stages developed within the apical cytoplasm of the enterocytes of the middle and posterior jejunum. The asexual development comprised two generations of meronts. Immature and mature meronts were found in groups up to five per host cell. The first fully developed macrogametes and mature microgamonts were seen at 9 days post-infection (DPI). The prepatent period was 10 days, and the patent period lasted 6–8 days. Sporulation of oocysts was completed within 12 days at 25°C, and 16 days at 20°C. E. neodebliecki infection produced clinical signs of coccidiosis in weaned pigs which developed frothy or mucoid diarrhea from 9 to 12 DPI. Pathological changes were situated in the second half of the small intestine, with the predilection for the posterior jejunum. At 9 and 10 DPI, macroscopically, ranged from catarrhal to focal, pseudomembranous inflammatory lesions. Histopathological and SEM examinations revealed moderate villous atrophy with focal epithelial erosions and fibrinonecrotic material at the villous tips. E. neodebliecki is pathogenic for pigs and can be associated with clinical manifestation of diarrhea, stunted growth and poor condition in pigs.     

First fossil fruits of Elaeocarpus (Elaeocarpaceae) in East Asia: Implications for phytogeography and paleoecology

The genus Elaeocarpus contains approximately 360 species and occurs in mesic forest communities from India, through to China, Southeast Asia, New Guinea, Australia, and New Caledonia. Elaeocarpus fossils are best known from the Eocene to the Miocene of Australia and the late Pliocene–early Pleistocene of India, but have not been documented from East Asia before. Here we describe six new species of Elaeocarpus, E. nanningensis sp. nov. from the late Oligocene Yongning Formation of the Nanning Basin, E. presikkimensis sp. nov. from the Miocene Erzitang Formation of the Guiping Basin, E. prerugosus sp. nov., E. prelacunosus sp. nov., E. preserratus sp. nov., and E. preprunifolioides sp. nov. from the late Miocene Foluo Formation of the Nankang Basin in Guangxi, South China. This is the first reliable report for the genus occurring in East Asia, and the fossils indicate that Elaeocarpus had colonized this region by the late Oligocene and represented by a morphologically diverse group of species by the late Miocene. This sheds new insights into the timing and migration patterns of the genus in East Asia. Elaeocarpus is typically a rainforest genus occurring in mesic forests. Based on the habitat of their morphologically similar modern relatives we propose that these three sedimentary basins were warm and wet adjacent to mountainous regions with the evergreen or rain forests during the late Oligocene to Miocene.     

Plasmodium gallinaceum: mechanisms of anemia in infected chickens

Erythrocyte labeling by random and cohort techniques was used to study erythrocyte survival in normal chickens and chickens infected with Plasmodium gallinaceum. Occurrence of erythrocyte destruction during the prepatent period was apparent in infected chickens by both techniques. Treatment with the antimalarials chloroquine and quinacrine not only cleared the circulation of parasites promptly but brought about an equally prompt cessation of disease-related erythrocyte destruction. Plasmodium gallinaceum infection caused a transitory decrease in blood volume at the time of rapid decrease in packed cell volume. The blood volume returned to preinfection values before the packed cell volume returned to normal. Parasitized erythrocytes were present in capillaries of the spleen, liver, and bone marrow during the entire prepatent period of the infection, thus providing a reasonable explanation for erythrocyte destruction observed in the absence of parasitemia during the prepatent period.     

不同大花类群淫羊藿属植物的花部特征及生殖特征研究

淫羊藿是我国重要的草本植物,在中药、功能性食品和园林观赏等领域都具有重要用途,受地域差异性影响,淫羊藿属植物的植株形态、花部特征、开花物候和生殖特征各不相同。本研究对四川不同产地野生种群大花类淫羊藿（巫山淫羊藿、粗毛淫羊藿和宝兴淫羊藿）的开花动态、花部特征、访花昆虫与繁殖系统进行了对比研究。结果表明：（1）3种淫羊藿属植物的开花期较为集中,巫山淫羊藿和粗毛淫羊藿的花期为3月下旬～4月下旬,单花花期均为3～4 d;宝兴淫羊藿的花期为4月中旬～5月中旬,单花花期5～6 d;（2）膜翅目和双翅目昆虫为3种淫羊藿属植物的有效访花者,有效访花者访花特性与花部特征之间具有明显的相关性;（3）3种淫羊藿属植物的花序、花和果实的数量受所处环境影响较大,自然结实率：巫山淫羊藿>宝兴淫羊藿>粗毛淫羊藿;温度和光照是最主要的影响因子。     

Histopathology and cellular response in Enteromyxum leei (Myxozoa) infections of Diplodus puntazzo (Teleostei)

Enteromyxum leei is an intestinal parasite responsible for serious outbreaks in Mediterranean sharpsnout sea bream Diplodus puntazzo. E. leei infection was experimentally transmitted to healthy D. puntazzo (R) by cohabitation with infected donor fish. Haematological changes and histopathological damage were evaluated in relation to the course of infection. The prevalence of infection in R fish was 100% from day 10 post-exposure (p.e.) onwards, and the infection intensity and histopathological damage increased progressively. Different developmental stages were found in the infected intestines, including proliferative (stages 1–3) and sporogonic (stages 4 and 5) stages. Intestinal damage consisted of vacuolation, necrosis, detachment and sloughing of epithelium, and was correlated with the progression of the infection and with the development of the parasite. Sporogonic stages appeared from day 20 p.e. onwards. Initially, D. puntazzo seems to counteract the infection through the increase in leucocyte numbers, respiratory burst activity, haematopoietic activity and MMC. Two types of eosinophilic granular cells (EGC1 and EGC2) were detected in the intestinal epithelium and lamina propria. EGC1 numbers decreased with the progression of infection, whereas an increase in EGC2 occurred, mainly in the lamina propria. The involvement of the cellular immunity in the response of D. puntazzo to E. leei was demonstrated. The depletion of this response at a certain point of the infection could contribute to the high virulence of this myxozoan in this fish species.     

Time gap between oocyst shedding and antibody responses in mice infected with Cryptosporidium parvum

We observed the time gap between oocyst shedding and antibody responses in mice (3-week-old C57BL/6J females) infected with Cryptosporidium parvum. Oocyst shedding was verified by modified acid-fast staining. The individually collected mouse sera were assessed for C. parvum IgM and IgG antibodies by enzyme-linked immunosorbent assay from 5 to 25 weeks after infection. The results showed that C. parvum oocysts were shed from day 5 to 51 post-infection (PI). The IgM antibody titers to C. parvum peaked at week 5 PI, whereas the IgG antibody titers achieved maximum levels at week 25 PI. The results revealed that IgM responses to C. parvum infection occurred during the early stage of infection and overlapped with the oocyst shedding period, whereas IgG responses occurred during the late stage and was not correlated with oocyst shedding. Hence, IgM antibody detection may prove helpful for the diagnosis of acute cryptosporidiosis, and IgG antibody detection may prove effective for the detection of past infection and endemicity.     

Mouse strain variation and effects of oocyst dose in infection of mice with Eimeria falciformis, a coccidian parasite of the large intestine

Stockdale P. G. H., Stockdale M. J., Rickard M. D. and Mitchell G. F. 1985. Mouse strain variation and effects of oocyst dose in infection of mice with Eimeria falciformis, a coccidian parasite of the large intestine, International Journal for Parasitology15: 447–452. Five inbred strains of mice and three hypothymic (nude) strains were infected orally with different doses of E. falciförmis oocysts. After resolution of primary infection as determined by faecal oocyst output, mice were challenged orally with a second dose of E. falciformis. Amongst the intact mice, BALB/c proved the most resistant to primary infection, while C3H/He mice were most susceptible, in terms of faecal oocyst production. Resistance was far more dramatic in BALB/c mice given high numbers of challenge oocysts. In terms of mortality at high oocyst doses, CBA/H were the most susceptible. All of the strains of mice were highly resistant to reinfection. In the case of nude mice, BALB/c. nu/nu were more susceptible than CBA/H.nu/nu or C57BL/6.nu/nu both in terms of faecal oocyst production and mortality. Thus the most resistant inbred mouse strain (BALB/c) is the least resistant in the absence of T cells. Unlike intact mice, nude mice showed no resistance to reinfection, this result being in line with previous work on this and other Eimeria spp. in nude mice.     

Effect of F-2 and T-2 fusariotoxins on experimental Cryptosporidium baileyi infection in chickens

The course of Cryptosporidium baileyi infection in chickens fed with different doses of fusariotoxins was compared with that of control groups. F-2 toxin levels of 0.187–1.5 mg kg⁻¹ and T-2 toxin levels of 0.187–6.0 mg kg⁻¹ were investigated. The experimental amimals were orally infected with 6 × 10⁵ C. baileyi oocysts at 1 week of age. Total daily oocyst output was monitored by a quantitative method. Acquired immunity was tested at the age of 4 weeks, by ELISA and by a challenge infection with an equal number of oocysts, upon recovery from the primary infection. The results show that in chickens kept on the lower doses of F-2 and T-2 toxins, the parasite infection ran a similar course to that in the control groups, and the animals became resistant to re-infection. However, when higher doses (2.0–6.0 mg kg⁻¹) of T-2 toxin were used, a depression of weight gain was observed with some other physiological parameters (PCV, weight of bursa, weight of thymus, skin thickness in PHA-P skin test) also indicating toxic effect and, simultaneously, the oocyst output decreased significantly and the patent period was slightly prolonged. Although certain modifications of the immune response could be revealed, the chickens became resistant to re-infection. Only early (1 week of age) parasite infection and 6 mg kg⁻¹ T-2 toxin in the feed significantly depressed body weight gain and immunity.     

Oocyst-induced Toxoplasma gondii infections in cats

To investigate the oocyst-induced cycle with a 21+ day prepatent period, 32 cats were fed 5 x 10(5) to 2 x 10(7) sporulated oocysts of Toxoplasma gondii and necropsied between 4 hr and 41 days thereafter. The presence of the earliest stages in 7 cats was tested in mice. The tissues of 25 cats were studied histologically; 17 were bioassayed by feeding them to cats to determine, by the length of the prepatent period, whether bradyzoites were present. Based on previous studies, a short (3-10 days) prepatent period indicated that bradyzoites were present in an oral inoculum and a long (greater than 21 days) prepatent period indicated the presence of tachyzoites only. Tissues from 14 cats were also bioassayed in mice for the presence of bradyzoites, using their resistance to pepsin as indicator. Six were studied by both methods. Based on these criteria, tachyzoites predominated in extraintestinal organs during the first 14 days after infection. They were found as early as 4 hr in mesenteric lymph nodes where their number reached 10(4) after 6 and 9 days; they were present after 1 day in all levels of the small intestine and after 6 days in the liver, lung, and blood. Bradyzoites were first detected 10 days after oocyst feeding; they predominated by the third week of infection and were present up to 41 days. Enteroepithelial stages were found histologically only in 2 cats, 24 and 41 days after inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aspects of the life cycle and pathogenesis of Elaphostrongylus cervi in red deer (Cervus elaphus)

Aspects of the migratory life cycle and pathogenesis of Elaphostrongylus cervi were studied in red deer (Cervus elaphus) using 2 farmed calves experimentally infected with 450 third-stage larvae killed 40 and 45 days postinfection and using 3 wild calves and 3 wild yearlings with natural infections killed during autumn hunting. A full necropsy was carried out on the experimental calves, but only the head, eviscerated carcass, and lungs were examined from the naturally infected animals. Histological examination included extensive studies of the central nervous system (CNS), spinal nerve roots, and lungs. The experimental calves had prepatent infections, with many immature adult nematodes in the CNS, whereas the wild calves showed CNS lesions indicating a very recent E. cervi infection. The yearlings had patent infections, with many mature E. cervi in their skeletal muscles, reflecting acquisition of infection during the previous summer. Our findings showed that E. cervi develop to the adult stage in the CNS (subarachnoid spaces) and subsequently migrate into the skeletal muscles, where the mature nematodes live in reproductive pairs and groups. In the nervous system, the nematode caused encephalomyelitis, focal encephalomalacia and gliosis, meningitis, radiculitis, ganglionitis, and perineuritis.     

Population dynamics of the harmful diatom Eucampia zodiacus Ehrenberg causing bleachings of Porphyra thalli in aquaculture in Harima-Nada, the Seto Inland Sea, Japan

The diatom Eucampia zodiacus Ehrenberg is one of the harmful diatom species which indirectly cause bleachings of Nori (Porphyra thalli) in aquaculture through competitive utilizing of nutrients (especially nitrogen) and resultant nutrient depletion in water columns during the bloom events. The seasonal changes in environmental factors, cell density and cell size of E. zodiacus were investigated for 4 years (April 2002–December 2005) to understand the population ecology of this diatom in Harima-Nada, the eastern part of the Seto Inland Sea, Japan. Vegetative cells of E. zodiacus were usually detected year-round. Total cell densities of E. zodiacus annually peaked from mid-February to early April, and high cell densities were observed in the whole water columns during the bloom-period. Nutrient concentrations decreased with the increase of cell density of E. zodiacus, and low nutrients concentrations continued throughout the E. zodiacus bloom-period. The average cell size (length of apical axis) of E. zodiacus populations ranged from 10.8 μm to 81.2 μm, and the restoration of cell size occurred once in autumn every year just after reaching the minimum cell size. In addition, its great seasonal regularity was confirmed by the decrease and restoration of its cell size through 4-year study period. Temperature and nutrients were suitable in autumn for the growth of E. zodiacus, its blooms never occur in that season. These results strongly suggest that E. zodiacus did not have a resting stage, and it spends autumn for size restoration and starts to bloom thereafter in Harima-Nada in winter and spring, causing fishery damage to Nori aquaculture by resulting nutrient deprivation.     

Atoxoplasma Associated with an Isosporan Oocyst in Canaries*

SYNOPSIS Coccidia-free and mite-free domestic canaries Serinus canarius given sporulated Isospora oocysts of canary origin and autopsied at intervals thereafter had numerous organisms in cells of the macrophage system structurally similar to organisms called Atoxoplasma. Judging from the sequence of intensity of infection, the mucosa of the upper intestine was parasitized first and the organisms spread from there down the intestine and to the internal organs. The prepatent period of 10 days for the Isospora infection could be started in one bird by oocyst administration and completed by transfer of infected liver cells from this bird to a 2nd bird. Isospara oocysts from sparrows were not infective for a canary.