Detection of prions in the faeces of sheep naturally infected with classical scrapie

ABSTRACT: Classical scrapie is a naturally transmitted prion disease of sheep and goats. Contaminated environments may contribute to the spread of disease and evidence from animal models has implicated urine, blood, saliva, placenta and faeces as possible sources of the infection. Here we sought to determine whether sheep naturally infected with classical scrapie shed prions in their faeces. We used serial protein misfolding cyclic amplification (sPMCA) along with two extraction methods to examine faeces from sheep during both the clinical and preclinical phases of the disease and showed amplification of PrPSc in 7 of 15 and 14 of 14 sheep respectively. However PrPSc was not amplified from the faeces of 25 sheep not exposed to scrapie. These data represent the first demonstration of prion shedding in faeces from a naturally infected host and thus a likely source of prion contamination in the environment.     

Detection of Mycobacterium paratuberculosis antigen with colloidal immunogold in naturally infected sheep.

The anti-Mycobacterium paratuberculosis polyclonal serum is proved useful for labelling Mycobacterium paratuberculosis in glutaraldehyde-osmium-fixed and epon-embedded intestinal samples from sheep with clinical symptoms of paratuberculosis. M. paratuberculosis marked with antibody-coated colloidal gold stain was seen in macrophages, epithelioid cells, giant cells and neutrophils throughout intestinal mucosa. In large macrophages with a low lysosomal content, a great number of intact mycobacteria was seen within phagosomes. In macrophages with average lysosomal content, very few intact mycobacteria or mycobacterial debris were present and lysosome-phagosome fusions were observed. Mycobacteria within neutrophils were scanty. These results show the usefulness of colloidal immunogold techniques for studies of the pathogenesis of paratuberculosis.     

羊附红细胞体巢式PCR检测方法的建立及临床应用

根据GenBank上发表的羊附红细胞体16SrRNA基因序列（登录号AF338268）设计2对引物,用巢式PCR方法扩增16SrRNA的部分序列,将目的片段克隆并测序。测序结果与AF338268相似性达99%以上,只有3bp的差异。该方法与猪附红细胞体、羊肺炎支原体、链球菌、葡萄球菌和大肠杆菌均无交叉反应,能检测到的最低DNA量为25fg。用于检测的28份临床样本中,23份为阳性。所建立的巢式PCR检测方法具有较高的敏感性和特异性,可用于羊附红细胞体病急性感染和隐性感染的早期诊断,为该病的临床检测、流行病学调查、进出口检疫和实验室研究提供了新的技术手段。     

Characterization of Anaplasma phagocytophilum and A. ovis infection in a naturally infected sheep flock with poor health condition

Anaplasma species are transmitted by ticks and cause diseases in humans and animals. These pathogens infect sheep, an economically important domestic animal worldwide. The current study was designed to characterize in 200 animals the infection with Anaplasma phagocytophilum and Anaplasma ovis and the genetic diversity of A. ovis strains collected from a naturally infected sheep flock with poor health condition. Sheep had 98% seroprevalence to Anaplasma spp. antibodies. PCR results confirmed the presence of A. phagocytophilum and A. ovis DNA in 11.5% and 37% of the sheep, respectively. Concurrent infections were detected in 6.5% of the sheep. Seventy-one adult ticks were collected from 45 sheep with infestations ranging from one to 15 ticks per animal. The analysis of A. ovis msp4 sequences demonstrated a previously unreported polymorphism for this pathogen with 17 different haplotypes in infected sheep. These results demonstrated that, although A. ovis msp4 haplotypes may be less variable when compared with Anaplasma marginale and A. phagocytophilum strains on a global scale, genetic polymorphisms occur in this locus in strains obtained from an infected sheep flock with poor health condition.     

应用套式PCR方法检测猪圆环病毒2型

根据Genbank中发表的猪圆环病毒2型(PCV2)全基因序列,设计2对PCV2特异性引物,建立套式PCR检测方法。外测引物p1、p2扩增片段长度为647bp(92-738),内测引物p3、p4扩增长度为219bp(319-537)。其中用外部引物可扩增DNA含量到10-5mg/mL,而套式PCR则比普通PCR灵敏性还要提高103倍。用该方法对山东、安徽和河北10省的899份临床发病猪的肺脏和淋巴结样品进行检测,结果有329份样品检测出阳性,平均阳性检测率达36.6%。由此可见,PCV2感染在全国范围内的发病猪群中普遍存在。     

Comparison of nested PCR and real-time PCR for the detection of Toxoplasma gondii in biological samples from naturally infected cats

There are different protocols of molecular diagnosis methods available including DNA extraction methods to diagnose of Toxoplasma gondii, being necessary to perform comparative studies in biological samples. The aim of this study is to compare real-time PCR (rtPCR) and nested PCR (nPCR) to evaluate the detection of T. gondii in naturally infected cats. Biological samples of Toxoplasma-seropositive cats were assayed for detection of T. gondii DNA - extracted by both the lysis buffer and proteinase K (LB proteinase K) method and the acid guanidinium thiocyanate (GuSCN) method - using rtPCR and nPCR. T. gondii DNA was detected by nPCR in 43.6% and 40.8% of the samples from which it was extracted by the LB proteinase K and the GuSCN method, respectively. With rtPCR these figures fell significantly to 33.8% and 14.1%. Despite of nPCR showed higher sensitivity, the agreement observed between two PCRs was good; this agreement, however, was affected by the DNA extraction method used, LB proteinase K method showed better results.     

Clinicopathologic observations on Coenurus cerebralis in naturally infected sheep

Twelve sheep from 7 different flocks consisting of approximately 150-250 animals each were diagnosed with coenurosis caused by the larval stage of Taenia multiceps. Ataxia, incoordination, drowsiness, hind leg paralysis and coma were the most prominent clinical symptoms. Monocytosis and lymphocytosis were observed upon hematological examination. Creatin kinase (CKBB) levels of the animals varied between 421 and 495 U/l. Cysts were commonly localized in the parietal and frontal lobes of the brain and in the cerebellum. In two cases, cysts were found on the lumbar aspect of the medulla spinalis. Symptoms were related to cyst localization. Depression, tilting of the head either to the right or left and head pressing were seen when cysts were located in the cerebrum. Incoordination and hyperexcitability were noted if the cysts were involved with the cerebellum and when located in the spinal cord, hind leg paralysis was the typical clinical sign. On microscopic examination, atrophy was observed in the central nervous system (CNS) organs due to pression by the bladderworms. Nonpurulent meningoencephalitis with perivascular cuffings were the most common histopathological findings. In periodic acid Schiff staining (PAS), positive reaction was observed in protoscoleces. Neurons were the most affected cell type when stained by the Klüver Barrera method.This method also showed that in the CNS, Coenurus cerebralis caused a prominent glial reaction. When parasites were localized in the nervous system treatment was impossible. Animals without neurologic sings were treated with praziquantel (Tenikur tablet-Topkim A.S.) 50-100 mg/kg/day for three days.     

Detection of toxoplasmosis in experimentally infected goats by PCR

PCR was used to diagnose toxoplasmosis in two pairs of Barbari goats infected by oral administration of doses of either 10(4) or 10(5) oocysts of Toxoplasma gondii. Blood and lymph node aspirates were collected from the infected goats and control goat at intervals, and tissues were also collected from a fetus that was aborted and a doe that died during the trial. Both processed and unprocessed samples were used for the PCR, using primers directed to the multicopy B1 gene. None of the blood samples was positive, but a specific signal was obtained from the lymph node aspirates after partial DNA extraction. Direct PCR of the lung, muscle and mesenteric lymph node of the doe and lung tissue of the aborted fetus yielded the target fragment. The simplified PCR protocols, including partial DNA extraction and direct assay of lung tissue, were effective for the diagnosis of toxoplasmosis.     

Feasibility of genus-specific real-time PCR for the differentiation of larvae from gastrointestinal nematodes of naturally infected sheep

Results of real-time PCR analysis of coproculture third stage larvae (L3) using genus specific TaqMan minor groove binder probes were compared with the results of morphological differentiation of L3 after coprocultured and direct morphological worm differentiation from gastrointestinal samples of eight sheep with naturally acquired nematodes infections. Faecal egg counts prior to postmortem confirmed infections with trichostrongyles with a geometric mean count of 4828 eggs per gram for all sheep. Individual egg counts correlated positively with total worm counts (correlation coefficient 0.794). Five different nematode species and one genus were found in the abomasi and small intestines: Cooperia curticei, Haemonchus contortus, Nematodirus spp., Teladorsagia (Ostertagia) circumcincta, Trichostrongylus axei and Trichostrongylus colubriformis. Coproculture of faecal eggs yielded five of these, Cooperia spp., Haemonchus spp., Ostertagia/Teladorsagia spp. and Trichostrongylus spp. Comparison between morphological L3 and worm differentiation data showed high congruence (94%). The agreement between PCR analysis of L3 after coproculture and direct morphological worm differentiation was 84%. Thus, real-time PCR was found to be suitable as a speedy and reliable diagnostic tool for the assessment of gastrointestinal nematode infections of ruminants in the field.     

用套式PCR方法对几种猪用冻干疫苗的PCV2检测

参照Genbank已发表的猪圆环病毒2型的基因序列,取其比较保守的基因片段ORF2,利用引物设计软件Oli-go5.0设计两对PCV2型特异的引物,其中第一对引物扩增跨度为ORF2全基因片段,长度为702 bp,第二对引物扩增ORF2中间的一个小片段,跨度大小为494 bp.首先用第一对引物扩增阳性DNA,阳性DNA的浓度从10-1稀释到10-11,然后以第一次PCR产物为模板,用第二对引物进行PCR,发现这种套式PCR的方法比用普通PCR灵敏度提高102倍.同这种套式PCR方法对广东省市场上出售的几种猪用弱毒疫苗进行猪圆环病毒2型检测,结果发现这几种疫苗全部为阴性,本实验说明目前广东市场上出售的弱毒疫苗在制作过程中没有受到PCV2的污染,解除了广大猪场主的顾虑.     

Detection of Brucella species in the milk of infected cattle,sheep, goats and camels by PCR

One hundred and three milk samples were collected from 52 cows, 21 ewes, 18 goats and 12 camels. The animals tested positive to at least one of the following: (1) standard tube agglutination test (SAT); (2) Rose Bengal plate test (RBPT); (3) milk ring test (MRT). All milk samples were examined by culture and single-step polymerase chain reaction (PCR) techniques for detection of Brucella species. The PCR assay amplified Brucella-DNA from 29 bovine milk samples, 10 from sheep, 13 from goats and one from a camel. The direct culture method detected Brucella organisms from 24 samples of cows' milk, 12 from sheep, 10 from goats and failed to detect any Brucella organisms from camels' milk. PCR detected up to 100 colony forming units (CFU) of B. abortus per millilitre of milk in 100% of diluted milk samples, and 1000 CFU of B. melitensis from 70% of milk samples. Although the overall sensitivity of the PCR was higher than the culture method, it should be possible to increase the sensitivity to detect lower numbers of Brucella organisms in field samples. The speed and sensitivity of the PCR assay suggest that this technique could be useful for detection of Brucella organisms in bovine milk, as well as in sheep, goat, and camels milk.     

Evaluation of antioxidant status and oxidative stress in cattle naturally infected with Theileria annulata

To assess the antioxidant status and oxidative stress in bovine theileriosis due to Theileria annulata blood samples were collected from 35 clinically affected cattle referred to Veterinary Teaching Hospital, School of Veterinary Medicine, Urmia University, Urmia, Iran. Complete blood count, piroplasm parasitemia percentage, erythrocyte glutathione peroxidase, superoxide dismutase, catalase and glucose-6-phosphate dehydrogenase activities, malondialdehyde concentration, osmotic fragility test and median corpuscular fragility were determined and the results were compared with those of 50 healthy controls. Of 35 affected cattle, 12 (34.28%) had severe anemia and 23 had mild to moderate anemia and parasitemia varied from 5 to 40%. The activities of erythrocyte glutathione peroxidase, superoxide dismutase and glucose-6-phosphate dehydrogenase were significantly lower (P<0.0001) and the activity of catalase was significantly higher in the affected cattle than in healthy ones (P<0.001). Malondialdehyde concentration in erythrocytes of affected cattle was significantly more than those of healthy cattle (P<0.001). The affected cattle showed increased fragility of erythrocytes, so that median corpuscular fragility (MCF) in affected group was significantly lower than those of healthy group (P<0.0001). Median corpuscular fragility showed a positive correlation with the severity of parasitemia (r=0.81, P<0.0005) and a negative correlation with the activities of GSH-Px (r=-0.78, P<0.0001), SOD (r=-0.71, P<0.0005), catalase (r=-0.53, P<0.018) and G6PD (r=-0.58, P<0.0005). The results of this study suggest that oxidative damage to RBCs may contribute to the pathogenesis of anemia in bovine theileriosis.     

Detection and genotyping of porcine circovirus in naturally infected pigs by oligo-microarray

A rapid and reliable method for the identification of porcine circovirus (PCV) genotypes based on oligonucleotide microarray hybridization has been developed. The genotype-specific oligonucleotides (22-30 mer) immobilized on the surface of glass slides were selected to bind to the multiple target sites within the replication gene that are conserved among individual PCV genotypes. Cy5-labeled DNA targets were amplified in a PCR with primers common to both genotypes. The identification of PCV genotype was based on hybridization with several individual genotype-specific oligonucleotides. This approach combines the high sensitivity of PCR with the selectivity of DNA-DNA hybridization. The utility and feasibility of oligonucleotide microarray hybridization was evaluated by testing standard and 87 clinical isolates. Analysis of the specimens showed that this microarray-based method is capable of unambiguous identification of both genotypes and fivefold more sensitive than gel electrophoresis. Our results indicated that the oligonucleotide array is useful for the identification and discrimination of PCV from clinical isolates and specimens in a clinical laboratory.     

Characterization of local immune response against lungworms in naturally infected sheep

This study describes the immunohistochemical and histochemical phenotypes of inflammatory cells in sheep lungs infected with lungworms. A total of 20 naturally infected sheep lungs were used. Protostrongylus spp., Muellerius capillaris, Neostrongylus linearis, and Cystocaulus ocreatus were the chief organisms determined from such lesions, which were of a chronic nature. All the lungs had many developmental stages of the parasites and a similar inflammatory response, which included numerous mast cells, eosinophils, T cells, B cells, dendritic cells, and macrophages. In the bronchial and interstitial tissues, the inflammatory cells were dominated by MHCII, CD1, CD4, CD5, CD14, CD21, IgM, and CD172a positive cells, whereas CD2 and WC1 positive cells were detected less. The data provided additional evidence that subsets of inflammatory cells were included within ovine lungs infected with lungworms; however, understanding the entire immune-response process and development of resistance to lungworms in sheep remain to be clearly elucidated.     

Detection of respiratory herpesviruses in foals and adult horses determined by nested multiplex PCR

A nested multiplex PCR was developed as a rapid (<12h), sensitive test for the simultaneous identification of equine herpesviruses (EHV1, EHV4, EHV2 and EHV5) in clinical samples from horses. Peripheral blood and nasal swab (NS) samples from 205 weanling Thoroughbred foals on 6 different studs over 3 consecutive seasons and from 92 adult horses without clinical signs of respiratory disease were examined using direct multiplex PCR of clinical samples (direct PCR) and conventional cell culture with differentiation of EHV in cell cultures by multiplex PCR. Multiplex PCR proved a sensitive and specific technique for the detection of EHV in cell culture and clinical samples. The technique described appeared equally sensitive as one using a single set of primers for individual EHV but reduced labour and reagent costs. Cell cultures showing cytopathic effect (CPE) were always positive for EHV on PCR. EHV were also detected by multiplex PCR in 11 samples which failed to show CPE. By a combination of multiplex PCR and cell culture or direct multiplex PCR, the presence of up to three EHV in the same sample was detected. Overall, EHV5 was detected by direct multiplex PCR of peripheral blood mononuclear cells (PBMC) and/or NS samples from 78% of foals and 47% of adult horses. Repeated sampling or cell culture in combination with multiplex PCR and with the incorporation of IL-2 in culture medium increased the sensitivity for detection of EHV in PBMC and demonstrated that EHV5 DNA could be identified in PBMC from 89% of foals and 100% of adult horses. EHV2 was identified from approximately 30% of foals, but was more frequently identified in samples from 17 foals with mild respiratory disease and was isolated infrequently from adult horses. EHV1 and EHV4 were identified uncommonly in any population in the current study.     

Immune responses to Staphylococcus aureus and Psoroptes ovis in sheep infected with P. ovis--the sheep scab mite

In sheep, lesions caused by Psoroptes ovis, the sheep scab mite, may become colonized by Staphylococcus aureus. The present study compares clinical signs, lesional area and the immune response to P. ovis and S. aureus in P. ovis-infested sheep with and without secondary S. aureus infection. No differences were detected in the clinical signs or lesional areas in the S. aureus-positive and -negative sheep. However, 6 weeks after infestation an IgG but not IgE isotype antibody response to S. aureus was detected in the S. aureus-positive but not the S. aureus-negative group of sheep. This response targeted S. aureus antigens with molecular weights of approximately 36, 38, 50 and 65 kDa. In addition, 6 weeks after infestation an IgE response to P. ovis was detected in the S. aureus-positive but not the S. aureus-negative group of sheep.