A Combined Approach for the Characterization of Small Ruminant Lentivirus Strains Circulating in the Islands and Mainland of Greece.

Small ruminant lentiviruses are a group of viruses infecting goat and sheep worldwide. These viruses exhibit an extraordinary degree of genetic and antigenic variability that severely influence in vivo and in vitro features, as well as diagnostic test results. Small ruminant farming is the most important animal farming business in Greece, with a high impact on the Greek primary economy. Although SRLV infection and its impact on animal production are well established in the country, little is known about the circulating SRLV strains and their prevalence. The aim of this study was to characterize SRLVs circulating in Greece with a combined serological and molecular approach, using the bulk milk matrix collected from 60 farms in different municipalities. This study allowed us to estimate a seroprevalence of around 52% at the herd level. The B1, B2 and A3 subtypes and a novel A viral cluster were identified. Moreover, the amplicon sequencing method allowed us to identify more than one viral subtype in a sample. These results again confirm the high variability of these viruses and highlight the importance of the constant monitoring of viral evolution, in particular in antigens of diagnostic interest.

Genetic Characterization of Small Ruminant Lentiviruses Isolated from Dairy Sheep in Greece.

The high genetic heterogeneity of small ruminant lentiviruses (SRLV) renders the genetic characterization of the circulating strains crucial for the epidemiological investigation and the designation of effective diagnostic tools. In Greece, research data regarding the genetic diversity of the circulating SRLV strains is scarce, hindering the implementation of efficient surveillance and control programs. The objective of the study was to genetically characterize SRLV strains isolated from intensive dairy sheep farms in Greece and evaluate the variability of the immunodominant regions of the capsid protein. For this reason, a total of 12 SRLV-infected animals from four intensive dairy sheep farms with purebred Chios and Lacaune ewes were used for the amplification and sequencing of an 800 bp gag-pol fragment. The phylogenetic analyses revealed a breed-related circulation of strains; Chios ewes were infected with strains belonging exclusively to a separate group of genotype A, whereas strains belonging to subtype B2 were isolated from Lacaune ewes. Immunodominant epitopes of capsid protein were quite conserved among the strains of the same genotype, except for the Major Homology Region which showed some unique mutations with potential effects on viral evolution. The present study contributes to the extension of the current knowledge regarding the genetic diversity of SRLV strains circulating in sheep in Greece. However, broader genetic characterization studies are warranted for the exploration of possible recombinant events and the more comprehensive classification of the circulating strains.

A Longitudinal Cohort Study of Risk Factors Associated with Small Ruminant Lentivirus Seropositivity in Intensively Reared Dairy Ewes in Greece.

A two-year longitudinal cohort study was conducted on a total of 407 purebred Chios and Lacaune ewes from four intensive dairy sheep farms to assess potential risk factors for small ruminant lentiviruses (SRLVs) seropositivity. Ewes were serologically tested semiannually at pre-mating and pre-lambing, and their age, breed, and body condition score (BCS) were recorded. Εwes were categorized as constantly seronegative, constantly seropositive, seroconverted, seroreverted, or animals with an intermittent presence of antibodies. Mixed binary logistic regression models were used to estimate the adjusted relative risks of the studied risk factors for (i) the individual ewes' seropositivity, (ii) the manifestation of specific serological patterns, and (iii) the occurrence of seroconversion and seroreversion incidents. Increased age was associated with seropositivity and constantly seropositive status (p < 0.001 in both cases). On the other hand, age was negatively associated with constantly seronegative pattern, seroconversion incident, and the intermittent presence of antibodies (p < 0.05 in all cases). Moreover, breed was recognized as a risk factor: Lacaune ewes demonstrated increased seropositivity, whereas Chios ewes were more likely to demonstrate an intermittent presence of antibodies (p < 0.01 in both cases). Seropositive status (p < 0.001), seropositivity in animals with an intermittent presence of antibodies (p = 0.001), and seroconversion incidents (p < 0.001) were significantly increased at pre-lambing compared to pre-mating. The risk factors recognized in our study contribute to a better understanding of SRLVs epidemiology and the evidence-based designation of SRLVs' control programs in intensive dairy sheep farms in Greece.

Seroepidemiology of Maedi-Visna in Intensively Reared Dairy Sheep: A Two-Year Prospective Study.

The objective of this study is to prospectively evaluate the seroepidemiology of maedi-visna (MV) infections in intensively reared dairy sheep. A total of 407 purebred Chios and Lacaune ewes from four farms were surveyed for two consecutive years and were serologically tested semiannually with an indirect ELISA at pre-mating and pre-lambing. The farms' structure and management practices were similar and animal traits (age, breed, and production stage) were recorded. Based on the serological status, morbidity frequency measures were estimated, and ewes were categorized as constantly seronegative, constantly seropositive, seroconverted, seroreverted, or as animals with an intermittent presence of antibodies. During the study, period seroprevalence, incidence rate, and cumulative incidence were 84.8%, 33.6 new cases per 100 sheep-semesters, and 64.2%. Point-seroprevalence ranged from 48.5% to 96.0% among the studied farms and sampling occasions, and they increased by age. Increased morbidity frequency measures indicate the significance of horizontal transmission in intensive dairy sheep farms. A remarkable percentage of infected animals seroreverted (8.1%) or presented an intermittent presence of antibodies (10.3%) during the study, confirming the risk of misdiagnosis in cross-sectional studies and in the currently implemented testing and elimination programs. The serological patterns observed in our study need to be considered when studying MV epidemiology and for the designing of efficient MV elimination programs.

Circulation of Pestiviruses in Small Ruminants from Greece: First Molecular Identification of Border Disease Virus.

The incidence of small ruminant pestivirus infections in Greece remains unknown as they have not been diagnosed in the country since 1974 when the most recent Border Disease Virus (BDV) outbreak was reported. The objective of our study was to explore the possible occurrence of pestiviral infections among sheep and goat farms in Greece and to further determine the variants of major concern. Thus, serum samples were collected from 470 randomly selected animals belonging to 28 different flocks/herds. ELISA on p80 antibody revealed the existence of seropositive animals in four out of the 24 studied sheep flocks, whereas all the goats in the four studied herds were seronegative. Viral RNA and antigens were detected in two sheep out of the four seropositive flocks by RT-PCR and ELISA, respectively. Sequencing and phylogenetic analysis showed that the newly identified Greek variants were closely related to the strains of the BDV-4 genotype. One of the BDV-positive sheep demonstrated the diagnostic profile of a persistently infected (PI) animal, providing additional information regarding the source of the infection. This is the first molecular identification of BDV isolates in Greece. Our findings indicate that BDV infections are likely to remain undiagnosed, highlighting the need for further epidemiological studies and active surveillance programs to determine the prevalence and impact of BDV infections on a countrywide level.

Modulation of immune responses using adjuvants to facilitate therapeutic vaccination.

Therapeutic vaccination offers great promise as an intervention for a diversity of infectious and non-infectious conditions. Given that most chronic health conditions are thought to have an immune component, vaccination can at least in principle be proposed as a therapeutic strategy. Understanding the nature of protective immunity is of vital importance, and the progress made in recent years in defining the nature of pathological and protective immunity for a range of diseases has provided an impetus to devise strategies to promote such responses in a targeted manner. However, in many cases, limited progress has been made in clinical adoption of such approaches. This in part results from a lack of safe and effective vaccine adjuvants that can be used to promote protective immunity and/or reduce deleterious immune responses. Although somewhat simplistic, it is possible to divide therapeutic vaccine approaches into those targeting conditions where antibody responses can mediate protection and those where the principal focus is the promotion of effector and memory cellular immunity or the reduction of damaging cellular immune responses as in the case of autoimmune diseases. Clearly, in all cases of antigen-specific immunotherapy, the identification of protective antigens is a vital first step. There are many challenges to developing therapeutic vaccines beyond those associated with prophylactic diseases including the ongoing immune responses in patients, patient heterogeneity, and diversity in the type and stage of disease. If reproducible biomarkers can be defined, these could allow earlier diagnosis and intervention and likely increase therapeutic vaccine efficacy. Current immunomodulatory approaches related to adoptive cell transfers or passive antibody therapy are showing great promise, but these are outside the scope of this review which will focus on the potential for adjuvanted therapeutic active vaccination strategies.

Expert opinion on livestock antimicrobial usage indications and patterns in Denmark, Portugal and Switzerland.

We aimed at describing antimicrobial usage patterns throughout livestock production cycles, and comparing them across three countries from Northern, Central and Southern Europe. Given the difficulties to collect such detailed usage data, an expert opinion was deemed the most appropriate study design. This study provides new insights into the time periods and indications for which specific antimicrobial substances are used in different livestock sectors. Veterinary experts (n=67) from different livestock sectors (broilers, pigs, dairy cattle and veal/fattening calves) and countries (Denmark, Portugal and Switzerland) replied to a questionnaire focusing on the time periods in the production cycle when antimicrobial substances were administered, and the respective indications for treatment. Our results showed that for several antimicrobials, between-country and within-country variations exist regarding the temporal distributions of treatments and indications for use. These differences were also true for several critically important antimicrobials, which is a matter of concern. Furthermore, differences between countries were also evident regarding the antimicrobial substances licensed. Based on our results, it is recommended to establish and promote treatment guidelines, invest in the prevention of diseases during critical moments of the production cycle and target undifferentiated use of antimicrobials. Moreover, discrepancies between countries should be further investigated to better understand the factors underlying the identified patterns and to distinguish prudent from non-prudent use. The results can inform decision-making with the aim to foster antimicrobial prudent use in the veterinary setting and, therefore, protect public health from the threat of antimicrobial resistance.

Accurate and precise real-time RT-PCR assays for the identification of astrovirus associated encephalitis in cattle.

A novel bovine astrovirus genotype species (BoAstV-CH13/NeuroS1) was recently identified in brain tissues of cattle as a plausible cause of encephalitis. The purpose of the present study was to develop and validate real time RT-PCR assays for the detection of BoAstV-CH13/NeuroS1 in brain tissues of cattle. Three different primer-probe combinations were designed based on BoAstV-CH13/NeuroS1 full-genome sequences of 11 different strains identified in cattle, and established in three distinct one-step real time RT-PCR protocols. These protocols were compared regarding their diagnostic performance using brain tissues of cattle with and without astrovirus associated encephalitis. The limit of detection (LOD) of all three assays was between 1.34 × 101 and 1.34 × 102 RNA copies, leading to an analytical sensitivity two orders of magnitude superior compared to a conventional pan-astrovirus RT-PCR protocol (LOD 1.31 × 104 RNA copies). Amplification efficiency was in the range of 97.3% to 107.5% with linearity (R2) > 0.99. The diagnostic sensitivity and specificity of the assays was determined as 100%, and all three revealed good intra- and inter-test repeatability. In conclusion, the newly developed RT-qPCRs are sensitive, specific, and reliable test formats that will facilitate BoAstV-CH13/NeuroS1 detection in routine diagnostics as well as in research settings.

Development and validation of an immunohistochemistry procedure for the detection of a neurotropic bovine astrovirus.

Members of the Astroviridae family are best known to cause diarrhea in different mammalian species. Lately, some strains have been associated with encephalitis in humans, minks and cattle. In this study, we developed an immunohistochemistry (IHC) procedure for the detection of a neurotropic bovine astrovirus (BoAstV-CH13/NeuroS1), which is associated with non-suppurative encephalitis in cattle. We expressed five recombinant antigens corresponding to different putative viral proteins of BoAstV-CH13/NeuroS1. Antigens were then used for the production of hyperimmune sera in rabbits. Out of the five hyperimmune sera, the one directed against the conserved N-terminus of the viral capsid protein, termed ORF2-con, clearly surpassed the others in the detection of viral antigens in IHC in terms of strong signal intensity and low background staining. The accuracy of the ORF2-con IHC protocol was then evaluated using different sets of brain tissue samples: 30 samples from 9 animals with confirmed BoAstV-CH13/NeuroS1 infection, 30 samples from 8 animals with non-suppurative encephalitis of another etiology and 30 samples from apparently healthy slaughtered animals. The IHC was positive only with tissue samples from animals with a known positive BoAstV-CH13/NeuroS1 status, but not with those from negative ones, indicating a good diagnostic sensitivity and specificity of the assay. The ORF2-con IHC procedure is therefore an adequate tool for the detection of BoAstV-CH13/NeuroS1 infections in cattle.

Detection of Astrovirus in Historical Cases of European Sporadic Bovine Encephalitis, Switzerland 1958-1976.

European sporadic bovine encephalitis is a frequent diagnosis in neurologically diseased cattle, but its etiology remained unresolved. Using in situ hybridization, we have detected a recently discovered neurotropic bovine astrovirus in historical tissues in a high proportion of brain samples of affected cattle. Our results suggest that astroviruses were already involved in the pathogenesis of the disease several decades ago, but have gone undetected.

Full-genome based molecular characterization of encephalitis-associated bovine astroviruses.

Novel types of astrovirus have been identified recently in association with neurological disease in cattle. Among those viruses is bovine astrovirus CH13 (BoAstV CH13) that has been identified in Switzerland in a cow with encephalitis. Molecular testing by a combination of reverse transcription (RT-) PCR and in situ hybridization (ISH) indicated that astrovirus infection accounts for around one quarter of viral encephalitis cases of unknown etiology in cattle. Yet, it remained to be explored whether these animals were infected by BoAstV CH13 or other astrovirus species. In the present study we sequenced the entire astrovirus genome in brain tissues of eight RT-PCR and/or ISH positive cattle. Phylogenetic comparison of the genomic RNA and the encoded non-structural and structural proteins revealed that all these astrovirus strains were very similar to BoAstV CH13 as well as to a bovine encephalitis strain reported from the USA (BoAstV NeuroS1), and clearly distinct from other previously reported astroviruses. Conserved 5' and 3' untranslated regions (UTRs) were predicted to display distinct secondary RNA structures, which likely play a role in viral RNA replication and/or protein translation. Based on these data we propose that BoAstV CH13/NeuroS1 represents a new genotype species within the genus Mammastrovirus. The high degree of similarity between the strains and their relative distance to other genotype species suggest that during evolution some astroviruses acquired factors that specifically contribute to neuroinvasion.

Exploring the virome of cattle with non-suppurative encephalitis of unknown etiology by metagenomics.

Non-suppurative encephalitis is one of the most frequent pathological diagnosis in cattle with neurological disease, but there is a gap in the knowledge on disease-associated pathogens. In order to identify viruses that are associated with non-suppurative encephalitis in cattle, we used a viral metagenomics approach on a sample set of 16 neurologically-diseased cows. We detected six virus candidates: parainfluenza virus 5 (PIV-5), bovine astrovirus CH13/NeuroS1 (BoAstV-CH13/NeuroS1), bovine polyomavirus 2 (BPyV-2 SF), ovine herpesvirus 2 (OvHV-2), bovine herpesvirus 6 (BHV-6) and a novel bovine betaretrovirus termed BoRV-CH15. In a case-control study using PCR, BoAstV-CH13 (p=0.046), BoPV-2 SF (p=0.005) and BoHV-6 (p=4.3E-05) were statistically associated with the disease. These data expand our knowledge on encephalitis-associated pathogens in cattle and point to the value of NGS in resolving complex infection scenarios in a clinical disease setting.

Neurotropic astrovirus in cattle with nonsuppurative encephalitis in Europe.

Encephalitis is a frequently diagnosed condition in cattle with neurological diseases. Many affected animals present with a nonsuppurative inflammatory reaction pattern in the brain. While this pattern supports a viral etiology, the causative pathogen remains unknown in a large proportion of cases. Using viral metagenomics, we identified an astrovirus (bovine astrovirus [BoAstV]-CH13) in the brain of a cow with nonsuppurative encephalitis. Additionally, BoAstV RNA was detected with reverse transcription-PCR and in situ hybridization in about one fourth (5/22 animals) of cattle with nonsuppurative encephalitis of unknown etiology. Viral RNA was found primarily in neurons and at the site of pathology. These findings support the notion that BoAstV infection is a common cause of encephalitis in cattle. Phylogenetically, BoAstV-CH13 was closely related to rare astrovirus isolates from encephalitis cases in animals and a human patient. Future research needs to be directed toward the pathogenic mechanisms, epidemiology, and potential cross-species transmission of these neurotropic astroviruses.

Isolation of Mycobacterium avium subspecies paratuberculosis from Ugandan cattle and strain differentiation using optimised DNA typing techniques.

BACKGROUND: The occurrence of paratuberculosis in Ugandan cattle has recently been reported but there is no information on the strains of Mycobacterium avium subspecies paratuberculosis (MAP) responsible for the disease. The aim of this study was to isolate and characterise MAP from seropositive cattle and paratuberculosis lesions in tissues obtained from slaughtered cattle in Uganda. RESULTS: Twenty one isolates of MAP were differentiated into 11 genotype profiles using seven genotyping loci consisting of Insertion Sequence 1311(IS1311), Mycobacterial interspersed repeat units (MIRU) (loci 2, 3), Variable number tandem repeats (VNTR) locus 32 and Short sequence repeats (SSR) (loci 1, 2 and 8). Three different IS1311 types and three MIRU 2 profiles (7, 9, 15 repeats) were observed. Two allelic variants were found based on MIRU 3 (1, 5 repeats), while VNTR 32 showed no polymorphism in any of the isolates from which it was successfully amplified. SSR Locus 1 revealed 6 and 7 G1 repeats among the isolates whereas SSR locus 2 revealed 10, 11 and 12 G2 repeats. SSR locus 8 was the most polymorphic locus. Phylogenetic analysis of SSR locus 8 sequences based on their single nucleotide polymorphisms separated the isolates into 8 genotypes. We found that the use of Ethylene glycol as a PCR additive improved the efficiency of the PCR reactions for MIRUs (2, 3), VNTR 32 and SSR (loci 1 and 2). CONCLUSIONS: There is a high strain diversity of MAP in Uganda since 21 isolates could be classified into 11 genotypes. The combination of the seven loci used in this study results into a very precise discrimination of isolates. However analysis of SNPs on locus alone 8 is very close to this combination. Most of the genotypes in this study are novel since they differed in one or more loci from other isolates of cattle origin in different studies. The large number of MAP strains within a relatively small area of the country implies that the epidemiology of paratuberculosis in Uganda may be complicated and needs further investigation. Finally, the use of Ethylene glycol as a PCR additive increases the efficiency of PCR amplification of difficult templates.

Distinct proteinase K-resistant prion protein fragment in goats with no signs of disease in a classical scrapie outbreak.

Considerable efforts have been directed toward the identification of small-ruminant prion diseases, i.e., classical and atypical scrapie as well as bovine spongiform encephalopathy (BSE). Here we report the in-depth molecular analysis of the proteinase K-resistant prion protein core fragment (PrP(res)) in a highly scrapie-affected goat flock in Greece. The PrP(res) profile by Western immunoblotting in most animals was that of classical scrapie in sheep. However, in a series of clinically healthy goats we identified a unique C- and N-terminally truncated PrP(res) fragment, which is akin but not identical to that observed for atypical scrapie. These findings reveal novel aspects of the nature and diversity of the molecular PrP(res) phenotypes in goats and suggest that these animals display a previously unrecognized prion protein disorder.

Caprine PRNP polymorphisms at codons 171, 211, 222 and 240 in a Greek herd and their association with classical scrapie.

The association between PRNP variation and scrapie incidence was investigated in a highly affected Greek goat herd. Four mutations were identified at codons 171Q/R, 211R/Q, 222Q/K and 240P/S. Lysine at codon 222 was found to be associated with the protection from natural scrapie (P=0.0111). Glutamine at codon 211 was observed in eight animals, all of them being scrapie-negative, indicating a possible protective role of this polymorphism although statistical analysis failed to support it (P=0.1074). A positive association (P=0.0457) between scrapie-affected goats and the wild-type Q(171)R(211)Q(222)S(240) allele is presented for the first time. In addition, a novel R(171)RQS allele, which is identical to the A(136)R(154)R(171) allele that has been associated with resistance to classical scrapie in sheep, was observed in low frequency. Resistant alleles that include K(222) and Q(211) are absent or rare in sheep and can provide the basis for the development of a feasible breeding programme for scrapie eradication in goats.