Development of recombinant chicken IgY from single chain fragment of variable region for diagnosis of BSE.

We generated two recombinant chicken IgYs, designated Ab3-15 and Ab4-19, against mammalian prion protein (PrP) from the single chain fragment of variable region (scFv) antibodies. These two antibodies recognized PrP(Sc) from bovine spongiform encephalopathy (BSE) in cattle and were more sensitive than the corresponding scFv antibodies. These antibodies also recognized PrP(Sc) from other scrapie-infected mammals. These results indicate that Ab3-15 and Ab4-19 are useful for diagnosis of BSE as well as other prion diseases.     

Intraspecies transmission of L‐type‐like bovine spongiform encephalopathy detected in Japan

It has been assumed that the agent causing BSE in cattle is a uniform strain (classical BSE); however, different neuropathological and molecular phenotypes of BSE (atypical BSE) have been recently reported. We demonstrated the successful transmission of L‐type‐like atypical BSE detected in Japan (BSE/JP24 isolate) to cattle. Based on the incubation period, neuropathological hallmarks, and molecular properties of the abnormal host prion protein, the characteristics of BSE/JP24 prion were apparently distinguishable from the classical BSE prion and closely resemble those of bovine amyloidotic spongiform encephalopathy prion detected in Italy.     

Autoregulation of mouse BMP-2 gene transcription is directed by the proximal promoter element

The prion proteins (PrP) from sheep and mouse were produced in large quantities of full-length protein in Escherichia coli after fusion with a carboxy-terminal hexahistidine sequence. Both recombinant proteins were recognized, at variable levels, in ELISA using a panel of antibodies recognizing different parts of the PrP molecules, from the octo-repeat region (79-92 human sequence), to the C terminal end of the protein. We show that these recombinant proteins enable polyclonal antisera to be produced in PrP0/0 mice, the sheep prion protein being strongly immunogenic, using either native or guanidium hydrochloride-treated recombinant protein. Sera produced against the sheep protein also reacted in Western blot with bovine, ovine, and murine PrP res, but showed higher reactivity with sheep PrP res. Interestingly, when compared to an antiserum produced against bovine 106-121 peptidic sequence (RB1), we found strikingly different ratios of the PrP res glycoforms, in both cattle with BSE and sheep with natural scrapie, but not in scrapie infected mice. Such results further demonstrate that the assessment of PrP res glycoform ratios, using different antibodies, may depend on antibodies species-specificities.     

Comparison of DNA variants in the PRNP and NF1 regions between bovine spongiform encephalopathy and control cattle

DNA from 252 bovine spongiform encephalopathy (BSE) cattle and 376 non-diseased control cattle were genotyped for nine loci in the prion protein (PRNP) gene region, three loci in the neurofibromin 1 (NF1) region and four control loci on different chromosomes. The allele and genotype frequencies of the control loci were similar in BSE and control cattle. In the analysed 7.4 Mb PRNP region, the largest differences between BSE and control cattle were found for the loci REG2, R16 and R18, which are located between +300 and +5600 bp, spanning PRNP introns 1 to 2. Carriers of the REG2 genotype 128/128 were younger at BSE diagnosis than those with the other genotypes (128/140 or 140/140). The predominant haplotype REG2 128 bp-R18 173 bp occurred more frequently (P < 0.001), and the second-most frequent haplotype (REG2 140 bp-R18 175 bp) occurred less frequently (P < 0.05) in BSE than in control cattle. The largest frequency differences between BSE and control groups were observed in the Brown Swiss breed. Across all breeds, most of the same alleles and haplotypes of the PRNP region were associated with BSE. In the 23-cM NF1 region, associations with BSE incidence were found for the RM222 allele and for the DIK4009 genotype frequencies. Cattle carrying RM222 genotypes with the 127- or 129-bp alleles were about half a year older at BSE incidence than those with other genotypes. Across the breeds, different alleles and genotypes of the NF1 region were associated with BSE. The informative DNA markers were used to localize the genetic disposition to BSE and may be useful for the identification of the causative DNA variants.     

PRNP gene variation in Pakistani cattle and buffaloes

Bovine spongiform encephalopathy (BSE) is a neurodegenerative prion protein misfolding disorder of cattle. BSE is of two types, classical BSE and atypical BSE which in turn is of two types, H-type BSE and L-type BSE. Both H-type BSE and L-type BSE are primarily sporadic prion disorders. However, one case of H-type BSE has recently been associated with E211K polymorphism in the prion protein gene (PRNP). Two polymorphisms in the bovine PRNP are also associated with susceptibility to classical BSE: a 23 bp insertion/deletion (indel) in the PRNP promoter region and a 12 bp indel in the first intron. No information regarding BSE susceptibility in Pakistani cattle is available. The present study aimed at achieving this information. A total of 236 cattle from 7 breeds and 281 buffaloes from 5 breeds were screened for E211K polymorphism and 23 bp and 12 bp indels employing triplex PCR. The E211K polymorphism was not detected in any of the animals studied. The 23 bp insertion allele was underrepresented in studied cattle breeds while the 12 bp insertion allele was overrepresented. Both 23 bp and 12 bp insertion alleles were overrepresented in studied buffalo breeds. Almost 90% of alleles were insertion alleles across all studied buffalo breeds. The average frequency of 23 bp and 12 bp insertion alleles across all studied cattle breeds was found to be 0.1822 and 0.9407, respectively. There were significant differences between Pakistani and worldwide cattle in terms of allele, genotype and haplotype frequencies of 23 bp and 12 bp indels. The higher observed frequency of 12 bp insertion allele suggests that Pakistani cattle are relatively more resistant to classical BSE than European cattle. However, the key risk factor for classical BSE is the dietary exposure of cattle to contaminated feedstuffs.     

Proteomic Selection of Immunodiagnostic Antigens for Trypanosoma congolense

Animal African Trypanosomosis (AAT) presents a severe problem for agricultural development in sub-Saharan Africa. It is caused by several trypanosome species and current means of diagnosis are expensive and impractical for field use. Our aim was to discover antigens for the detection of antibodies to Trypanosoma congolense, one of the main causative agents of AAT. We took a proteomic approach to identify potential immunodiagnostic parasite protein antigens. One hundred and thirteen proteins were identified which were selectively recognized by infected cattle sera. These were assessed for likelihood of recombinant protein expression in E. coli and fifteen were successfully expressed and assessed for their immunodiagnostic potential by ELISA using pooled pre- and post-infection cattle sera. Three proteins, members of the invariant surface glycoprotein (ISG) family, performed favorably and were then assessed using individual cattle sera. One antigen, Tc38630, evaluated blind with 77 randomized cattle sera in an ELISA assay gave sensitivity and specificity performances of 87.2% and 97.4%, respectively. Cattle immunoreactivity to this antigen diminished significantly following drug-cure, a feature helpful for monitoring the efficacy of drug treatment.     

Production of cattle lacking prion protein

Prion diseases are caused by propagation of misfolded forms of the normal cellular prion protein PrP(C), such as PrP(BSE) in bovine spongiform encephalopathy (BSE) in cattle and PrP(CJD) in Creutzfeldt-Jakob disease (CJD) in humans. Disruption of PrP(C) expression in mice, a species that does not naturally contract prion diseases, results in no apparent developmental abnormalities. However, the impact of ablating PrP(C) function in natural host species of prion diseases is unknown. Here we report the generation and characterization of PrP(C)-deficient cattle produced by a sequential gene-targeting system. At over 20 months of age, the cattle are clinically, physiologically, histopathologically, immunologically and reproductively normal. Brain tissue homogenates are resistant to prion propagation in vitro as assessed by protein misfolding cyclic amplification. PrP(C)-deficient cattle may be a useful model for prion research and could provide industrial bovine products free of prion proteins.     

PRNP and SPRN genes polymorphism in atypical bovine spongiform encephalopathy cases diagnosed in Polish cattle

Polymorphisms in the coding region of the prion protein gene (PRNP) have been associated with the susceptibility and incubation period of prion diseases in humans and sheep. However, polymorphisms in this part of the bovine PRNP gene do not affect the classical bovine spongiform encephalopathy (BSE) susceptibility in cattle. Studies carried out in Germany have shown that insertion/deletion-type polymorphisms located in the promoter region of the bovine prion gene are possible genetic factors modulating BSE susceptibility by changing the level of PRNP expression. No such association was observed for atypical BSE cases; however, due to the rare nature of the disease, these results should be confirmed. Additionally, a single nonsynonymous mutation in PRNP codon 211 (E211K) was described in one H-type BSE case in the USA; however, it was not found in any other cases. Here, we performed genetic characterization of PRNP promoter indel variations and determined the polymorphism of open reading frames (ORFs) of PRNP and bovine prion-like Shadoo (SPRN) genes in six Polish atypical BSE cases and compared these results to the population of clinically healthy Polish Holstein cattle. No potentially pathogenic mutations were found in the PRNP ORF in atypical BSE-affected cattle, but our study showed a high frequency of deletions at the indel loci of PRNP promoter in these animals. Additionally, a rare sequence variation in the SPRN protein-coding sequence was found in one L-type atypical BSE-affected animal.     

Polymorphism of the prion protein gene (<Emphasis Type="Italic">PRNP</Emphasis>) in Polish cattle affected by classical bovine spongiform encephalopathy

Recent attempts to discover genetic factors affecting cattle resistance/susceptibility to bovine spongiform encephalopathy (BSE) have led to the identification of two insertion/deletion (indel) polymorphisms, located within the promoter and intron 1 of the prion protein gene PRNP, showing a significant association with the occurrence of classical form of the disease. Because the effect of the polymorphisms was studied only in few populations, in this study we investigated whether previously described association of PRNP indel polymorphisms with BSE susceptibility in cattle is also present in Polish cattle population. We found a significant relation between the investigated PRNP indel polymorphisms (23 and 12 bp indels), and susceptibility of Polish Holstein-Friesian cattle to classical BSE (P < 0.05). The deletion variants of both polymorphisms were related to increased susceptibility, whereas insertion variants were protective against BSE.     

Paramphistomum cervi: antigenic profile of adults as recognized by infected cattle sera

An antigenic profile of adult Paramphistomum cervi was revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from cattle naturally infected with P. cervi, Fasciola gigantica and strongylids. SDS-PAGE of whole worm extracts exhibited 26 distinct protein bands. Immunoblotting analysis of these proteins showed five major antigenic bands which were recognized by serum of individual cattle naturally infected with P. cervi. These antigenic proteins had molecular weights ranging from 23 to 116kDa. One antigenic protein with a molecular weight of 52kDa exhibited a consistent reaction with sera from all infected cattle. It's diagnostic sensitivity, specificity and accuracy using this test were 100%, 98% and 98.9%, respectively. The positive and negative predictive values were 97.6% and 100%, respectively. This finding suggests that the 52kDa protein may be a diagnostic antigen for paramphistomosis.     

Immunoproteomics of Brucella abortus reveals differential antibody profiles between S19-vaccinated and naturally infected cattle

Brucella abortus is a Gram-negative intracellular bacterium that causes infectious abortion in food-producing animals and chronic infection in humans. This study aimed to characterize a B. abortus S19 antigen preparation obtained by Triton X-114 (TX-114) extraction through immunoproteomics to differentiate infected from vaccinated cattle. Three groups of bovine sera were studied: GI, 30 naturally infected cows; GII, 30 S19-vaccinated heifers; and GIII, 30 nonvaccinated seronegative cows. One-dimensional (1D) and two-dimensional electrophoretic profiles of TX-114 hydrophilic phase antigen revealed a broad spectrum of polypeptides (10-79 kDa). 1D immunoblot showed widespread seroreactivity profile in GI compared with restricted profile in GII. Three antigenic components (10, 12, 17 kDa) were recognized exclusively by GI sera, representing potential markers of infection and excluding vaccinal response. The proteomic characterization revealed 56 protein spots, 27 of which were antigenic spots showing differential seroreactivity profile between GI and GII, especially polypeptides <20 kDa that were recognized exclusively by GI. MS/MS analysis identified five B. abortus S19 proteins (Invasion protein B, Sod, Dps, Ndk, and Bfr), which were related with antigenicity in naturally infected cattle. In conclusion, immunoproteomics of this new antigen preparation enabled the characterization of proteins that could be used as tools to develop sensitive and specific immunoassays for serodiagnosis of bovine brucellosis, with emphasis on differentiation between S19 vaccinated and infected cattle.     

How was the French BSE epidemic underestimated?

In a previous study, we showed that estimates of the BSE epidemic in France were censored by cattle mortality and by a lack of diagnosis. Indeed, we estimated that 51 300 cattle were infected by the BSE agent between 1987 and 1997, whereas only 103 clinical BSE cases were detected by the passive surveillance system up to June 2000. The question thus arises as to the part played by each form of censorship in this underestimation. Here, using an updated cattle survival distribution, we estimated that 44 800 cattle were infected by the BSE agent between 1987 and 1997, and that 7100 of them showed clinical signs of BSE up to June 2000, showing the low efficiency of the surveillance system. Moreover, between 2087 and 5980 'infectious' cattle, with clinical or preclinical BSE, entered the human food chain before July 1996, the date of the ban on specified bovine offal.     

A comprehensive proteome map of bovine cerebrospinal fluid

Cerebrospinal fluid (CSF) is considered as the most promising body fluid target for the discovery of biomarkers for early diagnosis of neurodegenerative diseases such as Creutzfeldt–Jakob disease in humans and bovine spongiform encephalopathy in cattle. For the recognition of disease‐associated changes in bovine CSF protein patterns, a detailed knowledge of this proteome is a prerequisite. The absence of a high‐resolution CSF proteome map prompted us to determine all bovine CSF protein spots that can be visualised on 2‐D protein gels. Using state‐of‐the‐art 2‐DE technology for proteome mapping of bovine ante mortem CSF combined with sensitive fluorescent protein staining and MALDI‐TOF/TOF MS for protein identification, a highly detailed 2‐DE map of the bovine CSF proteome was established. Besides the proteins mapped by earlier studies, this map contains 66 different proteins, including 58 which were not annotated in bovine 2‐DE CSF maps before.     

Identification of an anti-aldolase autoantibody as a diagnostic marker for diabetic retinopathy by immunoproteomic analysis

Circulating autoantibodies specific for retinal proteins are associated with retinal destruction in patients with diabetic retinopathy (DR). In this study, we screened diabetic sera for the presence of anti-retinal autoantibodies with an aim of developing diagnostic markers for DR. Immunoblot analysis of DR patients' sera with human retinal cytosolic proteins revealed a higher incidence of anti-retinal autoantibodies, compared to normal blood donors or diabetic patients without DR. Anti-retinal protein autoantibody profiles of DR patient sera were obtained by 2-DE immunoblot analysis. Specifically, 20 protein spots reactive with DR patient sera were identified by ESI-MS/MS. Of these spots, 14 were specific for DR patients, and 4 reacted with both non-proliferative DR (non-PDR) and PDR sera. The anti-aldolase autoantibody was selected as a DR marker candidate, and specific reactivity of DR patient sera was confirmed by immunoblot analysis with rabbit aldolase. The serum anti-aldolase autoantibody level was measured by ELISA. DR patients showed significantly higher autoantibody levels than normal donors or diabetic patients without retinopathy. However, no significant differences were observed between non-PDR and PDR patients, suggesting that the level of anti-aldolase autoantibody is not determined by the severity of retinopathy in diabetic patients. Our data collectively demonstrate that the anti-aldolase autoantibody serves as a useful marker for DR diagnosis.     

ELISA detection of IgG antibody against a recombinant major surface antigen (Nc-p43) fragment of Neospora caninum in bovine sera

An ELISA was established to measure bovine IgG directed against the recombinant antigenic determinant of Nc-p43, a major surface antigen of Neospora caninum. In a previous study, two thirds of the C-terminal of the molecule was expressed as a 6 x His tagged protein (Ncp43P) for ELISA using 2/3 of the N-terminal of SAG1 from Toxoplasma gondii as a control (TgSAG1A). Among 852 cattle sera collected from stock farms scattered nation-wide, 103 sera (12.1%) were found to react with Ncp43P positively, but no positive reaction was observed with TgSAG1A. This study shows that Ncp43P could be available as an efficient antigen for the diagnosis of neosporosis in cattle. Furthermore, it together with TgSAG1A, could be useful for the differential diagnosis of N. caninum and T. gondii infections in other mammals.     

Polymorphisms of the prion protein gene (PRNP) in Hanwoo (Bos taurus coreanae) and Holstein cattle

Polymorphisms in the prion protein gene (PRNP) in humans and sheep correlate with susceptibility to transmissible spongiform encephalopathies (TSEs). Bovine spongiform encephalopathy (BSE) has been reported in British and Japanese cattle; it has occurred thus far in Holstein cattle. BSE in Hanwoo (Bos taurus coreanae) cattle has not been diagnosed up to now. To characterize the bovine PRNP polymorphisms in Korean cattle, we analyzed the open reading frame (ORF) of PRNP in 120 Hanwoo (beef) cattle and 53 Holstein (dairy) cattle. Three polymorphisms were found, the third position of codon 78 (G-->A), the third position of codon 192 (C-->T), and the deletion of a single octa-repeat. An analysis of codon 78 revealed no difference in the genotype (P = 0.2026) or allele (P = 0.7180) frequencies between Hanwoo and Holstein animals. However, there were significant differences in the genotype (P < 0.0001) and allele (P < 0.0001) frequencies at PRNP codon 192 between Hanwoo and Holstein animals. The rate of Holstein animals with deletion of a single octa-repeat was 91.5% undeleted homozygotes, 8.5% heterozygotes (with R3 deletion), and 0% deleted homozygotes. However, none of the 120 Hanwoo animals had any octa-repeat deletions. The genotype (P < 0.0001) and allele (P < 0.0001) frequencies of a single octa-repeat-deletion were also significantly different between Hanwoo and Holstein animals.     

Transmission barriers for bovine, ovine, and human prions in transgenic mice

Transgenic (Tg) mice expressing full-length bovine prion protein (BoPrP) serially propagate bovine spongiform encephalopathy (BSE) prions without posing a transmission barrier. These mice also posed no transmission barrier for Suffolk sheep scrapie prions, suggesting that cattle may be highly susceptible to some sheep scrapie strains. Tg(BoPrP) mice were also found to be susceptible to prions from humans with variant Creutzfeldt-Jakob disease (CJD); on second passage in Tg(BoPrP) mice, the incubation times shortened by 30 to 40 days. In contrast, Tg(BoPrP) mice were not susceptible to sporadic, familial, or iatrogenic CJD prions. While the conformational stabilities of bovine-derived and Tg(BoPrP)-passaged BSE prions were similar, the stability of sheep scrapie prions was higher than that found for the BSE prions but lower if the scrapie prions were passaged in Tg(BoPrP) mice. Our findings suggest that BSE prions did not arise from a sheep scrapie strain like the one described here; rather, BSE prions may have arisen spontaneously in a cow or by passage of a scrapie strain that maintains its stability upon passage in cattle. It may be possible to distinguish BSE prions from scrapie strains in sheep by combining conformational stability studies with studies using novel Tg mice expressing a chimeric mouse-BoPrP gene. Single-amino-acid substitutions in chimeric PrP transgenes produced profound changes in incubation times that allowed us to distinguish prions causing BSE from those causing scrapie.     

Conversion of the BASE prion strain into the BSE strain: the origin of BSE?

Atypical neuropathological and molecular phenotypes of bovine spongiform encephalopathy (BSE) have recently been identified in different countries. One of these phenotypes, named bovine "amyloidotic" spongiform encephalopathy (BASE), differs from classical BSE for the occurrence of a distinct type of the disease-associated prion protein (PrP), termed PrP(Sc), and the presence of PrP amyloid plaques. Here, we show that the agents responsible for BSE and BASE possess different biological properties upon transmission to transgenic mice expressing bovine PrP and inbred lines of nontransgenic mice. Strikingly, serial passages of the BASE strain to nontransgenic mice induced a neuropathological and molecular disease phenotype indistinguishable from that of BSE-infected mice. The existence of more than one agent associated with prion disease in cattle and the ability of the BASE strain to convert into the BSE strain may have important implications with respect to the origin of BSE and spongiform encephalopathies in other species, including humans.