Eve Laloy

Schmallenberg virus experimental infection of sheep.

Since late 2011, a novel orthobunyavirus, named Schmallenberg virus (SBV), has been implicated in many cases of severely malformed bovine and ovine offspring in Europe. In adult cattle, SBV is known to cause a mild transient disease; clinical signs include short febrile episodes, decreased milk production and diarrhoea for a few days. However, the knowledge about clinical signs and pathogenesis in adult sheep is limited. In the present study, adult sheep of European domestic breeds were inoculated with SBV either as cell culture grown virus or as virus with no history of passage in cell cultures. Various experimental set-ups were used. Sampling included blood collection at different time points during the experimental period and selected organ material at autopsy. Data from this study showed, that the RNAemic period in sheep was as short as reported for cattle; viral genome was detectable for about 3-5 days by real-time RT-PCR. In total, 13 out of 30 inoculated sheep became RNAemic, with the highest viral load in animals inoculated with virus from low cell culture passaged or the animal passaged material. Contact animals remained negative throughout the study. One RNAemic sheep showed diarrhoea for several days, but fever was not recorded in any of the animals. Antibodies were first detectable 10-14 days post inoculation. Viral RNA was detectable in spleen and lymph nodes up to day 44 post inoculation. In conclusion, as described for cattle, SBV-infection in adult sheep predominantly results in subclinical infection, transient RNAemia and a specific antibody response. Maintenance of viral RNA in the lymphoreticular system is observed for an extended period.

Schmallenberg Virus Infection among Red Deer, France, 2010–2012

Schmallenberg virus infection is emerging in European domestic and wild ruminants. We investigated the serologic status of 9 red deer populations to describe virus spread from September 2010 through March 2012 among wildlife in France. Deer in 7 populations exhibited seropositivity, with an average seroprevalence of 20%.

Dual Emergence of Usutu Virus in Common Blackbirds, Eastern France, 2015.

References 1. Yokota S, Sato T, Okubo T, Ohkoshi Y, Okabayashi T, Kuwahara O, et al. Prevalence of fluoroquinolone-resistant Escherichia coli O25:H4-ST131 (CTX-M-15-nonproducing) strains isolated in Japan. Chemotherapy. 2012;58:52–9. http://dx.doi.org/10.1159/000336129 2. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing (M100-S25). Wayne (PA): The Institute; 2015. 3. Colpan A, Johnston B, Porter S, Clabots C, Anway R, Thao L, et al.; VICTORY (Veterans Influence of Clonal Types on Resistance: Year 2011) Investigators. Escherichia coli sequence type 131 (ST131) subclone H30 as an emergent multidrug-resistant pathogen among US veterans. Clin Infect Dis. 2013;57:1256–65. http://dx.doi.org/10.1093/cid/cit503 4. Price LB, Johnson JR, Aziz M, Clabots C, Johnston B, Tchesnokova V, et al. The epidemic of extended-spectrumβ-lactamase-producing Escherichia coli ST131 is driven by a single highly pathogenic subclone, H30-Rx. mBio. 2013;4: e00377–13. http://dx.doi.org/10.1128/mBio.00377-13 5. Lau SH, Reddy S, Cheesbrough J, Bolton FJ, Willshaw G, Cheasty T, et al. Major uropathogenic Escherichia coli strain isolated in the northwest of England identified by multilocus sequence typing. J Clin Microbiol. 2008;46:1076–80. http://dx.doi.org/10.1128/JCM.02065-07 6. Peirano G, Schreckenberger PC, Pitout JD. Characteristics of NDM-1-producing Escherichia coli isolates that belong to the successful and virulent clone ST131. Antimicrob Agents Chemother. 2011;55:2986–8. http://dx.doi.org/10.1128/AAC.01763-10 7. Morris D, Boyle F, Ludden C, Condon I, Hale J, O’Connell N, et al. Production of KPC-2 carbapenemase by an Escherichia coli clinical isolate belonging to the international ST131 clone. Antimicrob Agents Chemother. 2011;55:4935–6. http://dx.doi.org/10.1128/AAC.05127-11 8. Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, Spencer J, et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis. 2016;16:161–8. http://dx.doi.org/10.1016/S1473-3099(15)00424-7 9. Xavier BB, Lammens C, Ruhal R, Kumar-Singh S, Butaye P, Goossens H, et al. Identification of a novel plasmid-mediated colistin-resistance gene, mcr-2, in Escherichia coli, Belgium, 2016. Euro Surveill. 2016;21. http://dx.doi.org/10.2807/1560-7917. ES.2016.21.2730280

Comparison of the Neuropathology Induced by Two West Nile Virus Strains

Some strains of West Nile virus (WNV) are neuroinvasive and may induce fatal encephalitis/meningitis in a variety of animal species including humans. Whether, however, there is a strain-specific signature in the brain is as yet unknown. Here we investigated the neuropathogenesis induced by two phylogenetically distant WNV strains of lineage 1, WNVIS98 and WNVKUN35 911. While four-week old C57Bl/6J mice were susceptible to both strains and succumbed rapidly after intraperitoneal inoculation, differences were observed in virulence and clinical disease. WNVKUN35 911, the less virulent strain as judged by determination of LD50, induced typical signs of encephalitis. Such signs were not observed in WNVIS98-infected mice, although they died more rapidly. Histological examination of brain sections also revealed differences, as the level of apoptosis and inflammation was higher in WNVKUN35 911- than WNVIS98-infected mice. Moreover, staining for cleaved caspase 3 showed that the two WNV strains induced apoptotic death through different molecular mechanisms in one particular brain area. Finally, the two strains showed similar tropism in cortex, striatum, brainstem, and cerebellum but a different one in hippocampus. In summary, our data show that, upon peripheral administration, WNVIS98 and WNVKUN35 911 strains induce partially distinct lesions and tissue tropism in the brain. They suggest that the virulence of a WNV strain is not necessarily correlated with the severity of apoptotic and inflammatory lesions in the brain.

Schmallenberg virus infection in dogs, France, 2012.

To the Editor: In 2011, Schmallenberg virus (SBV) emerged in Europe (1); the virus spread into France in January 2012 (2). During January–March 2012, a total of >1,000 cases were reported in France, mainly in stillborn and newborn lambs with congenital malformations. n nIn March 2012, neurologic disorders were detected in five 15-day-old puppies (Belgian shepherd) from a dog breeding kennel in northwestern France (Orne). We report data suggesting that these puppies were infected with SBV. n nIn June 2012, the kennel veterinarian contacted a veterinary school (Unite de Medecine de l’Elevage et du Sport Breeding and Sport Medicine Unit, Maisons-Alfort, France) after neurologic signs of ataxia, exotropia, a head tilt, and stunted growth were observed in a litter of 5 puppies. Four of the puppies had died at 5–6 weeks of age. The veterinarian collected blood samples from the surviving puppy at 3 months of age, and the puppy was euthanized for necropsy. Severe torticollis was observed during the necropsy, but no other macroscopic signs were detected. The brain, including the cerebellum; a part of the spine; and cerebrospinal fluid (CSF) were collected for further investigation. Specific PCR analyses for canine coronavirus, Neospora caninum, Toxoplasma gondii, and canine minute virus were performed on CSF; all test results were negative. The brain tissue was fixed in formalin and processed for histologic examination. Features of degenerative encephalopathy, including neuronal vacuolation, neuropil vacuolation, and minimal gliosis, were observed. n nBecause some clinical signs were evocative of SBV infection and the puppy was born in an area where the virus was circulating actively in cattle and sheep, veterinarians decided to investigate SBV as a possible etiology. Serum samples from the 3-month-old puppy and the dam were tested by virus neutralization test (VNT), according to the protocol used for ruminant serum. The results were negative for the puppy but positive (titer 128) for the mother. Specific competitive SBV ELISA (IDVet, Montpellier, France) against the SBV N protein showed similar results. n nReal-time reverse transcription PCR (RT-PCR) was performed (3) to detect the presence of the SBV genome in the cerebellum. Because the sample was paraffin-embedded, RNA was extracted from 5-µm sections, as described (4). All of the extracted cerebellum sections had positive test results (cycle threshold range 33−36); the extraction and PCR controls all showed negative results. To confirm these positive results, conventional RT-PCR was used to amplify a 573-nt sequence of the SBV S segment. The amplification product was sequenced, and a BLAST analysis was performed (www.ncbi.nlm.nih.gov/BLAST). An identity of 100% was obtained with the SBV small gene segment from a ruminant (GenBank accession no. {type:entrez-nucleotide,attrs:{text:KC108860,term_id:443498566,term_text:KC108860}}KC108860). An immunohistochemical assay was also performed; the result was negative. n nThe remaining 7 female dogs in the breeding kennel were tested for SBV in October 2012; 1 showed positive test results by VNT (titer 256), which confirmed that SBV was circulating in the kennel. This positive dam had a litter of puppies in December 2012, but no signs developed, and the puppies were not tested. In March 2013, repeat testing was done on serum samples from the 2 dogs that had shown positive results. Results for both animals were positive by VNT (titers 32 for the dam and 128 for the other dog) and ELISA. n nTaken together, specific SBV antibodies in the mother and the SBV genome in her puppy suggest that these dogs experienced SBV infection. The absence of detectable SBV antibodies in the puppy in this investigation suggests that transplacental infection occurred before the onset of fetal immune competence. Maternal infection probably occurred in January or February 2012; entomologic monitoring conducted in France showed the presence of Culicoides spp. midges, a vector of SBV, during this period in northwestern France. In addition, because the puppies were born in March 2012 and SBV antibodies were still detectable in the mother in March 2013, the duration of SBV antibodies in dogs appears to be >1 year. In cattle and sheep, the SBV genome persists in an infected fetus and is detectable after birth by real-time RT-PCR, despite gestation length (5,6). n nFew reports on orthobunyavirus infections in dogs are available. Two serologic studies from the United States (7) and Mexico (8) found antibodies against La Crosse virus, South River virus, and Jamestown Canyon virus in dogs. Two other reports described cases in which La Crosse virus was detected in canine littermates who had clinical encephalitis (9) or neurologic disorders (10). n nIt is unclear if the apparent SBV infection we detected in these dogs was an isolated event or if other cases occurred elsewhere but were not detected because they were not investigated. Further serologic and clinical surveys are needed to estimate SBV prevalence in dogs and the virus’ involvement in the occurrence of neurologic signs in puppies.

Schmallenberg Virus in Zoo Ruminants, France and the Netherlands.

1. Regional action plan for the conservation of western lowland gorillas and central chimpanzees, 2015–2025 [cited 2016 Aug 2]. http://static1.1.sqspcdn.com/static/f/1200343/25932483/1423326166303/ WEA_apes_plan_2014 2. Bermejo M, Rodríguez-Teijeiro JD, Illera G, Barroso A, Vilà C, Walsh PD. Ebola outbreak killed 5,000 gorillas. Science. 2006;314:1564. http://dx.doi.org/10.1126/science.1133105 3. Nagel M, Dischinger J, Türck M, Verrier D, Oedenkoven M, Ngoubangoye B, et al. Human-associated Staphylococcus aureus strains within great ape populations in central Africa (Gabon). Clin Microbiol Infect. 2013;19:1072–7. http://dx.doi.org/10.1111/14690691.12119 4. Liu W, Li Y, Learn GH, Rudicell RS, Robertson JD, Keele BF, et al. Origin of the human malaria parasite Plasmodium falciparum in gorillas. Nature. 2010;467:420–5. http://dx.doi.org/10.1038/ nature09442 5. Ochman H, Worobey M, Kuo C-H, Ndjango J-B, Peeters M, Hahn BH, et al. Evolutionary relationships of wild hominids recapitulated by gut microbial communities. PLoS Biol. 2010;8:e1000546. http://dx.doi.org/10.1371/journal.pbio.1000546 6. Subtil A, Collingro A, Horn M. Tracing the primordial Chlamydiae: extinct parasites of plants? Trends Plant Sci. 2014;19:36–43. http://dx.doi.org/10.1016/j.tplants.2013.10.005 7. Ehricht R, Slickers P, Goellner S, Hotzel H, Sachse K. Optimized DNA microarray assay allows detection and genotyping of single PCR-amplifiable target copies. Mol Cell Probes. 2006;20:60–3. http://dx.doi.org/10.1016/j.mcp.2005.09.003 8. Lienard J, Croxatto A, Aeby S, Jaton K, Posfay-Barbe K, Gervaix A, et al. Development of a new Chlamydiales-specific real-time PCR and its application to respiratory clinical samples. J Clin Microbiol. 2011;49:2637–42. http://dx.doi.org/10.1128/ JCM.00114-11 9. Baud D, Thomas V, Arafa A, Regan L, Greub G. Waddlia chondrophila, a potential agent of human fetal death. Emerg Infect Dis. 2007;13:1239–43. http://dx.doi.org/10.3201/ eid1308.070315 10. Baud D, Goy G, Osterheld M-C, Croxatto A, Borel N, Vial Y, et al. Role of Waddlia chondrophila placental infection in miscarriage. Emerg Infect Dis. 2014;20:460–4. http://dx.doi.org/10.3201/eid2003.131019

Validation of a new experimental model for assessing drug efficacy against infection with Trypanosoma equiperdum in horses

Trypanosoma equiperdum, the causative agent of dourine, may affect the central nervous system, leading to neurological signs in infected horses. This location protects the parasite from most (if not all) existing chemotherapies. In this context, the OIE terrestrial code considers dourine as a non-treatable disease and imposes a stamping-out policy for affected animals before a country may achieve its dourine-free status. The use of practices as drastic as euthanasia remains controversial, but the lack of a suitable tool for studying a treatments efficacy against dourine hampers the development of an alternative strategy for dourine infection management. The present study reports on the development of an experimental infection model for assessing drug efficacy against the nervous form of dourine. The model combines the infection of horses by Trypanosoma equiperdum and the search for trypanosomes in the cerebrospinal fluid (CSF) through an ultrasound-guided cervical sampling protocol. After a development phase involving four horses, we established an infection model that consists of inoculating 5u2009×u2009104T. equiperdum OVI parasites intravenously into adult Welsh mares (Equus caballus). To evaluate its efficacy, eight horses were infected according to this model. In all these animals, parasites were observed in the blood at 2 days post-inoculation (p.i.) and in CSF (12.5u2009±u20091.6 days p.i.) and seroconversion was detected (8.25u2009±u20090.5 days p.i.). All eight animals also developed fever (rectal temperature > 39u2009°C), low hematocrit (< 27%), and ventral edema (7.9u2009±u20092.0 days p.i.), together with other inconstant clinical signs such as edema of the vulva (six out of eight horses) or cutaneous plaques (three out of eight horses). This model provides a robust infection protocol that induces an acute trypanosome infection and that allows parasites to be detected in the CSF of infected horses within a period of time compatible with animal experimentation constraints. We conclude that this model constitutes a suitable tool for analyzing the efficacy of anti-Trypanosoma drugs and vaccines.

Safe and cost-effective protocol for shipment of samples from Foot-and-Mouth Disease suspected cases for laboratory diagnostic

An essential step towards the global control and eradication of foot-and-mouth disease (FMD) is the identification of circulating virus strains in endemic regions to implement adequate outbreak control measures. However, due to the high biological risk and the requirement for biological samples to be shipped frozen, the cost of shipping samples becomes one of major obstacles hindering submission of suspected samples to reference laboratories for virus identification. In this study, we report the development of a cost-effective and safe method for shipment of FMD samples. The protocol is based on the inactivation of FMD virus (FMDV) on lateral flow device (LFD, penside test routinely used in the field for rapid immunodetection of FMDV), allowing its subsequent detection and typing by RT-PCR and recovery of live virus upon RNA transfection into permissive cells. After live FMDV collection onto LFD strip and soaking in 0.2% citric acid solution, the virus is totally inactivated. Viral RNA is still detectable by real-time RT-PCR following inactivation, and the virus strain can be characterized by sequencing of the VP1 coding region. In addition, live virus can be rescued by transfecting RNA extract from treated LFD into cells. This protocol should help promoting submission of FMD suspected samples to reference laboratories (by reducing the cost of sample shipping) and thus characterization of FMDV strains circulating in endemic regions.