Advances in Detecting Cystic Echinococcosis in Intermediate Hosts and New Diagnostic Tools: A Literature Review.

Cystic echinococcosis (CE) is a zoonotic disease affecting humans and animals. Despite a lack of clarity about many details of parasite-intermediate host interactions, the nature of the immune responses triggered by hydatid infection has revealed new perspectives. This study discusses the latest advances in elucidating the immunologic mechanism of echinococcosis and its detection and potential approaches to enhance serodiagnosis accuracy. Moreover, nanobiosensors have been evaluated according to their potential to improve treatment efficiency and aid in an early diagnosis of cystic echinococcosis. The serum of an intermediate host can diagnose CE by analyzing antibodies induced by Echinococcus granulosus. Among the most notable features of this method are its noninvasive ability and high sensitivity, both of which make it an excellent tool for clinical diagnosis. Several serological tests, including ELISAs and immunoblotting, can detect these antibodies to assess the disease's state and determine the treatment outcome. A thorough understanding of what cross-reactivity means and the stage of the disease are crucial to interpreting serological results. Nanobiosensors have also proven better than conventional biosensors in detecting hydatid cysts. Additionally, they are highly sensitive and versatile when detecting specific biomarkers, improving diagnostic accuracy. These immunomodulatory molecules, induced by E. granulosus, are a good candidate for diagnosing cystic echinococcosis because they alter intermediate host immune responses. Hydatid cyst detection is also enhanced through nanobiosensors, which provide better accuracy.

Immunohistochemistry as a Reliable Tool for the Diagnosis of Cystic Echinococcosis in Patients from Sardinia, Italy-A Confirmatory Study.

Cystic Echinococcosis (CE) is a zoonotic disease caused by the larval stage of the tapeworm Echinococcus granulosus sensu lato (s.l.). This study aims to investigate the use of two monoclonal antibodies (mAbEmG3 and mAbEm2G11) by immunohistochemistry (IHC) to confirm the diagnosis of CE in human patients, in particular in those cases in which other techniques fail to provide a correct or conclusive diagnosis. For this purpose, a survey on 13 patients was performed. These subjects were referred to Sardinian hospitals (Italy) from 2017 to 2022 and were suspected to be affected by CE. Our findings from these 13 patients showed the detection of E. granulosus sensu stricto by IHC in 12 of 13 echinococcal cysts, as one sample was of a non-parasitological origin. The results confirmed that IHC, by means of the mAbEmG3 and mAbEm2G11, is a reliable diagnostic tool that showed a very high performances when tested on strain of E. granulosus s.l. from Sardinia.

Diagnosis and treatment of unusual multiorgan Echinococcus hydatid cysts.

Background: Echinococcosis is a zoonotic disease caused by Echinococcus granulosus. Usually, the liver is the most affected organ, accounting for approximately 70% to 85% of cases. The lungs represent 20% of the cases. Fewer than 10% are found in other sites, including the bone, brain, spleen, and kidneys. Case presentation: A young man was referred to a local hospital for dull pain in the right flank, fever, and mild cough. Computed tomography showed 2 large cysts: the first involved the left lung and measured 130 × 90.5 × 120 mm, whereas the second cyst was located in the right kidney and measured 130 × 100 × 120 mm. Surgery was performed to remove both lesions in 2 separate surgical sessions. Conclusions: Echinococcosis is a compulsorily notifiable disease. Collaboration between medical doctors from different specializations is necessary. A multidisciplinary approach is important for the correct therapeutic management of the disease. Furthermore, the high possibility of recurrence makes the long-term follow-up mandatory.

Investigation of patients with new infection of echinococcal cyst in Sardinia, Italy.

Cystic Echinococcosis (CE) is a zoonotic disease caused by the larval stage of a tapeworm of Taeniidae family, genus Echinococcus and species Echinococcus granulosus sensu lato (s.l.). CE is a worldwide public health problem and is highly incident in all Mediterranean areas. Following clinical, image techniques and serological investigations all 83 subjects involved in the study were diagnosed for CE. General and clinical data were entered into a database and evaluated. The 43.37% were female and 56.63% male, mean age was 50.71 while the range most represented (22.7%) was between 61->70 years. The purposes of our survey were to investigate these 83 patients enrolled in the study and to deeply examine 20 (24.10%) patients that developed a new echinococcal cyst. Moreover, the causes at the basis of the onset of a new cyst were analysed, together with a possible correlation with different treatments related to primary infection corresponding to surgery (n=7), albendazole (n=6), PAIR (n=3) and watch and wait (n=4). A possible link with medical treatments of the primary infection was observed in the subjects who underwent surgery or PAIR and a likely correlation attributable to high environmental contamination in subjects managed with drugs or watch and wait was detected. Moreover, our analysis evidenced that patients with a new infection presented a more severe diagnosis along with a major mortality rate. Finally, these data may have an important contribution for an epidemiological point of view concerning the percentage of CE in a specific geographical endemic area, such as Sardinia.

Environmental Influence on the Occurrence of Multi-Organ Cystic Echinococcosis Infection in a Patient from Sardinia, Italy.

An uncommon clinical case of an adult woman who was referred to the hospital with severe symptoms attributable to cystic echinococcosis (CE) is described in this report. According to a questionnaire, the subject was exposed to a high risk of infection since she was employed on a farm about 20 years before diagnosis. She lived close to several animal species and handled vegetables in inadequate hygienic conditions. Medical and laboratory investigations confirmed the presence of massive echinococcal cystic lesions in each lung and in the liver. Given the peculiarity of the case, pharmacological and surgical treatments were the only conceivable option. The association of pharmacological treatment, surgery, and interventional radiology procedure represented a reliable and effective way to handle a complex case of human hydatidosis. A multi-disciplinary approach was mandatory, resulting in a clear and conclusive diagnosis of CE caused by the zoonotic parasite E. granulosus sensu stricto of the G1 genotype.

Genetic Characterization of Echinococcus granulosus sensu stricto Isolated from Human Cysts from Sardinia, Italy.

This study involved 20 patients affected by cystic echinococcosis (CE) who were referred to different hospitals of Sardinia (Italy) from 2017 to 2022. By means of a multidisciplinary approach, diagnosis was confirmed for CE in 18 patients and for different aetiologies in two subjects. Moreover, serology was positive for 15 subjects. Since multiple CE cysts were found in five patients, a total of 27 lesions were collected; however, only one for each patient was investigated for genetic characterization of E. granulosus s.s. DNA isolates. Our results included 15 fertile cysts that underwent DNA extraction and amplification by three different PCRs targeting nuclear (calreticulin) and mitochondrial genes (cox1 and nad5). DNA was sequenced, and by neighbour-joining phylogenetic trees we determined 10 G1 and five G3 genotypes previously reported in Sardinia. These sequences were used to construct a network, along with those circulating in Mediterranean areas. The haplotype network calculated on cox1 evidenced seven different haplotypes of the 15 isolates, with SAR2 the most represented, carried by seven cysts, and SAR17 never described in the Mediterranean area. Meanwhile, the nad5 sequences showed the most common haplotype as nd5SAR7, as well as two new haplotypes not previously described, nd5SAR13, isolated from a Sardinian patient, and nd5SAR14, isolated from a Romanian patient.

Comparison and evaluation of analytic and diagnostic performances of four commercial kits for the detection of antibodies against Echinococcus granulosus and multilocularis in human sera.

Cystic echinococcosis (CE) is a disease caused by Echinococcus granulosus sensu lato (s.l.), an ubiquitous worldwide zoonotic agent affecting humans and animals. Diagnosis of CE in humans is usually performed by imagine techniques along with immunoassays. The aim of our study was to evaluate and compare four commercial diagnostic kits, based on the detection of IgG antibodies against E. granulosus and E. multilocularis. The study was performed on a total of 259 sera: the positive (n = 74) and the negative (n = 185) group. The following analytic and diagnostic performances of the four kits were evaluated: operator skills, specificity, sensitivity, repeatability, reproducibility, accuracy, positive and negative predictive values. Based on the parameters evaluated, all four tests demonstrated excellent quality and proved to be reliable diagnostic tools to support the clinical evaluation of human patients suspected of having CE. The four commercial assays, in our hands, presented altogether, a range of performances from good to excellent, being immunoblotting (IB) the most reliable, used as gold standard, followed by the immunochromatographic test (ICT) and finally the two enzyme linked immunosorbent assay (ELISAs).

Serological, Molecular Prevalence and Genotyping of Coxiella burnetii in Dairy Cattle Herds in Northeastern Algeria.

In Algeria, data on the epidemiology of coxiellosis in cattle are still lacking. In this study, bulk tank milk (BTM) samples from 200 randomly selected dairy cattle herds from Setif province of Algeria were analyzed by an indirect enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). Results highlighted that 37% (95% CI: 30.31-43.69%) and 9% (95% CI: 5.03-12.96%) of BTM samples contained Coxiella burnetii antibodies and DNA, respectively. Based on Cohen's kappa coefficient, a very low agreement between the ELISA and PCR results was found (k = 0.0849) (95% CI: 0.00-0.189). For a second experiment, 186 whole blood samples of cows from farms with reproduction disorders were analyzed by molecular tools to detect C. burnetii. This study revealed an overall prevalence of 6.98% (95% CI: 3.32-10.65%). All positive samples determined by conventional PCR were analyzed by real-time quantitative PCR (qPCR). Eleven samples with cycle threshold (Ct) values lower than 35 were selected for genotyping by the multispacer sequence typing (MST) method. The MST12 genotype in BTM samples, the MST32 genotype and a new MST genotype (partial profile) in whole blood samples were identified. Obtained results have allowed us to better understand the epidemiology of bovine coxiellosis in the region of Setif.

Proteomic characterization of Echinococcus granulosus sensu stricto, Taenia hydatigena and Taenia multiceps metacestode cyst fluids.

Cystic echinococcosis (CE) diagnosis by means of serological assays is hampered by the presence of parasites closely related to Echinococcus granulosus sensu lato (s.l.), responsible of the zoonotic disease and with which share cross-reacting antigens. Thus, improvements on the characterization of Echinococcus specific antigens expressed in the larval stage are required, in order to provide useful information for the development of immunological assays for the serodiagnosis of CE in sheep. Here, the proteome of the hydatid cyst fluids (HFs) of Echinococcus granulosus (hydatid fluid, EgHF) and other ovine parasites cyst fluids (CFs), Taenia hydatigena (ThCF) and Taenia multiceps (TmCF) were analyzed by a shotgun proteomic approach. Parasite and host protein profiles in the three types of cyst fluids were characterized and compared. Among the identified proteins, differential parasitic markers with serodiagnostic potential, due to their well-known immunoreactivity in human, included Ag5, AgB proteins, 8-kDa glycoproteins, hydatid disease diagnostic antigen P29 and major egg antigen P40. In particular, seven proteoforms of AgB and 8-kDa glycoprotein resulted to be the most promising diagnostic biomarkers, as they might predict CE in ovine and discriminate between different types of parasites.

Identification of Echinococcus granulosus Genotypes G1 and G3 by SNPs Genotyping Assays.

Echinococcus granulosus sensu lato (s.l.) is the causative agent of cystic echinococcosis in animals and humans. Different E. granulosuss.l. genotypes exhibit great diversity in their life cycle, host selectivity and pathogenicity. For this reason, the study of genetic variation within Echinococcus species is of importance for their epidemiological implication. We employed two SNP genotyping technologies to distinguish G1 and G3 E. granulosus sensu stricto (s.s.). genotypes. The genotypes of DNA samples (n = 28) extracted from hydatid cysts of different animal species were identified by amplification and sequencing of a fragment of the mitochondrial nad5 gene. Two SYBR green and three TaqMan real time PCR assays were developed for targeting of three nad5 informative positions (SNP758, 1123, and 1380) known to be able to discriminate G1 from G3. Genotyping by SYBR Green PCR based on cycle threshold (Ct) with melting temperature (Tm) analysis and performed on SNP1123 and SNP1380 failed to identify one DNA sample. TaqMan assays for SNP758, 1123 and 1380 effectively confirmed genotype identification obtained by Sanger sequencing. Our results demonstrated that the combination of the three Taqman assays developed in this study represents a valuable and cost effective tool alternative to DNA sequencing for E. granulosus s.s. genotyping.

Validation of a Novel Commercial ELISA Test for the Detection of Antibodies against Coxiella burnetii.

Q fever is a zoonosis caused by Coxiella burnetii, a Gram-negative pathogen with a complex life cycle and a high impact on public and animal health all over the world. The symptoms are indistinguishable from those belonging to other diseases, and the disease could be symptomless. For these reasons, reliable laboratory tests are essential for an accurate diagnosis. The aim of this study was to validate a novel enzyme-linked immunosorbent assay (ELISA) test, named the Chorus Q Fever Phase II IgG and IgM Kit (DIESSE, Diagnostica Senese S.p.A), which is performed by an instrument named Chorus, a new device in medical diagnostics. This diagnostic test is employed for the detection of antibodies against C. burnetii Phase II antigens in acute disease. Our validation protocol was performed according to the Italian Accreditation Body (ACCREDIA) (Regulation UNI CEI EN ISO/IEC 17025:2018 and 17043:2010), OIE (World Organization for Animal Health), and Statement for Reporting Studies of Diagnostic Accuracy (STARD). Operator performance was evaluated along with the analytical specificity and sensitivity (ASp and ASe) and diagnostic accuracy of the kit, with parameters such as diagnostic specificity and sensitivity (DSp and DSe) and positive and negative predictive values (PPV and NPV), in addition to the repeatability. According to the evaluated parameters, the diagnostic ELISA test was shown to be suitable for validation and commercialization as a screening method in human sera and a valid support for clinical diagnostics.

Cystic Echinococcosis: Clinical, Immunological, and Biomolecular Evaluation of Patients from Sardinia (Italy).

Cystic echinococcosis (CE), a zoonotic disease caused by the larval stage of the tapeworm Echinococcus granulosus sensu lato (s.l.), is a worldwide public health problem. Echinococcus granulosus sensu stricto (s.s.), associated with G1 and G3 genotypes, is endemic with high prevalence in the Mediterranean basin. The parasite's life cycle comprises definitive hosts (canids) and intermediate hosts (ruminants) and can occasionally involve humans. The main aim of this research was to confirm the diagnosis of 13 patients suspected of CE who presented different complications and needed the surgical removal of the cysts. We also wanted to understand and clarify more the diagnosis of echinococcosis in humans. For this purpose, the patients first underwent cyst evaluation by ultrasound (US), immunological analysis, and then total pericystectomy, followed by parasitological, histopathological, and molecular biology examinations of the cysts. US stadiated one CE1, one CE2, eight CE3b, one CE4, and two CE5; immunology evidenced nine positives; histopathology confirmed 11 CE cysts, of which 8 fertile presenting protoscoleces were identified as E. granulosus s.s. by molecular biology, genotyped as three G1 and four G3 by neighbor-joining (NJ) phylogenetic tree. In conclusion, the results showed that 11 patients were affected by E. granulosus s.s. G1 orG3, and 2 cystic neoformations were of non-parasitic origin.

Genetic diversity of Echinococcus granulosus sensu stricto in Sardinia (Italy).

Cystic echinococcosis (CE) is a severe parasitic zoonosis caused by the metacestode of the tapeworm Echinococcus granulosus sensu lato (s.l.). The disease has a global distribution representing a significant public health concern. Based on mitochondrial DNA analysis E. granulosus s.l. has been subdivided into five species: E. granulosus sensu stricto (s.s.) (G1, G3 genotype), E. equinus (G4 genotype), E. ortleppi (G5 genotype), E. canadensis (G6-G8, G10 genotype) and E. felidis. E. granulosus s.s., and in particular G1, is the most widespread genotype and the major responsible of human CE cases worldwide. In Italy G1 genotype is higly represented with larger percentages in some hyperendemic areas such as Sardinia. Molecular studies represent a valuable tool to improve our understanding of the E. granulosus epidemiology and CE control strategies. In the present study we investigated genetic variability of E. granulosus s.s. in Sardinia. To this purpose 83 hydatid cysts were collected from different animal species including humans and the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was partially sequenced (720 bp). Nucleotide sequences from Mediterranean basin were also analyzed for comparison. The phylogenetic network revealed 30 haplotypes grouped around a predominant isolate that had been already reported from other world regions. Haplotype diversity (0.8495 ± 0.0336) and nucleotide diversity (0.003305 ± 0.002014) were similar in Sardinia respect to other Mediterranean countries. Neutrality indices obtained by Tajima's D and Fu's Fs test were significantly negative (p ≤ .01) suggesting expansion of Sardinian population. Low Fixation indices (Fst), ranging from negative values (Algeria, Greece, Spain, other part of Italy) to 0.089 (Albania, France), indicated absence of genetic differentiation, and gene flow between Sardinia and other Mediterranean countries.

Seroprevalence and risk factors of Coxiella burnetii infection in cattle in northeast Algeria.

A cross-sectional study was conducted to determine the seroprevalence and the risk factors associated with C. burnetii infection in cattle in the state of Setif in northeastern Algeria from March 2016 to April 2018. A total of 678 cows animals aged at least 24 months and belonging to 90 herds were randomly selected. A serum sample from each cow was tested for antibodies against C. burnetii using an indirect enzyme-linked immunosorbent assay (ELISA). A structured questionnaire focusing on risk factors for C. burnetii infection was administered to farm owners involved in the study. The individual animal prevalence was 11.36% (77/678) (95%CI 8.97-13.75%), the herd prevalence was 45.56% (41/90) (95%CI 35.27-55.84%), and the within-herd prevalence ranged from 9.09 to 57.14% (mean 23.71%; Q1 11.11%, Q2 or median 20%, Q3 30%). Multivariable logistic regression analysis revealed that contact with other herds (odds ratio (OR) 1.95, 95 CI 1.12-3.42) and purchased animals (OR 2.05, 95 CI 1.14-3.68) was identified as risk factors for seropositivity to C. burnetii, while the use of disinfectants (OR 0.32, 95 CI 0.14-0.72) was identified as protective factor. The results from the present study indicate that C. burnetii is circulating into cattle herds in the region of Setif in Northeastern of Algeria. It is recommended to implement good hygienic practices and measures of biosecurity to reduce the spread of infection between cattle herds and possible exposure of humans.

Validation of a one-step PCR assay for the molecular identification of Echinococcus granulosus sensu stricto G1-G3 genotype.

The Italian National Reference Center for Echinococcosis (CeNRE, Sassari, Italy) set up a diagnostic protocol of "one-step-PCR" useful for the detection of E. granulosus sensu stricto (E.g.s.s.) and the identification of its genotype (G1-G3). The purpose of this work was to perform the validation of the "PCR E.g.s.s." method. The procedures were performed employing the criteria of the World Organization for Animal Health as well as of the Italian Accreditation Body (ACCREDIA) based on the Regulation UNI CEI EN ISO/IEC 17025. Positive DNA samples belonging to E. granulosus, E. ortleppi, E. multilocularis, E. canadensis species were used for the experiments. Analytical specificity evidenced primer pairs Cal (Calreticulin l gene of 1001 bp) with an specificity higher respect to Ef1 (Elongation-Factor 1 Alpha gene of 706 bp) and NAD (Dehydrogenase-subunit 1 gene of 219 bp). The analytical sensitivity presented the capability to detect a very low amount of parasite DNA corresponding to a concentration of 12.5 pg/µl; accuracy and precision related to the operator performance, along with repeatability and reproducibility, evidenced high concordance among results and demonstrated an excellent κ values of Cohen. According to the good performance related to the evaluated parameters, the method "PCR E.g.s.s." was suitable for the validation procedure, and consequently, to be undergone to the accreditation process. In conclusion, the results demonstrated an elevated robustness and reliable features of the "PCR E.g.s.s." able to perform a rapid diagnosis of E. granulosus in only "one step", hence, it is likely to avoid the sequencing step.

Cystic echinococcosis in a domestic cat (Felis catus) in Italy.

Echinococcus granulosus sensu lato is a zoonotic agent with a life cycle consisting of definitive hosts (dogs and wild carnivores), and intermediate hosts (usually ungulates). Other animals and humans may accidentally ingest eggs and contract cystic echinococcosis, acting as aberrant hosts. A 3-year-old neutered female cat was brought to a veterinary practice in Sassari (Italy) with abdominal distension. Ultrasound showed multiple intraperitoneal vesicles, which on laparotomy were found to be metacestodes of E. granulosus. Videos of the extraction of cysts are provided. Phylogenetic analysis based on a fragment of the cytochrome oxidase subunit 1 (cox1) mitochondrial gene identified the isolate as E. granulosus sensu stricto genotype G1, the most common genotype circulating in Europe and the Mediterranean basin. This is the first case report of cystic echinococcosis in domestic cats from Italy.

Structural and Immunodiagnostic Characterization of Synthetic Antigen B Subunits From Echinococcus granulosus and Their Evaluation as Target Antigens for Cyst Viability Assessment.

Background: Several tools have been proposed for serodiagnosis of cystic echinococcosis (CE), but none seems promising for cyst viability assessment. Antigens with stage-specific diagnostic value have been described, but few studies with well-characterized antigens and human serum samples have been performed. Antigen B (AgB) proteoforms hold promise as markers of viability, due to their differential stage-related expression and immunoreactivity. Methods: Four AgB subunits (AgB1, AgB2, AgB3, AgB4) were synthesized and structurally characterized. Based on the preliminary evaluation of the subunits by western immunoblotting and enzyme-linked immunosorbent assay (ELISA), AgB1 and AgB2 were further tested in two ELISA setups and extensively validated on 422 human serum samples. Results: All subunits showed a high degree of spontaneous oligomerization. Interacting residues within oligomers were identified, showing that both the N-terminal and C-terminal of each subunit are involved in homo-oligomer contact interfaces. No hetero-oligomer was identified. AgB1 and AgB2 ELISAs revealed different sensitivities relative to cyst stage. Of note, besides high specificity (97.2%), AgB1 revealed a higher sensitivity for active-transitional cysts (100% for CE1, 77.8% for CE2, 81.5% for CE3a, and 86.3% for CE3b) than for inactive cysts (41.7% for CE4 and 11.1% for CE5) and postsurgical patients (44%). Interestingly, 19 of 20 patients with spontaneously inactive cysts and 6 of 9 treated with albendazole >5 years earlier were negative on the AgB1 assay. Conclusions: The structural characterization of subunits provides insights into the synthetic antigen conformation. The stage-related sensitivity of synthetic AgB1 holds promise as part of a multiantigen setting and deserves further longitudinal evaluation as marker of cyst viability.

Yessotoxin detection in bivalve molluscs: A case study from coastal mussel farms (Sardinia, Italy).

This work reports the first communication relating to the presence of yessotoxins in Mytilus galloprovincialis from coastal mussel farms (Sardinia, western Mediterranean) detected during 2008 and 2013 through a monitoring programme. The paper emphasizes how the changes both in yessotoxin permitted limits and used methods, established by legislation, have influenced the interpretation of the obtained results. Consequently, the samples that resulted negative during 2008 would have been positive until August 2013 and negative from September 2013 up to now, and the samples that were positive in 2013 would have been positive in 2008 and negative nowadays, according to Regulation currently in force. Regular monitoring of biotoxins demonstrated that, although yessotoxins have been rarely present in the past in Sardinia, they may cause toxicity in shellfish. So, it's important to keep up on legislation's changing and laboratory methods.

Genetic and Pathological Follow-Up Study of Goats Experimentally and Naturally Exposed to a Sheep Scrapie Isolate.

UNLABELLED: Thirty-seven goats carrying different prion protein genotypes (PRNP) were orally infected with a classical scrapie brain homogenate from wild-type (ARQ/ARQ) sheep and then mated to obtain 2 additional generations of offspring, which were kept in the same environment and allowed to be naturally exposed to scrapie. Occurrence of clinical or subclinical scrapie was observed in the experimentally infected goats (F0) and in only one (F1b) of the naturally exposed offspring groups. In both groups (F0 and F1b), goats carrying the R154H, H154H, R211Q, and P168Q-P240P dimorphisms died of scrapie after a longer incubation period than wild-type, G37V, Q168Q-P240P, and S240P goats. In contrast, D145D and Q222K goats were resistant to infection. The immunobiochemical signature of the scrapie isolate and its pathological aspects observed in the sheep donors were substantially maintained over 2 goat generations, i.e., after experimental and natural transmission. This demonstrates that the prion protein gene sequence, which is shared by sheep and goats, is more powerful than any possible but unknown species-related factors in determining scrapie phenotypes. With regard to genetics, our study confirms that the K222 mutation protects goats even against ovine scrapie isolates, and for the first time, a possible association of D145 mutation with scrapie resistance is shown. In addition, it is possible that the sole diverse frequencies of these genetic variants might, at least in part, shape the prevalence of scrapie among naturally exposed progenies in affected herds. IMPORTANCE: This study was aimed at investigating the genetic and pathological features characterizing sheep-to-goat transmission of scrapie. We show that in goats with different prion protein gene mutations, the K222 genetic variant is associated with scrapie resistance after natural and experimental exposure to ovine prion infectivity. In addition, we observed for the first time a protective effect of the D145 goat variant against scrapie. Importantly, our results demonstrate that the phenotypic characteristic of the wild-type sheep scrapie isolate is substantially preserved in goats carrying different susceptible PRNP gene variants, thus indicating that the prion protein gene sequence, which is shared by sheep and goats, plays a fundamental role in determining scrapie phenotypes.

(1)H NMR brain metabonomics of scrapie exposed sheep.

While neurochemical metabolite modifications, determined by different techniques, have been diffusely reported in human and mice brains affected by transmissible spongiform encephalopathies (TSEs), this aspect has been little studied in the natural animal hosts with the same pathological conditions so far. Herein, we investigated, by high resolution (1)H NMR spectroscopy and multivariate statistical data analysis, the brain metabolite profile of sheep exposed to a scrapie agent in a naturally affected flock. On the basis of clinical examinations and western blotting analysis for the pathological prion protein (PrP(Sc)) in brain tissues, sheep were catalogued as not infected (H), infected with clinical signs (S), and infected without clinical signs (A). By discriminant analysis of spectral data, comparing S vs. H, we found a different metabolite distribution, with inosine, cytosine, creatine, and lactate being higher in S than in H brains, while the branched chain amino acids (leucine, isoleucine, and valine), phenylalanine, uracil, tyrosine, gamma-amino butyric acid, total aspartate (aspartate + N-acetyl aspartate) being lower in S. By a soft independent modelling of class analogy approach, 1 out of 3 A samples was assigned to class H. Furthermore, A brains were found to be higher in choline and choline-containing compounds. By means of partial least squares regression, an excellent correlation was found between the PrP(Sc) amount and the (1)H NMR metabolite profile of infected (S and A) sheep, and the metabolite mostly correlated with PrP(Sc) was alanine. The overall results, obtained using different chemometric tools, were able to describe a brain metabolite profile of infected sheep with and without clinical signs, compared to healthy ones, and indicated alanine as a biomarker for PrP(Sc) amounts in scrapie brains.