A simplified protocol for molecular identification of Eimeria species in field samples

This study aimed to find a fast, sensitive and efficient protocol for molecular identification of chicken Eimeria spp. in field samples. Various methods for each of the three steps of the protocol were evaluated: oocyst wall rupturing methods, DNA extraction methods, and identification of species-specific DNA sequences by PCR. We then compared and evaluated five complete protocols. Three series of oocyst suspensions of known number of oocysts from Eimeria mitis, Eimeria praecox, Eimeria maxima and Eimeria tenella were prepared and ground using glass beads or mini-pestle. DNA was extracted from ruptured oocysts using commercial systems (GeneReleaser, Qiagen Stoolkit and Prepman) or phenol-chloroform DNA extraction, followed by identification of species-specific ITS-1 sequences by optimised single species PCR assays. The Stoolkit and Prepman protocols showed insufficient repeatability, and the former was also expensive and relatively time-consuming. In contrast, both the GeneReleaser protocol and phenol-chloroform protocols were robust and sensitive, detecting less than 0.4 oocysts of each species per PCR. Finally, we evaluated our new protocol on 68 coccidia positive field samples. Our data suggests that rupturing the oocysts by mini-pestle grinding, preparing the DNA with GeneReleaser, followed by optimised single species PCR assays, makes a robust and sensitive procedure for identifying chicken Eimeria species in field samples. Importantly, it also provides minimal hands-on-time in the pre-PCR process, lower contamination risk and no handling of toxic chemicals.     

Sensitivity of field isolates of Eimeria species to monensin and lasalocid in the chicken

Isolates of Eimeria species obtained from broiler or from breeder farms were compared for their sensitivity to two ionophorous anticoccidial drugs, monensin and lasalocid. All of 25 isolates from broiler farms were resistant to 100 ppm monensin or 90 ppm lasalocid, while 14 of 16 isolates were resistant to monensin and seven of 16 to lasalocid from breeder farms (replacement layer and broiler breeder).     

Eimeria brunetti and Eimeria necatrix in chickens of Argentina and confirmation of seven species of Eimeria

Ten poultry farms (broiler breeder pullets, layer pullets, and broilers) in the provinces of Entre Rios and Buenos Aires in Argentina were examined for presence of Eimeria spp. Litter samples obtained from flocks 7-11 wk old were taken to the laboratory for oocyst counting and sporulation, then concentrated for inoculation into coccidia-free chickens. Species were identified by prepatent period, oocyst size, location and appearance of lesions in the intestine, microscopic examination of mucosal smears, and histology (to confirm Eimeria brunetti). On this basis, Eimeria praecox was found in two samples, Eimeria mitis in two, Eimeria acervulina in nine, Eimeria maxima in seven, Eimeria necatrix in three, Eimeria tenella in seven, and E. brunetti in four. These results confirm the presence of all seven recognized species of Eimeria in chickens in the Republic of Argentina.     

Diagnosis of hereditary diseases with molecular biology methods

The identification of phenotypically normal carriers of genetic defects is crucial for eliminating recessive defect genes from farm animal populations. Recombinant DNA techniques allow us to identify defect genes on the DNA-basis regardless of the animals age and time of gene expression. Genetic defects also are important as model systems to investigate the regulation of metabolic pathways and the mechanisms of embryonic development.     

Eimeria species in dairy goats in Brazil

The focus of this work is to determine the distribution and identify species of Eimeria parasites of dairy goats in the livestock of the National Goat and Sheep Research Center in Sobral, State of Ceará, Northeast Brazil. Results showed the presence of multiple species in 196 of 215 analyzed samples (91.2%). Fifty five out of these were from kids (28%) and 141 from adult goats (72%). Eight different Eimeria species were identified and their prevalence in the herd was: Eimeria alijevi Musaev, 1970 (26.7%), E. arloingi (Marotel, 1905) Martin, 1909 (20.6%), E. hirci Chevalier, 1966 (18%), E. ninakohlyakimovae Yakimoff & Rastegaieff, 1930 (16.2%), E. jolchijevi Musaev, 1970 (8.7%), E. christenseni Levine, Ivens & Fritz, 1962 (6%), E. caprovina Lima, 1980 (2.8%) and E. caprina Lima, 1979 (1%). Moreover, E. ninakohlyakimovae showed higher prevalence in kids (97%), followed by E. arloingi and E. alijevi (88%). On the other hand, E. alijevi (77%) was more common in adult goats followed by E. hirci (74%) and E. ninakohlyakimovae (70%). The species E. caprina had low frequency in both kids (27%) and adult goats (13%). Data indicated that infection was relatively common among kids and adult goats. The implementation of a routine diagnostic strategy can be useful in maintaining Eimeria populations under monitoring and will enable the determination of its potential impact on dairy goat herds in Northeast Brazil.     

Prevalence of Eimeria species in cattle in Kenya

A total of 620 bovine faecal samples collected from unselected animals brought for post-mortem to the Department of Veterinary Pathology and Microbiology or from animals in the Kabete (Kenya) practice area of the Faculty of Veterinary Medicine were examined to determine the types and prevalence of Eimeria spp. present. Coccidian oocytes were detected in 67.4% of the samples and eight different species of Eimeria were recognized. The species detected (and their prevalence) were E. bovis (79.0%), E. zuernii (60.2%), E. ellipsoidalis (26.1), E. cylindrica (13.4%), E. auburnensis (28.4%), E. alabamensis (10.3%), E. subspherica (5.0%) and E. wyomingensis (6.1%). E. bovis and E. zuernii led to few cases of clinical coccidiosis and the greatest number of E. bovis in one of the samples from the clinical cases was 30,600 oocysts per gram of faeces (OPG). Age and seasonal variation appeared to have an influence on the intensity of infection.     

Attenuation of Eimeria species: further characterisation of two lines of Eimeria mitis

The immunogenicity of a 'precocious' and attenuated line (HP10) of Eimeria mitis was studied and the stability of attenuation of two precocious lines was compared with that of an embryo-adapted line. Chicks housed in wire-floored cages and given 1 X 10(5) oocysts of the HP10 line were protected against challenge with the parent Houghton strain and two field strains, but remained partially susceptible to the Durham and one other field strain. However, when chicks were kept in litter pens so that reinfection could occur freely, inoculation with as few as 1 X 10(3) oocysts of the precocious line resulted in complete immunity to both the Houghton and Durham strains for at least five weeks afterwards. The stability of attenuation of the precocious and embyro-adapted lines was examined by relaxing the selection pressures used to produce attenuation and then testing the virulence of the resulting lines. The precocious lines remained attenuated after several consecutive relaxed passages, in contrast to the embyro-adapted line which showed a marked reversion to virulence after six passages in chickens.     

绒山羊艾关耳球虫病的诊断与治疗

关于绒山羊球虫病的报道不多，2005年9月，某农场饲养的绒山羊相继发生以精神沉郁、机体消瘦、食欲减退和排褐色血便为主要特征的疾病，通过临床症状观察、病理剖检和实验室检查，确诊为羊的柯氏艾美耳球虫（FAmeria christenseni）感染，经采用特效药治疗，使病情得到了控制。     

绒山羊艾美耳球虫病的诊断与治疗

关于绒山羊球虫病的报道不多,2005年9月,某农场饲养的绒山羊相继发生以精神沉郁、机体消瘦、食欲减退和排褐色血便为主要特征的疾病,通过临床症状观察、病理剖检和实验室检查,确诊为羊的柯氏艾美耳球虫(Eim eria christenseni)感染,经采用特效药治疗,使病情得到了控制。1基本情     

Differentiation of mycoplasma gallisepticum strains using molecular methods

Increasing use of Mycoplasma gallisepticum (MG) live vaccines has led to a need for the differentiation of MG strains. The MG strains MK-7, MS-16, S6, FS-9 and R strains and the MG live vaccine strain F were compared by random amplification of polymorphic DNA (RAPD) in this study. Using RAPD, different patterns were found among the MG strains. In addition to this, we examined the differentiating potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) primers targeted at the crmA, crmB, crmC, gapA, mgc2 and pvpA genes encoding cytadherence-related surface proteins. These proteins may take part in the pathogenesis of MG-induced disease. Differentiation of strain F is based on the identification of restriction enzyme sites in the PCR amplicons. Using HphI enzyme, crmC PCR amplicons produced different RFLP patterns. Digestion of amplicons of gapA-specific PCR with MboI enzyme also produced distinct patterns. Differences were observed among strains R and F by digestion of mgc2 PCR amplicons with HaelIl and VspI enzymes and digestion of pvpA PCR amplicons with AccI, PvulI and ScrFI endonucleases. This method can be used for the rapid differentiation of vaccine strain from wild strains. Differentiation of MG strains is a great advantage for diagnosticians or practitioners and it is useful for epidemiological studies.     

三株柔嫩艾美球虫野外分离株的致病性分析

用每鸡4×104和8×104个孢子化卵囊的剂量感染黄羽肉雏,以临床症状、肉眼病变、平均增重、病变记分和死亡率为判定指标,对柔嫩艾美球虫EtCZ-1、EtCZ-2、EtCZ-3株的致病性进行了比较分析。结果显示:各虫株引起的临床症状和肠道肉眼病变基本相似,致病性随着感染剂量的增加而增强。各感染组的增重与对照组相比均有显著差异(P<0.50),相对增重率分别下降了74.4%～83.2%、68.7%～72.2%和70.6%～74.4%;感染后鸡出现血便的时间较一致,即第108小时开始排血便,在120h～144h达高峰;病变值和卵囊值均随着感染剂量的增加而增大;除EtCZ-1株高剂量组的存活率是90%以外,其余各组均为100%。显示3株球虫的致病性相似。     

Vaccination of rabbits against coccidiosis using precocious lines of Eimeria magna and Eimeria media in Benin

Three groups of twelve 35-day-old rabbits were used for the experiment. Two groups were vaccinated with a mixture of precocious lines of Eimeria magna and Eimeria media originating from corresponding wild strains isolated in Benin. One group benefited of a booster whereas the second one was kept without booster. A third non-vaccinated group was used as control. All groups were challenged per os with an equal mixture of the wild strains of E. magna and E. media at a dose of 104 oocysts per animal. Three weeks after the challenge inoculation, no case of diarrhoea was recorded in the two groups of vaccinated rabbits, as compared to the non-vaccinated rabbits that developed diarrhoea. No mortality was recorded in the three groups. During the patent period, oocyst output of vaccinated rabbits was significantly lower than that of control animals (P < 0.01), confirming a good immunogenic characteristic of the precocious lines. No booster effect was noticed for the boost vaccinated group. The daily weigh gain of the two groups of vaccinated rabbits was significantly higher than that of the non-vaccinated rabbits (P < 0.05). Consequently the precocious lines of Benin origin turned out to be immunogenic and therefore constitute good potential candidates for vaccine production for this country.     

PCR-based differentiation of three porcine Eimeria species and Isospora suis

Isospora suis and Eimeria are frequent coccidian parasites of pigs. The unsporulated oocysts of Eimeria species and of I. suis are difficult to differentiate. Therefore, a species-specific PCR was developed. PCR products were amplified from Eimeria polita, Eimeria porci, and Eimeria scabra using primers from the conserved 18S rRNA regions and were subsequently sequenced. Based on variable sequence regions, primers were constructed for the differentiation of the three Eimeria species and I. suis. Using a combination of PCRs detecting one or two species, all four coccidian species were detected (theoretical lower detection level: DNA content of 250 oocysts of each Eimeria species or 25 oocysts of Isospora in 1microl) and differentiated. The PCR-based differentiation of the above mentioned species provides a useful alternative to microscopy.     

Enzyme variation and pathogenicity of recent field isolates of Eimeria tenella

Seventy isolates of Eimeria tenella, obtained from commercial poultry farms worldwide and four reference laboratory strains were characterised by studies on the electrophoretic mobility of up to three enzymes. All populations possessed the same electrophoretic form of lactate dehydrogenase and malate dehydrogenase and one of two forms of glucose phosphate isomerase. One isolate was characterised by both forms of glucose phosphate isomerase. Studies on several isolates indicated that there was no correlation between the form of glucose phosphate isomerase found and the pathogenicity of an isolate.     

Flock-level prevalence of Eimeria species among broiler chicks in northern Jordan

Six chicks (3–6 weeks of age) were taken randomly from each of 200 broiler farms in northern Jordan, these chicks were submitted for post-mortem and parasitological examinations. Seven Eimeria spp. were identified: E. acervulina, E. brunetti, E. maxima, E. necatrix, E. mivati, E. mitis, and E. tenella. Half (50%) of the farms surveyed had all six chicks infected, 23% of the farms were free of the infection. E. tenella was the most prevalent species (39%) followed by E. necatrix (12%), E. brunitti (12%), and E. maxima (10%). Prevalences did not vary by flock size. Also, neither the use of coccidiostat nor previous coccidiosis clinical outbreaks was associated with the prevalence of coccidiosis.     

Diagnosis of canine leishmaniasis: conventional and molecular techniques using different tissues

Serology, bone marrow (BM)-, lymph node (LN)- and whole blood-polymerase chain reaction (PCR) were evaluated as potential reference tests for the diagnosis of canine leishmaniasis. A high degree of agreement (91.0%) was observed between Leishmania cultures and serology or BM/LN-PCR. In the light of these results as well as the access to biological test material and the cost of each method, LN-PCR is recommended for the diagnosis or therapeutic control of canine leishmaniasis, but BM-PCR is a suitable alternative in dogs without detectable adenomegaly. For large-scale epidemiological field studies, antibody detection is appropriate and whole blood-PCR can be used to complement the serological results.