Persistent infections after natural transmission of bovine viral diarrhoea virus from cattle to goats and among goats

Bovine viral diarrhoea virus (BVDV) is an economically important pathogen of cattle worldwide. Infection of a pregnant animal may lead to persistent infection of the foetus and birth of a persistently infected (PI) calf that sheds the virus throughout its life. However, BVD viruses are not strictly species specific. BVDV has been isolated from many domesticated and wild ruminants. This is of practical importance as virus reservoirs in non-bovine hosts may hamper BVDV control in cattle. A goat given as a social companion to a BVDV PI calf gave birth to a PI goat kid. In order to test if goat to goat infections were possible, seronegative pregnant goats were exposed to the PI goat. In parallel, seronegative pregnant goats were kept together with the PI calf. Only the goat to goat transmission resulted in the birth of a next generation of BVDV PI kids whereas all goats kept together with the PI calf aborted. To our knowledge, this is the first report which shows that a PI goat cannot only transmit BVD virus to other goats but that such transmission may indeed lead to the birth of a second generation of PI goats. Genetic analyses indicated that establishment in the new host species may be associated with step-wise adaptations in the viral genome. Thus, goats have the potential to be a reservoir for BVDV. However, the PI goats showed growth retardation and anaemia and their survival under natural conditions remains questionable.

Development of a novel marker vaccine platform for protection against Bluetongue Virus (BTV)

Bluetongue virus (BTV) is an economically important member of the genus Orbivirus and closely related to African horse sickness virus (AHSV) and Epizootic hemorrhagic disease virus (EHDV). Currently, 26 different serotypes of BTV are known. The virus is transmitted by blood-feeding Culicoides midges and causes disease (bluetongue [BT]) in ruminants. In 2006/2007, BTV serotype 8 (BTV-8) caused widespread outbreaks of BT amongst livestock in Europe, which were eventually controlled employing a conventionally inactivated BTV vaccine. However, this vaccine did not allow the discrimination of infected from vaccinated animals (DIVA) by the commonly used VP7 cELISA. RNA replicon vectors based on propagation-incompetent recombinant vesicular stomatitis virus (VSV) represent a novel vaccine platform that combines the efficacy of live attenuated vaccines with the safety of inactivated vaccines. Our goal was to generate an RNA replicon vaccine for BTV-8, which is safe, efficacious, adaptable to emerging orbivirus infections , and compliant with the DIVA principle. The VP2, VP5, VP3 and VP7 genes encoding the BTV-8 capsid proteins, as well as the non-structural proteins NS1 and NS3 were inserted into a VSV vector genome lacking the essential VSV glycoprotein (G) gene. Infectious virus replicon particles (VRP) were produced on a transgenic helper cell line providing the VSV G protein in trans. Expression of antigens in vitro was analysed by immunofluorescence using monoclonal and polyclonal antibodies. In a pilot study, sheep were immunized with two different VRP-based vaccine candidates, one comprising the BTV-8 antigens VP2, VP5, VP3, VP7, NS1, and NS3, the other one containing antigens VP3, VP7, NS1, and NS3. Control animals received VRPs containing an irrelevant antigen. Virus neutralizing antibodies and protection after BTV-8 challenge were evaluated and compared to animals immunized with the conventionally inactivated vaccine. Full protection was induced only when the two antigens VP2 and VP5 were included in the vaccine. To further evaluate if VP2 alone, a combination of VP2 and VP5 or VP5 alone were necessary for complete protection, we performed a second animal trial. Interestingly, VP2 as well as the combination of VP2 and VP5 but not VP5 alone conferred full protection in terms of neutralizing antibodies, and protection from clinical signs and viremia after BTV-8 challenge. These results show that the VSV replicon system represents a safe, efficacious and DIVA-compliant vaccine against BTV as well as a possible platform for protection against other Orbiviruses, such as AHSV and EHDV.

Independent polled mutations leading to complex gene expression differences in cattle

The molecular regulation of horn growth in ruminants is still poorly understood. To investigate this process, we collected 1019 hornless (polled) animals from different cattle breeds. High-density SNP genotyping confirmed the presence of two different polled associated haplotypes in Simmental and Holstein cattle co-localized on BTA 1. We refined the critical region of the Simmental polled mutation to 212 kb and identified an overlapping region of 932 kb containing the Holstein polled mutation. Subsequently, whole genome sequencing of polled Simmental and Holstein cows was used to determine polled associated genomic variants. By genotyping larger cohorts of animals with known horn status we found a single perfectly associated insertion/deletion variant in Simmental and other beef cattle confirming the recently published possible Celtic polled mutation. We identified a total of 182 sequence variants as candidate mutations for polledness in Holstein cattle, including an 80 kb genomic duplication and three SNPs reported before. For the first time we showed that hornless cattle with scurs are obligate heterozygous for one of the polled mutations. This is in contrast to published complex inheritance models for the bovine scurs phenotype. Studying differential expression of the annotated genes and loci within the mapped region on BTA 1 revealed a locus (LOC100848215), known in cow and buffalo only, which is higher expressed in fetal tissue of wildtype horn buds compared to tissue of polled fetuses. This implicates that the presence of this long noncoding RNA is a prerequisite for horn bud formation. In addition, both transcripts associated with polledness in goat and sheep (FOXL2 and RXFP2), show an overexpression in horn buds confirming their importance during horn development in cattle.

Identification and characterization of IS1296 in Mycoplasma mycoides subsp. mycoides SC and presence in related mycoplasmas

IS1296, a new insertion sequence belonging to the IS3 family of insertion elements has been identified in Mycoplasma mycoides subsp. mycoides (Mmm) biotype small colony (SC), the agent of contagious bovine pleuropneumonia (CBPP). IS1296 is 1485-bp long and has 30-bp inverted repeats. It contains two open reading frames, ORFA and ORFB, which show significant similarities to the ORFs which encode the transposase function of IS elements of the IS3 family, in particular IS150 of Escherichia coli. IS1296 is present in 19 copies in Mmm SC-type strain PG1 and in 18 copies in a recently isolated field strain L2. It seems to transpose at low frequency in Mmm SC. IS1296 is also present in 5 copies in Mmm biotype large colony (LC)-type strain Y-goat, and in two copies in Mycoplasma sp. 'bovine group 7' reference strain PG50. It is, however, not present in other species of the 'mycoides cluster' or other closely related Mycoplasma sp. of ruminants.

Patho-genetics of Clostridium chauvoei

The genomic sequence of Clostridium chauvoei, the etiological agent of blackleg, a severe disease of ruminants with high mortality specified by a myonecrosis reveals a chromosome of 2.8 million base-pairs and a cryptic plasmid of 5.5 kilo base-pairs. The chromosome contains the main pathways like glycolysis/gluconeogenesis, sugar metabolism, purine and pyrimidine metabolisms, but the notable absence of genes of the citric acid cycle and deficient or partially deficient amino acid metabolism for Histidine, Tyrosine, Phenylalanine, and Tryptophan. These essential amino acids might be acquired from host tissue damage caused by various toxins and by protein metabolism that includes 57 genes for peptidases, and several ABC transporters for amino acids import.

Laboratory diagnosis of ruminant abortion in Europe.

Abortion in ruminants is a major cause of economic loss worldwide, and the management and control of outbreaks is important in limiting their spread, and in preventing zoonotic infections. Given that rapid and accurate laboratory diagnosis is central to controlling abortion outbreaks, the submission of tissue samples to laboratories offering the most appropriate tests is essential. Direct antigen and/or DNA detection methods are the currently preferred methods of reaching an aetiological diagnosis, and ideally these results are confirmed by the demonstration of corresponding macroscopic and/or histopathological lesions in the fetus and/or the placenta. However, the costs of laboratory examinations may be considerable and, even under optimal conditions, the percentage of aetiological diagnoses reached can be relatively low. This review focuses on the most commonly occurring and important abortifacient pathogens of ruminant species in Europe highlighting their epizootic and zoonotic potential. The performance characteristics of the various diagnostic methods used, including their specific advantages and limitations, are discussed.

Outbreak investigation identifies a single Listeria monocytogenes strain in sheep with different clinical manifestations, soil and water.

Listeria (L.) monocytogenes causes orally acquired infections and is of major importance in ruminants. Little is known about L. monocytogenes transmission between farm environment and ruminants. In order to determine potential sources of infection, we investigated the distribution of L. monocytogenes genetic subtypes in a sheep farm during a listeriosis outbreak by applying four subtyping methods (MALDI-TOF-MS, MLST, MLVA and PFGE). L. monocytogenes was isolated from a lamb with septicemia and from the brainstem of three sheep with encephalitis. Samples from the farm environment were screened for the presence of L. monocytogenes during the listeriosis outbreak, four weeks and eight months after. L. monocytogenes was found only in soil and water tank swabs during the outbreak. Four weeks later, following thorough cleaning of the barn, as well as eight months later, L. monocytogenes was absent in environmental samples. All environmental and clinical L. monocytogenes isolates were found to be the same strain. Our results show that the outbreak involving two different clinical syndromes was caused by a single L. monocytogenes strain and that soil and water tanks were potential infection sources during this outbreak. However, silage cannot be completely ruled out as the bales fed prior to the outbreak were not available for analysis. Faeces samples were negative, suggesting that sheep did not act as amplification hosts contributing to environmental contamination. In conclusion, farm management appears to be a crucial factor for the limitation of a listeriosis outbreak.

Evaluation of Swiss slaughterhouse data for integration in a syndromic surveillance system

BACKGROUND: We evaluated Swiss slaughterhouse data for integration in a national syndromic surveillance system for the early detection of emerging diseases in production animals. We analysed meat inspection data for cattle, pigs and small ruminants slaughtered between 2007 and 2012 (including emergency slaughters of sick/injured animals); investigating patterns in the number of animals slaughtered and condemned; the reasons invoked for whole carcass condemnations; reporting biases and regional effects. RESULTS: Whole carcass condemnation rates were fairly uniform (1-2‰) over time and between the different types of production animals. Condemnation rates were much higher and less uniform following emergency slaughters. The number of condemnations peaked in December for both cattle and pigs, a time when individuals of lower quality are sent to slaughter when hay and food are limited and when certain diseases are more prevalent. Each type of production animal was associated with a different profile of condemnation reasons. The most commonly reported one was "severe lesions" for cattle, "abscesses" for pigs and "pronounced weight loss" for small ruminants. These reasons could constitute valuable syndromic indicators as they are unspecific clinical manifestations of a large range of animal diseases (as well as potential indicators of animal welfare). Differences were detected in the rate of carcass condemnation between cantons and between large and small slaughterhouses. A large percentage (>60% for all three animal categories) of slaughterhouses operating never reported a condemnation between 2007 and 2012, a potential indicator of widespread non-reporting bias in our database. CONCLUSIONS: The current system offers simultaneous coverage of cattle, pigs and small ruminants for the whole of Switzerland; and traceability of each condemnation to its farm of origin. The number of condemnations was significantly linked to the number of slaughters, meaning that the former should be always be offset by the later in analyses. Because this denominator is only communicated at the end of the month, condemnations may currently only be monitored on a monthly basis. Coupled with the lack of timeliness (30-60 days delay between condemnation and notification), this limits the use of the data for early-detection.

Short communication: Timing of first milking affects serotonin (5-HT) concentrations

Hormonal signals differentially regulate the timing of parturition, as well lactogenesis and, potentially, colostrum formation in the mammary gland. Non-neuronal serotonin (5-HT) is a homeostatic regulator of the mammary gland. In the current study, we manipulated the timing of first milking to investigate its effects on serum 5-HT and calcium concentrations in the maternal and calf circulation, as well as in colostrum. Twenty-three cows were randomly assigned to a control (CON; n=10) group, milked for the first time at 4h postcalving, or a treatment (TRT; n=13) group, milked for the first time approximately 1 d before calving in addition to 4h postcalving. Maternal blood samples were collected for 4 d precalving, 3 times daily, and 1 blood sample was taken 4h postcalving. Calf blood samples were collected 4 (before first colostrum feeding) and 12h after birth, and at 3 wk of age. Calves from both treatments were fed colostrum from their respective mothers. Serum 5-HT concentrations were greater in CON cows and decreased significantly in TRT cows after milking was initiated precalving (951 vs. 524 ± 111 ng/mL, respectively). Cow serum calcium concentrations were affected by time, beginning to decrease 1 d precalving until 4h postcalving, but this drop in serum calcium was more pronounced in TRT cows. Serum 5-HT and calcium concentrations were negatively correlated (r=-0.57) for the CON cows and positively correlated (r=0.6) for the TRT cows. Maternal calcium and 5-HT decreased similarly due to precalving milking. Calcium and 5-HT concentrations were greater in colostrum collected from TRT cows milked precalving. Overall, calves had higher circulating 5-HT concentrations than cows, and calves born to TRT cows had increased 5-HT concentrations compared with the CON. Precalving milking could affect 5-HT synthesis within the mammary gland and therefore affect maternal 5-HT and calcium concentrations. Further research is needed in ruminants to assess the extent of 5-HT placental transfer, its role on pre- and postnatal development of the calf, the importance of its presence in colostrum, and potential long-term effects on calf health

Serosurveillance of Schmallenberg virus in Switzerland using bulk tank milk samples.

Infections with Schmallenberg virus (SBV), a novel Orthobunyavirus transmitted by biting midges, can cause abortions and malformations of newborns and severe symptoms in adults of domestic and wild ruminants. Understanding the temporal and spatial distribution of the virus in a certain territory is important for the control and prevention of the disease. In this study, seroprevalence of antibodies against SBV and the spatial spread of the virus was investigated in Swiss dairy cattle applying a milk serology technique on bulk milk samples. The seroprevalence in cattle herds was significantly higher in December 2012 (99.5%) compared to July 2012 (19.7%). This high between-herd seroprevalence in cattle herds was observed shortly after the first detection of viral infections. Milk samples originating from farms with seropositive animals taken in December 2012 (n=209; mean 160%) revealed significantly higher S/P% ratios than samples collected in July 2012 (n=48; mean 103.6%). This finding suggests a high within-herd seroprevalence in infected herds which makes testing of bulk tank milk samples for the identification farms with past exposures to SBV a sensitive method. It suggests also that within-herd transmission followed by seroconversion still occurred between July and December. In July 2012, positive bulk tank milk samples were mainly restricted to the western part of Switzerland whereas in December 2012, all samples except one were positive. A spatial analysis revealed a separation of regions with and without positive farms in July 2012 and no spatial clustering within the regions with positive farms. In contrast to the spatial dispersion of bluetongue virus, a virus that is also transmitted by Culicoides midges, in 2008 in Switzerland, the spread of SBV occurred from the western to the eastern part of the country. The dispersed incursion of SBV took place in the western part of Switzerland and the virus spread rapidly to the remaining territory. This spatial pattern is consistent with the hypothesis that transmission by Culicoides midges was the main way of spreading.

Vesicular stomatitis virus replicon expressing the VP2 outer capsid protein of bluetongue virus serotype 8 induces complete protection of sheep against challenge infection.

Bluetongue virus (BTV) is an arthropod-borne pathogen that causes an often fatal, hemorrhagic disease in ruminants. Different BTV serotypes occur throughout many temperate and tropical regions of the world. In 2006, BTV serotype 8 (BTV-8) emerged in Central and Northern Europe for the first time. Although this outbreak was eventually controlled using inactivated virus vaccines, the epidemic caused significant economic losses not only from the disease in livestock but also from trade restrictions. To date, BTV vaccines that allow simple serological discrimination of infected and vaccinated animals (DIVA) have not been approved for use in livestock. In this study, we generated recombinant RNA replicon particles based on single-cycle vesicular stomatitis virus (VSV) vectors. Immunization of sheep with infectious VSV replicon particles expressing the outer capsid VP2 protein of BTV-8 resulted in induction of BTV-8 serotype-specific neutralizing antibodies. After challenge with a virulent BTV-8 strain, the vaccinated animals neither developed signs of disease nor showed viremia. In contrast, immunization of sheep with recombinant VP5 - the second outer capsid protein of BTV - did not confer protection. Discrimination of infected from vaccinated animals was readily achieved using an ELISA for detection of antibodies against the VP7 antigen. These data indicate that VSV replicon particles potentially represent a safe and efficacious vaccine platform with which to control future outbreaks by BTV-8 or other serotypes, especially in previously non-endemic regions where discrimination between vaccinated and infected animals is crucial.

Pestiviruses.

Pestiviruses cause economically important diseases among domestic ruminants and pigs, but they may also infect a wide spectrum of wild species of even-toed ungulates (Artiodactyla). Bovine viral diarrhea virus (BVDV) and Border disease virus of sheep infect their hosts either transiently or persistently. Cellular and humoral immunotolerance to the infecting strain is a unique feature of persistent infection (PI) by ruminant pestiviruses. Persistence, caused by transplacental infection early in fetal development, depends on virally encoded interferon antagonists that inactivate the host's innate immune response to the virus without globally interfering with its function against other viruses. At epidemiological equilibrium, approximately 1-2% of animals are PI. Successful BVDV control programs show that removal of PI animals results in viral extinction in the host population. The nucleotide sequences of ruminant pestiviruses change little during persistent infection. Nevertheless, they display large heterogeneity, pointing to a long history of virus-host coevolution in which avirulent strains are more successful.

Babesia spp. in European wild ruminant species: parasite diversity and risk factors for infection.

Babesia are tick-borne parasites that are increasingly considered as a threat to animal and public health. We aimed to assess the role of European free-ranging wild ruminants as maintenance mammalian hosts for Babesia species and to determine risk factors for infection. EDTA blood was collected from 222 roe deer (Capreolus c. capreolus), 231 red deer (Cervus e. elaphus), 267 Alpine chamois (Rupicapra r. rupicapra) and 264 Alpine ibex (Capra i. ibex) from all over Switzerland and analysed by PCR with pan-Babesia primers targeting the 18S rRNA gene, primers specific for B. capreoli and Babesia sp. EU1, and by sequencing. Babesia species, including B. divergens, B. capreoli, Babesia sp. EU1, Babesia sp. CH1 and B. motasi, were detected in 10.7% of all samples. Five individuals were co-infected with two Babesia species. Infection with specific Babesia varied widely between host species. Cervidae were significantly more infected with Babesia spp. than Caprinae. Babesia capreoli and Babesia sp. EU1 were mostly found in roe deer (prevalences 17.1% and 7.7%, respectively) and B. divergens and Babesia sp. CH1 only in red deer. Factors significantly associated with infection were low altitude and young age. Identification of Babesia sp. CH1 in red deer, co-infection with multiple Babesia species and infection of wild Caprinae with B. motasi and Babesia sp. EU1 are novel findings. We propose wild Caprinae as spillover or accidental hosts for Babesia species but wild Cervidae as mammalian reservoir hosts for B. capreoli, possibly Babesia sp. EU1 and Babesia sp. CH1, whereas their role regarding B. divergens is more elusive.

Impact de la fabrication de charbon de bois sur la production et la disponibilité fourragère de pâturage en zone subaride, cas de la commune de Soalara-Sud, Toliara ll: Rapport final

Ce projet a pour objectif de (1) décrire la filière charbon dans un terroir de la commune de Soalara, (2) d’identifier les effets des activités de fabrication de charbon de bois sur la végétation des pâturages qui est un fourré épineux et (3) d’estimer la quantité maximale de charbon pouvant être produite sans causer sa dégradation irréversible. Pour ce faire, des enquêtes auprès de la population locale et des relevés écologiques ont été entrepris. La majorité des ménages enquêtés (>80% ; N=56) pratiquent la fabrication de charbon de bois. Environ 41 000 sacs de charbons ont été produits dans le terroir en 2009. L’impact socio-économique de la filière charbon est positif puisqu’elle génère des revenus importants pour ses agents (producteur, transporteur et collecteur). Par contre, l’impact écologique est plutôt négatif puisque les activités charbonnières contribuent à réduire la densité d’arbres et d’arbustes de la végétation des fourrés épineux et sa richesse spécifique même si elles n’affectent pas trop sa disponibilité fourragère. De plus, la quantité de charbon produite (environ 820 t en 2009) dépasse la productivité de la végétation, estimée à 218 t.an-1. Par conséquent, une dégradation du site de production actuel, se traduisant par la disparition des espèces charbonnières, arriverait dans 17 ans au plus tard, si le rythme de production actuel continue. Des mesures de réduction des pressions sur la végétation (professionnalisation des éleveurs, amélioration des rendements de carbonisation et réglementation et contrôle plus strict de la filière charbon) et d’augmentation de sa production et productivité (restauration) doivent être prises pour éviter cette dégradation irréversible qui à terme pourrait être dommageable à l’élevage de petits ruminants même si ce n’est pas encore le cas actuellement.

Listeria monocytogenes spreads within the brain by actin-based intra-axonal migration

Listeria monocytogenes rhombencephalitis is a severe progressive disease despite a swift intrathecal immune response. Based on previous observations, we hypothesized that the disease progresses by intra-axonal spread within the central nervous system. To test this hypothesis, neuroanatomical mapping of lesions, immunofluorescence analysis, and electron microscopy were performed on brains of ruminants with naturally occurring rhombencephalitis. In addition, infection assays were performed in bovine brain cell cultures. Mapping of lesions revealed a consistent pattern with a preferential affection of certain nuclear areas and white matter tracts, indicating that Listeria monocytogenes spreads intra-axonally within the brain along interneuronal connections. These results were supported by immunofluorescence and ultrastructural data localizing Listeria monocytogenes inside axons and dendrites associated with networks of fibrillary structures consistent with actin tails. In vitro infection assays confirmed that bacteria were moving within axon-like processes by employing their actin tail machinery. Remarkably, in vivo, neutrophils invaded the axonal space and the axon itself, apparently by moving between split myelin lamellae of intact myelin sheaths. This intra-axonal invasion of neutrophils was associated with various stages of axonal degeneration and bacterial phagocytosis. Paradoxically, the ensuing adaxonal microabscesses appeared to provide new bacterial replication sites, thus supporting further bacterial spread. In conclusion, intra-axonal bacterial migration and possibly also the innate immune response play an important role in the intracerebral spread of the agent and hence the progression of listeric rhombencephalitis.