Sequence and Phylogenetic Analysis of rDNA ITS of Three Eimeria Species in Rabbit

In order to study whether the internal transcribed spacers (ITS) sequence could be used as a molecular marker for the species identification of rabbit coccidian, the rDNA ITS of Eimeria intestinalis, Eimeria flavescens and Eimeria magna were amplified by polymerase chain reaction (PCR), and were cloned into pGEM-T Easy vector subsequently. The positive recombinant plasmids were identified by PCR and then sequenced. By sequence comparison and comparative analysis with the relative sequences of rabbit Eimeria spp. available in GenBank, the results showed that the lengths of Eimeria intestinalis, Eimeria flavescens and Eimeria magna were 1065, 1009 and 1047 bp, respectively, and the sequence homologies with the same species sequences were 99.2%, 99.0% and 94.5%, respectively, while were 55.3% to 82.1% compared with corresponding sequences of other different species sequences. The phylogenetic analysis using software Mega 5.0 showed that all rabbit coccidia clustered together in a clade, which was divided into two sister lineages, corresponding to the presence or absence of oocyst residuum. The result demonstrated ITS could be used as a molecular marker for the species identification of rabbit coccidia.     

Effects of in ovo vaccination and anticoccidials on the distribution of Eimeria spp. in poultry litter and serum antibody titers against coccidia in broiler chickens raised on the used litters

The present study reports the effects of various field anticoccidial programs on the distribution of Eimeria spp. in poultry litter and serum antibody titers against coccidia in broiler chickens raised on the used litters. The programs included in ovo vaccination and various medications with either chemicals, ionophores, or both. In general, serum samples from these chickens showed anticoccidial antibody titers when tested at days 7 and 14 post hatch with the peak response at day 43. Serum anticoccidial titers were highest in birds fed a non-medicated diet compared with those vaccinated or fed medicated diets. Total number of Eimeria oocysts and the composition of Eimeria spp. present in the litter samples from different treatment groups varied depending on the type of anticoccidial program. Oocyst counts in general ranged from 3.7×10(3) to 7.0×10(4) per g of litter. Importantly, both morphological and molecular typing studies revealed four major predominant Eimeria spp., E. acervulina, E. maxima, E. praecox, and E. tenella in the litter samples. Collectively, these results indicate that the field anticoccidial programs influenced the type and abundance of Eimeria spp. present in the litter samples and also modulated host immune response to Eimeria.     

Health status of northern bobwhite quail (Colinus virginianus) in eastern Kansas

The health status of wild northern bobwhite quail (Colinus virginianus) from Lyon County, Kansas, was evaluated by conducting comprehensive health assessments on 25 birds. Gross lesions indicative of avian pox, ulcerative enteritis, and quail bronchitis were not present. Serologic tests for antibodies to Salmonella pullorum, Salmonella gallinarum, Pasteurella multocida, Mycoplasma gallisepticum, Mycoplasma synoviae, and avian adenoviruses were all negative. Intestinal coccidia (Eimeria spp.) were found in 36% of the birds. Only three species of helminth parasites were found: Dispharynx nasuta in two birds, Cyrnea colini in one bird, and larval Physaloptera sp. in four birds. Arthropod parasites (ticks, lice, mites, and/or chiggers) were present on 96% of the birds examined. Compared with wild bobwhite populations in the southeastern United States, the diversity, prevalence, and intensities of microbial and parasitic agents were low.     

Role of Coccidia in the occurrence of necrotic enteritis of chickens

Clostridium perfringens type A, Eimeria acervulina, and Eimeria necatrix were used to produce necrotic enteritis in chickens. The disease was produced in all groups of birds that received feed contaminated with C. perfringens. Mortality due to necrotic enteritis was highest (53%) in birds infected with E. acervulina before infection with clostridia. There was a significant difference in mortality rates between birds infected with E. acervulina and birds infected with E. necatrix before infection with C. perfringens. Mortality rates also differed significantly between the group infected with E. necatrix and the group that received only feed contaminated with C. perfringens. It was concluded that under field conditions, coccidia can play a significant role in the occurrence of necrotic enteritis when a sufficient number of toxigenic strain of C. perfringens type A is present. The pathological changes induced by clostridia and coccidia are described.     

The role of European starlings in the spread of coccidia within concentrated animal feeding operations

To investigate the relationship between European starlings and bovine coccidiosis we collected samples from European starlings, cattle feed bunks, cattle water troughs, and cattle feces within concentrated animal feeding operations (CAFOs). These samples were screened for coccidia spp. to investigate (i) the prevalence of coccidia in starlings using CAFOs; (ii) if there is a relationship between bovine coccidiosis and starling numbers; (iii) if coccidia contamination of cattle feed and water is related to the number of starlings observed on CAFOs. Coccidia belonging to the genus Eimeria were detected in cattle feces and one water sample but no Eimeria spp. were detected in European starlings or cattle feed. However, many European starling samples were positive for Isospora. Starling use of CAFOs did not appear to be associated with coccidia spp. shedding by cattle and there was no correlation between starling numbers and contamination of cattle feed and water, suggesting that starling do not contribute to the amplification and spread of Eimeria in CAFOs.     

斯氏艾美耳球虫ITS1-5.8S rRNA-ITS2序列的克隆及PCR检测方法的建立

通过对多种鸡球虫和松鼠球虫18SrRNA和28SrRNA进行序列比对分析,在18SrRNA 3′端和28SrRNA 5′端保守区设计艾美耳属通用引物,以斯氏艾美耳球虫洛阳分离株LY卵囊基因组DNA为模板首次成功克隆到斯氏艾美耳球虫完整的ITS1-5.8SrRNA-ITS2序列,其大小为1 178bp,其中ITS1序列长度为423bp,5.8SrRNA为155bp,ITS2为600bp,斯氏艾美耳球虫LY株ITS1/2序列高度变异,与鸡球虫、啮齿动物球虫的序列相似性低于60%。然后在斯氏艾美耳球虫ITS1/2序列超变区设计种特异引物,建立了灵敏、特异的PCR检测方法。本研究结果将为兔球虫强致病种的临床诊断和揭示兔球虫种群遗传特征提供有效的分子工具。     

基于ITS1-5.8S rRNA-ITS2序列兔肝球虫PCR检测方法的建立

通过对多种鸡球虫和松鼠球虫18S rRNA和28S rRNA进行序列比对分析,在18S rRNA 3’端和28S rRNA 5’端保守区设计艾美耳属通用引物,以斯氏艾美耳球虫洛阳分离株LY卵囊基因组DNA为模板首次成功克隆到斯氏艾美耳球虫完整的ITS1-5.8S rRNA-ITS2序列,其大小为1178bp,其中ITS1序列长度为423bp,5.8S rRNA为155 bp,ITS2为600 bp,斯氏艾美耳球虫LY株ITS1/2序列高度变异,与鸡球虫、啮齿动物球虫的序列同源性低于60%。然后在斯氏艾美耳球虫ITS1/2序列超变区设计种特异引物,建立了灵敏、特异的PCR检测方法。本研究结果将为兔球虫强致病种的临床诊断和揭示兔球虫种群遗传特征提供有效的分子工具。     

Occurrence of coccidiosis in calves at one to six months of age, which are housed in a large capacity barn]

One- to six-month-old calves, housed in the large-capacity calf house of the Nová Bystrice state farm in the district of Jindrich?v Hradec were studied for the occurrence of coccidia in the period from January 1974 to April 1975. The coccidia found in these animals included nine species of the genus Eimeria: E. zuernii, E. bovis, E. ellipsoidalis, E. subspherica, E. auburnensis. The species E. alabamensis, E. cylindrica, E. wyomingensis and E. bukidnonensis occurred just sporadically. Further, the findings included oocysts of Isopora spp. The oocysts of the coccidia E. wyomingensis and Isopora spp. were found for the first time in Czechoslovakia. The overall extensity of invasion (EI) and intensity of invasion (II) grow with age as follows: (chart: see text) The results suggest that even modern large-capacity calf houses are not free from invasion by coccidia; the calves in the house under study were invaded by ten species. Transition to vegetable feeding is accompanied by a marked rise of the EI of all the species of coccidia found in the animals.     

Notes on some coccidia of peafowl,pheasants and chickens

The coccidium Isospora mayuri was isolated from sick peafowl on a farm in Essex (Great Britain). This is the first British record. The oocyst morphology and an unsuccesful attempt to infect chickens are described. The degree of pathogenicity is uncertain. Oocyst production might be controlled by sulphaquinoxaline and diaveridine in the drinking water but further studies are necessary.The coccidia Eimeria phasiani, E. duodenalis and E. colchici were isolated from pheasants on an Essex estate over two years. Coccidiosis due to E. colchici in intensively raised pheasants was controlled by sulphaquinoxaline and diaveridine in the drinking water.Two attempts were made to infect pheasants with the chicken coccidium E. tenella using birds raised coccidia-free in cages. The pheasants showed no signs of infection, which contradicts the conclusions of previous authors based only on field studies.     

An outbreak of avian malaria in captive yellowheads/mohua (Mohoua ochrocephala)

CASE HISTORY: Eight mohua, or yellowheads (Mohoua ochrocephala), were held in a large open aviary over the summer months of 2003-2004, following their capture for captive breeding purposes. Two birds died of transportation trauma shortly after arrival, one became ill and died a month later, and another four died within a 2-week period in February 2004. The eighth bird also became ill at this time but survived for a year following treatment with chloroquine and doxycycline. CLINICAL AND PATHOLOGICAL FINDINGS: The affected birds were depressed, lethargic and dyspnoeic. Necropsy of three birds showed a slightly pale and swollen liver and spleen. Impression smears of the liver of one bird revealed schizonts resembling Plasmodium spp. within the cytoplasm of many hepatocytes, which was confirmed histopathologically. Similar protozoal organisms were seen within splenic histiocytes and pulmonary endothelial cells of 5/6 birds. Electron microscopy identified these as protozoal schizonts containing merozoites of similar size and structure to those of Plasmodium spp. DIAGNOSIS: The birds were infected with a protozoal haemoparasite resembling Plasmodium spp.; asexual stages within hepatocytes and endothelial cells of the lung and spleen were typical of this organism. CONCLUSIONS AND CLINICAL RELEVANCE: The mohua captured from west Otago were highly susceptible to avian malaria as they came from an isolated population that was likely to be na?ve and have had no previous contact with this organism. The birds were probably infected by bites from mosquitoes feeding off local populations of blackbirds subsequently found to be infected with Plasmodium spp.     

Efficacy evaluation of lasalocid plus roxarsone combination medication with different geographic field strains of Eimeria acervulina

Performance of broiler chickens medicated with lasalocid alone (at 125 ppm) or in combination with roxarsone (at 50 ppm) was evaluated in battery and floorpen trials after challenge with geographically different field strains of coccidia containing predominately the upper intestinal species Eimeria acervulina. No significant difference in bird performance measured at 6 days postinfection (PI) was observed between lasalocid plus roxarsone-medicated (L+RM) or lasalocid-medicated (LM) birds challenged in separate battery trials with mixed-species inocula from Alabama or Georgia containing 92% or 88% E. acervulina, respectively. In contrast, L+RM birds challenged in another battery trial with a Louisiana mixed-species inoculum containing 92% E. acervulina showed significant reduction in average weight gain at 6 days PI compared with LM-challenged birds. A floorpen trial done with the same Louisiana inoculum showed significant reduction in average bird weight gain at 27 and 35 days of age (6 and 14 days PI) for L+RM-challenged birds compared with both unmedicated-nonchallenged (UMNC) control and LM-challenged birds. The LM+R groups were significantly lower in average bird weight at 27 days of age than the unmedicated-challenged controls. Feed conversions (FCs) for L+RM birds were significantly higher than those for the UMNC control birds during time of challenge (21-27 days of age) and for the 1-to-27-day-of-age time period. No significant difference in FC was seen between the UMNC and LM groups. Results of this study showed that performance of broiler birds medicated with lasalocid plus roxarsone could vary for geographically different mixed-species challenge inocula that contained predominately E. acervulina.     

Behavioral changes in laying hens after introduction to battery cages, furnished cages and an aviary

A variety of investigations into alternative systems and furnished cages for laying hens have been conducted, mainly in the European Union. However, comparative studies about the behavior of laying hens just after introduction to these housing systems are few. Therefore, this study aimed to investigate the changes of location and behavior of laying hens just after introduction to three housing systems. In total, 181 White Leghorns were used. Thirty‐six birds were allocated to 12 battery cages with three birds per cage (613 cm²/bird); 36 birds were allocated to nine furnished cages with four birds per cage (1170 cm²/bird); and 109 birds were allocated to an aviary (1130 cm²/bird). Direct observations using scanning techniques were conducted over 2 weeks for 4 h/day from the day following the introduction. Scan samples of location and behavior were taken at 10 min intervals. The proportion of birds that stayed at each location in the furnished cages was stable during the observation period. In the aviary, 78% of birds were observed on the floor on the first day, and thereafter the proportion linearly decreased (P < 0.01). The proportions of birds eating in both the battery and furnished cages were stable, indicating that the caged birds would adjust to these environments within a short period. Display of aggression was lower in both the cages (both 0.3 ± 0.1%) than in the aviary (3.5 ± 1.0%, P < 0.001), which indicates the early establishment of social order in both cage systems. In the aviary, the birds were observed eating less frequently than in both the cages on the first day, and the proportion thereafter linearly increased (P < 0.01), and the use of tiered wire floors with feeders accompanied this (P < 0.01). Comfort behaviors, including dust bathing, were noted less in the aviary than in the furnished cages throughout the observation period (both P < 0.05). These results suggest that adjustment of aviary birds to their new environment had been delayed compared with caged birds because of the prerearing conditions and the environmental complexity of the aviary.     

An outbreak of avian malaria in captive yellowheads/mohua (Mohoua ochrocephala)

CASE HISTORY: Eight mohua, or yellowheads (Mohoua ochrocephala), were held in a large open aviary over the summer months of 2003–2004, following their capture for captivebreeding purposes. Two birds died of transportation trauma shortly after arrival, one became ill and died a month later, and another four died within a 2-week period in February 2004. The eighth bird also became ill at this time but survived for a year following treatment with chloroquine and doxycycline.

CLINICAL AND PATHOLOGICAL FINDINGS: The affected birds were depressed, lethargic and dyspnoeic. Necropsy of three birds showed a slightly pale and swollen liver and spleen. Impression smears of the liver of one bird revealed schizonts resembling Plasmodium spp. within the cytoplasm of many hepatocytes, which was confirmed histopathologically. Similar protozoal organisms were seen within splenic histiocytes and pulmonary endothelial cells of 5/6 birds. Electron microscopy identified these as protozoal schizonts containing merozoites of similar size and structure to those of Plasmodium spp.

DIAGNOSIS: The birds were infected with a protozoal haemoparasite resembling Plasmodium spp.; asexual stages within hepatocytes and endothelial cells of the lung and spleen were typical of this organism.

CONCLUSIONS AND CLINICAL RELEVANCE: The mohua captured from west Otago were highly susceptible to avian malaria as they came from an isolated population that was likely to be naïve and have had no previous contact with this organism. The birds were probably infected by bites from mosquitoes feeding off local populations of blackbirds subsequently found to be infected with Plasmodium spp.     

3种兔球虫ITS-1序列测定与系统发育分析

从河北省兔场分别单卵囊分离孢子化大型艾美耳球虫卵囊、黄艾美耳球虫卵囊及肠艾美耳球虫卵囊,接种无球虫兔后获得纯种卵囊,CTAB法提取孢子化卵囊基因组DNA。利用艾美耳属球虫18SrDNA和5．8SrDNA保守引物,PCR扩增3种兔球虫ITS-1片段,产物纯化后测序。将3种球虫ITS-1测序结果与GenBank发布的兔球虫ITS-1序列进行比对和遗传距离比较,绘制系统发育树。结果表明,大型艾美耳球虫、黄艾美耳球虫及肠艾美耳球虫河北株分别扩增出424、455、434bp的ITS-1片段。大型艾美耳球虫、黄艾美耳球虫及肠艾关耳球虫河北株与GenBank中发布的同种兔球虫ITS-1序列相似性分别为97．4％、97．9％和96．9％。系统发育树显示兔球虫ITS-1序列形成1个单系群,该单系群根据寄生部位分为2个姊妹群。     

Efficacy of selected coccidiostats in sandhill cranes (Grus canadensis) following challenge.

The anticoccidial efficacy of amprolium, clazuril, and monensin were studied in sandhill cranes (Grus canadensis) infected with a mixture of Eimeria spp. oocysts. Five groups of four 1-day-old sandhill crane chicks were maintained on a crumbled ration containing no coccidiostat, amprolium at 2.2 ppm, clazuril at 1.1 ppm, clazuril at 5.5 ppm, or monensin at 99 ppm. After 2 wk on their respective feeding regimens, birds in each of the five groups were administered 25 x 10(3) pooled sporulated Eimeria spp. oocysts per os and observed for another 3 wk. A sixth group of four chicks served as nonmedicated, nonchallenged control during the study. Clinical signs and lesions consistent with disseminated visceral coccidiosis were observed in all challenged controls and birds fed amprolium and clazuril. Birds in these groups died 9-10 days after challenge. In contrast, only one monensin-medicated bird had clinical signs of disseminated visceral coccidiosis, and it died 13 days after challenge (DAC). This and an asymptomatic bird that were necropsied at study termination had less-severe gross and microscopic lesions of disseminated visceral coccidiosis. Two of three monensin-treated birds that survived challenge passed from 50 to 500 coccidial oocysts 11 to 18 DAC but were negative at study termination. Of the coccidiostats tested, monensin, at the dietary level of 99 ppm, was the only anticoccidial drug that provided protection against experimentally induced disseminated visceral coccidiosis in sandhill cranes.     

Immunization of chukar partridges against coccidia (Eimeria kofoidi and Eimeria legionensis) with low doses of live oocysts

Experiments were conducted to determine whether chukar partridge (Alectoris chukar) chicks would develop protective immunity after inoculation with coccidia. Young chukar chicks in battery cages inoculated with 100 or more oocysts of Eimeria kofoidi or Eimeria legionensis had significant protection at challenge 4 wk later, as measured by greatly reduced oocyst shedding and improved weight gain as compared with unvaccinated, challenged controls. However, when birds were housed in litter pens and vaccinated by various regimens (including two species of chukar coccidia at 100/dose), coccidiosis rapidly spread through all treatments and caused significant mortality. Vaccination with Coccivac-T or with 100 oocysts of Eimeria dispersa did not prevent mortality resulting from accidental contamination, and feed treatment with a Lactobacillus competitive-exclusion product had no benefit. Most if not all of the mortality was from E. kofoidi. This study illustrated the natural fecundity of chukar coccidia in a floor-pen environment where multiplication rate and reinfection combine to produce clinical disease from a small original exposure. Further, these results cast doubt on the potential use of low doses of live oocysts as a vaccine in the chukar partridge.     

3种兔球虫18S rDNA部分序列测定与系统发育分析

采用单卵囊分离法从河北某兔场分离大型艾美耳球虫、黄艾美耳球虫及肠艾美耳球虫,接种无球虫兔后获得大量纯种卵囊,CTAB法提取孢子化卵囊基因组DNA。利用艾美耳属球虫18S rDNA保守引物,PCR扩增3种兔球虫18S rDNA片段,产物纯化后测序。将3种球虫18S rDNA测序结果与GenBank中发布的兔球虫18S rDNA序列用DNAStar软件进行比对。使用MEGA4.0软件对兔球虫18S rDNA进行同源性比较,并绘制遗传进化树。结果表明,大型艾美耳球虫扩增出大小为1 521bp的18S rDNA片段;黄艾美耳球虫及肠艾美耳球虫均扩增出大小为1 520bp的18S rDNA片段。序列比对结果显示,3种河北株兔球虫与GenBank中相应的3种兔球虫18S rD-NA(EF694016、EF694011、EF694012)相似性分别为99.6%、99.6%和100%。3种河北株兔球虫序列和GenBank中兔球虫18S rDNA序列(EF694007-EF694017)位于一个单系集群。     

Avian malaria in captive psittacine birds: detection by microscopy and 18S rRNA gene amplification

A cross-sectional survey was conducted to estimate the occurrence of malaria infection among captive psittacine birds (n=127) from three zoological gardens in Brazil. Malaria infection was evaluated by the association of direct examination of blood smears with amplification of the 18SSU rRNA gene of the Plasmodium genus, demonstrating an overall occurrence of 36%. Most infected bird species were Amazona aestiva (28/73), Ara ararauna (6/10), and Amazona amazonica (3/10). The low parasitemias observed among the infected birds suggest a chronic infection. The sequence analyses of 10 isolates indicate a potential occurrence of four distinct Plasmodium lineages. These findings provide new data on malarial infection in captive psittacine birds, and emphasize the need for better control of importation and exportation of these birds.     

Eimeria infections in litter-based, high stocking density systems for loose-housed laying hens in Sweden

1. Coccidiosis, caused by different Eimeria species, is believed to be a more prominent problem in loose-housed layers kept on litter than in battery cages. In this study, the impact and development of Eimeria infections were investigated in layers kept in litter-based, high stocking density systems for loose-housed hens. 2. Layers from 57 flocks on 26 farms were followed by necropsy of a representative sample of birds that died or had to be culled. Coccidiosis was diagnosed in 11 flocks (19.3%) from 9 (31%) of the farms. The outbreaks occurred when the birds were 19 to 32 weeks old. E. maxima was identified in 6 and E. tenella in 3 of the outbreaks. 3. Sixteen of the flocks were also monitored with faecal and litter samples collected at regular intervals. Oocysts were detected in samples from all these flocks. The pattern of oocyst excretion was similar in most of the flocks, with maximum counts at 4 to 8 weeks after introduction to the laying house. There was no significant correlation between the levels of oocysts in faeces and clinical coccidiosis. 4. Raising pullets without any coccidiostat, to increase their chance to develop immunity against coccidia, was not found to decrease the risk of coccidiosis during the production period when compared to the practice of giving amprolium and ethopabate during the rearing period.