The molecular characterization of an Eimeria and Cryptosporidium detected in Asian seabass (Lates calcarifer) cultured in Vietnam

An intestinal Eimeria was previously reported as a significant pathogen of Asian seabass (Lates calcarifer) in nurseries in Vietnam. In the present study, both Eimeria and Cryptosporidium were detected by sequence analyses of fragments of the 18S rRNA gene amplified from these Vietnamese L. calcarifer tissues. Based on these analyses, the Eimeria from the Vietnamese L. calcarifer formed clades with the Eimeria detected in L. calcarifer tissues from Australia, but clustered separately from other known Eimeria and Goussia species. The Cryptosporidium detected in L. calcarifer from Vietnam clustered closest with C. parvum and C. hominis. In situ hybridization using DIG-labeled DNA probes generated from 18S PCR products on the Vietnamese L. calcarifer wax block tissues showed that this method could not be used to distinguish between Eimeria and Cryptosporidium, due to the conserved nature of the 18S locus. A previously published study on the morphology of parasite developmental stages and oocysts in the Vietnamese L. calcarifer tissues showed only an intestinal Eimeria infection. The Cryptosporidium could be present at very low levels undetectable by microscopy in intestines, or being ubiquitous, was a possible contaminant from feed or water. While molecular analysis is a very useful tool in the study of disease and identification of aetiological agents, this study reiterates the importance of demonstrating organisms in situ in tissues.     

巨型艾美耳球虫感染鸡空肠粘膜肥大细胞数量和组织胺含量的变化

本文借助于改良甲苯胺蓝染色法和荧光测定法，分别检测了巨型艾美耳球虫感染鸡空肠粘膜肥大细胞（ＭＭＣ）数量和组织胺（ＨＡ）含量的变化。结果表明，初次感染巨型艾美耳球虫之后，空肠ＭＭＣ呈减少趋势，至感染后第１４天回复至不感染对照组水平。而初次感染组相应空肠ＨＡ水平呈现出不显著波动后，在感染后第４天达最大值。在再次攻毒试验中，免疫攻毒组ＭＭＣ呈现水升趋势，尔后回复至不感染对照组水平；免疫攻毒组织相应肠道Ｈ     

江苏部分地区奶牛球虫病流行情况调查

为了解江苏省奶牛感染球虫的情况,笔者对6个规模化奶牛场和3个奶牛小区9个牧场的奶牛球虫感染状况进行了随机抽样调查,分别于2008年11月和2009年4月按不同年龄阶段共采集粪样2100份。调查结果显示:9个牧场都有球虫感染,1个奶牛小区牧场的感染率最高达50%,其中11月份调查的感染率为30.10%,4月份调查的感染率为36.95%,两者没有明显的差异;从不同年龄阶段奶牛的感染情况来看,发现感染率最高的2个阶段为1～6月龄和6～12月龄,感染率高达53.81%和49.52%,感染率最低的年龄阶段为1月龄以内的奶牛,感染率为12.86%;从感染强度来看,大部分牧场平均OPG低于1000,占55.56%,只有一个牧场的平均OPG比较高,为2410;从本次调查结果发现9个牧场感染有8种艾美耳球虫(E.imeria),分别是牛艾美耳球虫(E.bovis)、邱氏艾美耳球虫(E.zumii)、椭圆艾美耳球虫(E.ellipsoidallis)、阿拉巴艾美尔球虫(E.alabamensis)、亚球形艾美尔球虫(E.subspherica)、怀俄明艾美尔球虫(E.wyomigensis)、柱状艾美尔球虫(E.cylindrica)、奥博艾美尔球虫(E.auburnensis)。优势虫种为牛艾美耳球虫、邱氏艾美耳球虫、椭圆艾美耳球虫,其他5种球虫所占比例较少。     

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.     

Husbandry risk factors associated with subclinical coccidiosis in young cattle

This paper describes an observational longitudinal study of cattle farms in England and Wales, which aimed to identify management practices associated with the presence of Eimeria spp. infection in young cattle. Thirty cattle farms situated in England and Wales were selected and one group of more than 20 young cattle aged 5-18 months of age was monitored on each farm. Three variables were identified as significantly associated with status in a multivariable model. The odds of finding Eimeria spp. were lower on farms that kept sheep on the same premises as the cattle, as was an increase in the maximum age within the sampled group. The latter probably reflects the development of post-infection immunity within the sampled animals. Good water-trough hygiene protected against Eimeria spp. oocyst excretion, with the odds of detection being higher on farms where it was reported that the water troughs were not cleaned and emptied more than once per month. The value of frequent emptying and cleaning of water troughs in reducing the exposure of calves to Eimeria spp. and thus lowering the impact of coccidiosis, both clinical and subclinical should be communicated to cattle farmers.     

布氏艾美耳球虫南宁株的分离及鉴定

本试验从南宁市郊区某鸡场感染球虫病的病鸡粪便中收集到混合卵囊,采用改良过的琼脂单卵囊分离法从中分离纯化出2个球虫虫株,并对其中的1个球虫分离株进行了鉴定,鉴定结果为:该株球虫卵囊呈椭圆形或卵圆形,卵囊平均大小为(22.51±0.4790)μm×(15.94±0.5599)μm,平均卵型指数为1.41212±0.06714,裂殖体平均大小为31.33 μm×27.46 μm,潜在期为120 h 50 min,排卵高峰期为感染后第6天,最短孢子化时间为18 h 50 min,寄生部位为小肠中、下段,属中等毒力的球虫虫株。通过将这些测定和观察到的指标与各类文献中所记载的各种鸡艾美耳球虫的特征进行了对比,经过综合分析后,将该分离株球虫鉴定为布氏艾美耳球虫,并且将它命名为布氏艾美耳球虫南宁株,记为Eimeria.brunetti-GXNN。对该虫株的成功分离鉴定,为进一步了解本地球虫虫株的生物学特性,开展鸡球虫病的免疫预防奠定了基础。     

Elevation of muscle and plasma 3-methylhistidine as a result of turkey coccidiosis

To assess muscle breakdown during avian coccidiosis, the level of the non-metabolizable amino acid 3-methylhistidine (3MH) was determined in muscle and plasma from turkey poults that received an infection with a field isolate containing a mixture of Eimeria species. The effect of increased levels of parasitism was evaluated at 6 days postinoculation (DPI) in birds receiving 2.5 x 10(4), 1 x 10(5), or 2 x 10(5) oocysts each. The changes in 3MH levels during recovery from acute infection were assessed at 6-29 DPI in animals given 1.9 x 10(5) oocysts per bird. In some experiments, uninoculated birds given the same amount of feed as infected birds (pair fed) were used to determine the impact of feed deprivation on weight loss and 3MH levels. Infected birds had significantly elevated plasma and muscle 3MH at 6 DPI after a single dose of Eimeria oocysts. The plasma and muscle 3MH returned to control levels after 14 DPI. The 3MH levels increased with increased dose of oocysts. Plasma and muscle 3MH levels were well correlated, and an inverse curvilinear relationship between weight gain and plasma 3MH concentrations levels was observed. Plasma and muscle 3MH levels were significantly elevated in pair-fed birds, but 3MH levels in infected birds were increased by 30% over pair-fed birds. The results suggested that muscle breakdown, as assessed by plasma and muscle levels of 3MH, increased during the acute stage of Eimeria infection in turkey poults.     

Kinetics of interleukin-2 production in chickens infected with Eimeria tenella

Interleukin (IL)-2 is a major cytokine of cell-mediated immunity (CMI). Because chickens infected with Eimeria, the causative agent of coccidiosis, develop a robust cell-mediated response against the parasite, we measured IL-2 concentrations in vivo and in vitro during the course of primary and secondary experimental Eimeria tenella infections. IL-2 levels in serum and culture supernatants of spleen lymphocytes stimulated with mitogen or E. tenella sporozoites were significantly increased on day 7 post-primary infection compared with control group. This peak in IL-2 coincided with the time of maximum intestinal lesions as measured by cecum lesion scores. By contrast, during secondary infection highest IL-2 concentrations preceded intestinal lesions by 5 days (day 2 versus day 7, respectively). These results confirmed that IL-2 production is augmented during experimental coccidiosis and suggested that cellular immunity elicited during an anamnestic response to parasite reinfection is mediated, at least in part, by IL-2.     

安徽省部分地区鸡球虫种类及感染情况调查

目的了解安徽省五个县（区）鸡球虫种类及感染情况。方法采用群体采样法分别从个调查点采集新鲜鸡粪，检查粪样。阳性粪样采用饱和盐水漂浮和离心沉淀法进行卵囊分离。显微镜下观察卵囊进行虫种鉴定，并统计各调查点的鸡球虫感染率和感染强度等。结果检查172份鸡的新鲜粪样，得出鸡球虫感染率为100％；共检出7种球虫，经鉴定均隶属于艾美耳属，分别为毒害艾关耳球虫（Eimeria necatrix），堆型艾关耳球虫（E．acervulina），巨型艾关耳球虫（E．maxima），柔嫩艾美耳球虫（E．tenella），和缓艾美耳球虫（E．mitis），哈氏艾美耳球虫（E．hagani）和早熟艾美耳球虫（E．praecox）。结论鸡球虫在安徽省的这5个县（区）普遍存在，且有的调查点感染强度很高。     

Estimation of the economical effects of Eimeria infections in Estonian dairy herds using a stochastic model

In this study, a stochastic predictive model stimulating a constant infection pressure of Eimeria was used to estimate production outcome, economic, and effects of treatment decisions in a dairy herd of 100 cows. The intestinal parasite cause problems mainly in calves, and is known to have long term effects on the growth rate, and in severe cases can result in mortalities. Due to the inconspicuous nature of the parasite, the clinical signs and sub-clinical manifestations it may produce can be overlooked. Acquired data from literature and Estonian dairy farms were implemented in the SimHerd IV model to simulate three scenarios of symptomatic treatment: no calves treated (NT), default estimate of the current treatment strategy (DT), and all calves treated (AT). Effects of metaphylactic treatment were studied as a lowering of the infection pressure. Delay in the age for beginning of insemination of heifers was the effect with the largest economic impact on the gross margin, followed by calf mortality and reduction in growth rate. Large expenses were associated with the introduction of replacement heifers and feeding of heifers as a result of the delay in reaching a specific body weight at calving. Compared to the control scenarios, with no effects and treatments of Eimeria, dairy farmers were estimated to incur annual losses ranging 8-9% in the balanced income. Providing metaphylactic drugs resulted in an increased gross margin of 6-7%. Purchase of new heifers compensated for some production losses that would otherwise have enhanced expenses related to Eimeria. The simulation illustrates how effects of Eimeria infections can have long lasting impact on interacting management factors. It was concluded that all three simulated symptomatic treatment regimes provided only small economic benefits if they were applied alone and not in combination with lowering of infection pressure.     

Interleukin-2 production in SC and TK chickens infected with Eimeria tenella

SC and TK inbred chicken strains display differential protective immunity to coccidiosis, SC being more resistant and TK susceptible to disease. In this study, the association between interleukin (IL)-2 and disease phenotype was assessed by cytokine quantification in serum, duodenum, cecum, and spleen cell cultures of SC and TK chickens experimentally infected with Eimeria tenella. In general, after primary infection, SC and TK strains produced equivalent amounts of IL-2 in all sources examined. However, after secondary infection, SC animals displayed significantly greater IL-2 levels in serum and the duodenum compared with strain TK. IL-2 production after reinfection with Eimeria may be an important factor contributing to the genetic differences in coccidiosis between SC and TK chickens and provides a rational foundation for cytokine-based immunotherapeutic approaches to disease control strategies.     

Immune responses to an Eimeria acervulina infection in different broilers lines

The (T-cell) immune responses of two different broiler lines to a primary Eimeria acervulina infection were investigated. The lines used were a commercial fast-growing broiler line and a slow-growing type of broiler as used in organic farming. Seven-day-old broilers of both lines were infected with 5 x 10(4) oocysts of E. acervulina. The animals were weighed and a species-specific real-time PCR was used to quantify the total amount of parasites in the duodenum. In the fast-growing line, a lower parasite load was seen from day 4 onwards compared to the slow-growing line. In both lines the intestinal peak of Eimeria DNA was observed at day 5 post infection (p.i.). In the duodenum no increase in CD4(+) T-cells was found in both infected lines, but a fast increase in CD8(+) T-cells was observed in the fast-growing line. At day 3 p.i. in the slow-growing broilers an IL-18 mRNA response was observed. At day 4 p.i. strong IFN-gamma and IL-8 mRNA responses were found in both lines. No IL-4 mRNA responses were found in the duodenum. In conclusion, both lines have different growth rates and control and infected conditions. Based on the kinetics of observed phenomena a primary infection with E. acervulina in 7-day-old broilers seems to generate an early CD8alpha(+) response in fast-growing broilers compared to the slow-growing broilers. This difference in immune reaction after an E. acervulina infection could result in a different Eimeria load in the duodenum.     

Optimized necrotic enteritis model producing clinical and subclinical infection of Clostridium perfringens in broiler chickens

In this study we assessed the roles of Eimeria infection and dietary manipulation (feeding a diet with a high level of fishmeal) in an Australian necrotic enteritis (NE) challenge model in broiler chickens. An experiment was designed to test the hypothesis that Eimeria infection and dietary manipulation, i.e., inclusion of fishmeal in the diet, are necessary to induce NE experimentally. The results showed that the combination of Eimeria administration and fishmeal feeding had a significant effect on induction of clinical and subclinical Clostridium perfringens infection. The majority of the mortality that occurred during the second week of the trial was due to an NE outbreak following the C. perfringens challenge. The mortality rate of the birds was 12.00% for the high-fishmeal (HFM; 500 g/kg) group and 9.33% for the low-fishmeal (LFM; 250 g/kg) group when the birds were subjected to C. perfringens and Eimeria. Fishmeal alone did not induce significant mortality in birds challenged only with C. perfringens but showed a significantly higher C. perfringens count than the non-fishmeal (NFM) control group. Eimeria administration had a significant effect on NE-related mortality but did not have an effect on the C. perfringens count. In accordance with the time course of bird mortality, it can be determined that of the 3 successive days of oral gavage with C. perfringens, the first inoculation was essential for inducing NE, but the third had no additional effect on NE-related mortality. Also, reducing the fishmeal level from 500 to 250 g/kg had no negative impact on the reproducibility of the model. It may be concluded that NE can be consistently induced under experimental conditions by feeding broilers a diet containing 250 g/kg fishmeal, using a single inoculation with low numbers of Eimeria, administering one or two oral C. perfringens inoculations, and maintaining appropriate ambient temperatures and diets.     

Gel-Bead Delivery of Eimeria oocysts protects chickens against coccidiosis

Vaccines composed of either virulent or attenuated Eimeria spp. oocysts have been developed as an alternative to medication of feed with ionophore drugs or synthetic chemicals. The purpose of this study was to evaluate the use of gel-beads containing a mixture of Eimeria acervulina, Eimeria maxima, and Eimeria tenella oocysts as a vaccine against coccidiosis. Newly hatched chicks (Gallus gallus domesticus) were either sprayed with an aqueous suspension of Eimeria oocysts or were allowed to ingest feed containing Eimeria oocysts-incorporated gel-beads. Control day-old chicks were given an equivalent number of Eimeria oocysts (10(4) total) by oral gavage. After 3 days, chicks were randomly assigned to individual cages, and feces were collected between days 5 and 8 postinfection. All samples were processed for total Eimeria oocysts. At 4 wk of age, all chickens and a control nonimmunized group received a high-dose E acervulina, E maxima, and E. tenella challenge infection. Oocyst excretion by chicks fed gel-beads or inoculated by oral gavage was 10- to 100-fold greater than that of chicks spray-vaccinated with the Eimeria oocysts mixture (log 6.3-6.6 vs. log 4.8). Subsequent protection against challenge as measured by weight gain and feed conversion efficiency was significantly greater (P < 0.05) in gel-bead and oral gavage groups compared with spray-vaccinated or nonimmunized groups. Also, gel-bead and oral gavage groups showed no significant difference (P > 0.05) in weight gain and feed conversion efficiency compared with nonchallenged controls. These findings indicate that incorporation of Eimeria spp. oocysts in gel-beads may represent an effective way to deliver live oocyst vaccines to day-old chicks for preventing subsequent outbreaks of coccidiosis in the field.     

Nitric oxide production by macrophages stimulated with Coccidia sporozoites, lipopolysaccharide, or interferon-gamma, and its dynamic changes in SC and TK strains of chickens infected with Eimeria tenella

Nitric oxide (NO) is an important mediator of innate and acquired immunities. In the studies reported here, we quantified NO produced in vitro by chicken leukocytes and macrophages and in vivo during the course of experimental infection with Eimeria, the causative agent of avian coccidiosis, and identified macrophages as the primary source of inducible NO. Eimeria tenella-infected chickens produced higher levels of NO compared with noninfected controls. In Eimeria-infected animals, SC chickens produced greater amounts of NO compared with infected TK chickens, particularly in the intestinal cecum, the region of the intestine infected by E. tenella. Macrophages that were isolated from normal spleen were a major source of NO induced by interferon (IFN)-gamma, lipopolysaccharide (LPS), and E. tenella sporozoites. Macrophage cell line MQ-NCSU produced high levels of NO in response to Escherichia coli or Salmonella typhi LPS, whereas the HD-11 macrophage cell line was more responsive to IFN-gamma. These findings are discussed in the context of the genetic differences in SC and TK chickens that may contribute to their divergent disease phenotypes.     

Selection for early (precocious) development of Eimeria meleagridis in the turkey

A precocious line of Eimeria meleagridis was developed by repeated propagation of the parasite in young turkeys and collection of the very first oocysts produced following infection. After 20 generations of selection, the prepatent period was reduced by 4-8 hr. Weight gain of poults given this line, from days 0-3 after infection, was significantly greater than that of poults given the parental strain, indicating that selection resulted in a loss of pathogenicity. Poults immunized with the precocious line produced no oocysts following challenge with the parental strain, indicating that immunogenicity was retained following selection. This is the first published report of the selection of a precocious line of Eimeria in the turkey.     

Coccidial infections in housed lambs: oocyst excretion, antibody levels and genetic influences on the infection

Faecal Eimeria oocyst excretion, body weights, humoral antibodies against E. ovinoidalis sporozoite antigen and related heritabilities were determined in housed Merinoland sheep lambs throughout a period of 100 days after birth in Germany. Altogether 10-11 Eimeria spp. were found. Cumulative incidences of E. ovinoidalis and E. weybridgensis/crandallis increased rapidly resulting in almost 100% incidence in 8 weeks old lambs. In the other species, the cumulative incidence increased more continuously. Except for E. granulosa oocysts of all species had been excreted at last once until day 30. By far the highest oocyst counts (OpG) were observed with E. ovinoidalis, followed by E. weybridgensis/crandallis. High counts were limited to the period of 5-8 weeks after birth. In the other Eimeria species oocyst counts persisted at comparatively low levels until the end of the observation period although their proportion of the total counts increased with age of the lambs. Time courses of oocyst excretion suggest an early onset of effective immunity to the major Eimeria spp., which differed for the minor species. Mean and maximum oocyst counts and body weights of the lambs were inversely correlated suggesting negative effects of the infection on the lamb's performance. High mean antibody levels on day 7 after birth dropped until day 40 and increased subsequently again. There were no indications that maternal antibodies were protective. Antibody levels on day 40 after birth were positively correlated with oocyst counts in the faeces whereas those determined on day 80 were independent of infection parameters. Heritabilities of log(10)OpG were not significantly different from 0 up to an age of 60 days. Later estimated values were between 0.54 and 0.79 suggesting that immune protective effects rather than innate effects determining disease susceptibility are under genetic influence.