Roser Velarde

Border Disease Virus among Chamois, Spain

Approximately 3,000 Pyrenean chamois (Rupicapra pyrenaica pyrenaica) died in northeastern Spain during 2005-2007. Border disease virus infection was identified by reverse transcription-PCR and sequencing analysis. These results implicate this virus as the primary cause of death, similar to findings in the previous epizootic in 2001.

Schmallenberg Virus Circulation in High Mountain Ecosystem, Spain

To the Editor: Schmallenberg virus (SBV) is an emerging vector-borne virus mainly associated with Culicoides spp. midges (1,2). Factors affecting the density and distribution of vectors may help determine the prevalence of SBV infection in particular areas. Altitude could be one limiting factor for virus transmission; however, little information is available regarding SBV in high-altitude regions. During December 29, 2012–February 21, 2013, morphologic anomalies were identified in 4 stillborn calves from different farms in northeastern Spain, and infection with SBV was suspected. The cases were clustered in the Ripolles and Garrotxa regions of Catalonia and appeared in beef cattle herds that spent the grazing season (May–November) in the alpine meadows (>2,000 m altitude) of the National Game Reserve of Freser-Setcases in the Eastern Pyrenees Mountains. The calves had severe arthrogryposis, ankylosis of several joints, abnormal curvature of the vertebral column, and severe muscle atrophy. Malformations of the central nervous system included bilateral hydrocephalus, cerebellar hypoplasia, and micromyelia, characterized by the presence of few neurons in the ventral horns and moderate to severe bilateral reduction of white matter in the ventral and lateral funiculi. SBV infection was confirmed by real-time reverse transcription qualitative PCR (RT-qPCR) (1,3) or serologic testing in 3 of the 4 calves and all 4 of the mothers (Table). Serum samples were tested by using a commercial indirect ELISA (ID.vet; Innovative Diagnostics, Montpellier, France) and a virus neutralization test using the BH80/11–4 isolate (provided by the Friedrich-Loeffler-Institut, Isle of Riems, Germany) (4). Consistent results were obtained from both of these techniques, and the proportions of calves positive by ELISA and RT-qPCR were similar to those found in previous studies (5). Table Results of serologic and molecular analyses of SBV in sympatric wild and domestic ruminants, Eastern Pyrenees, Spain, 2010–2013* The neurologic and musculoskeletal lesions found in the calves indicated that fetal infection probably occurred at 5–6 months’ gestation (6). Gestation started in mid-April to mid-May; therefore, maternal infection most probably occurred in late summer 2012 (September–October), when cows were grazing in the alpine meadows. We then performed a serologic study in domestic and sympatric wild ruminants from the National Game Reserve of Freser-Setcases, which comprises 20,200 ha of alpine and subalpine ecosystems. We analyzed serum samples from 355 wild ruminants hunted during August 2010–May 2013; species sampled included Pyrenean chamois (Rupicapra pyrenaica), European mouflon (Ovis aries musimon), and roe deer (Capreolus capreolus). We also analyzed samples from fetuses of these species obtained in April 2013 (Table), as well as animals from 8 cow herds and 4 sheep–goat mixed herds; a mean of 14 samples were collected per herd during 2 sampling periods (Table). Two of the mixed sheep–goat herds were sampled during both sampling periods. All serum samples underwent ELISA testing; positive results were confirmed by virus neutralization (4). Domestic ruminants sampled during October–November 2011 were seronegative, whereas all farms sampled during November 2012–April 2013 had infected animals (Table). High mean seroprevalence was found in cow herds; 105 (86.8% [95% CI 80.7%–92.8%]) of 121 herds tested were infected. Seroprevalance was lower but still high for mixed sheep–goat herds; 16 (41% [95% CI 25.6%–56.5%]) of 39 herds were infected. The earliest evidence of SBV in the study area came from a seropositive Pyrenean chamois hunted on September 3, 2012; this date coincides with the estimated months when cows that delivered stillborn calves were infected. For wild ungulates tested from September 2012 onwards, overall SBV seroprevalence was statistically higher (χ2 33.47, 2 d.f., p<0.0001) in roe deer (4/5, 80% [95% CI 44.9%–100%]) than in Pyrenean chamois (8/105, 7.6% [95% CI 2.5%–12.7%]) and mouflon (0/23). Differences in seroprevalence for summer through autumn 2012 compared with spring 2013 in Pyrenean chamois were not significant (Table). Roe deer seroprevalence was similar to the 88.9% reported in Belgium in December 2011, which contrasted with the lower seroprevalence observed in red deer, 54.6%, for the same month in the same study (7). Differences in seroprevalence between wild host species might be related to differences in exposure to SBV vectors depending on habitat selection, vector feeding habits, or host-specific factors; altitude might be an additional factor affecting exposure (8). Thus, the lower altitude habitat selection of roe deer and the housing of domestic ruminants in valley areas could explain the higher seroprevalence observed in these species compared with that in Pyrenean chamois and mouflon. All fetuses of wild ruminants had negative serologic test results for SBV, and no gross lesions indicating infection were observed (Table). However, the potential reproductive disorders that SBV infection can cause in these species are unknown. Our findings support the hypothesis that SBV can circulate in alpine meadows at >2,000 m altitude and confirm the appearance of SBV in late summer and autumn 2012 in the high mountain ecosystem of the Eastern Pyrenees in Spain. A variety of domestic and wild ruminants showed susceptibility to SBV infection, but differences in seroprevalence suggest different roles for sympatric ruminants in SBV epidemiology. The role of vector species in the transmission of SBV in alpine ecosystems should be analyzed.

Spillover Events of Infection of Brown Hares (Lepus europaeus) with Rabbit Haemorrhagic Disease Type 2 Virus (RHDV2) Caused Sporadic Cases of an European Brown Hare Syndrome-Like Disease in Italy and Spain

Summary Rabbit haemorrhagic disease virus (RHDV) is a lagovirus that can cause fatal hepatitis (rabbit haemorrhagic disease, RHD) with mortality of 80–90% in farmed and wild rabbits. Since 1986, RHDV has caused outbreaks in rabbits (Oryctolagus cuniculus) in Europe, but never in European brown hares (Lepus europaeus, EBH). In 2010, a new RHDV‐related virus, called RHDV2, emerged in Europe, causing extended epidemics because it largely overcame the immunity to RHDV present in most rabbit populations. RHDV2 also was identified in Cape hare (Lepus capensis subsp. mediterraneus) and in Italian hare (Lepus corsicanus). Here, we describe two distinct incidents of RHDV2 infection in EBH that occurred in Italy (2012) and Spain (2014). The two RHDV2 strains caused macroscopic and microscopic lesions similar to European brown hare syndrome (EBHS) in hares, and they were genetically related to other RHDV2 strains in Europe. EBHs are common in Europe, often sharing habitat with rabbits. They likely have been exposed to high levels of RHDV2 during outbreaks in rabbits in recent years, yet only two incidents of RHDV2 in EBHs have been found in Italy and Spain, suggesting that EBHs are not a primary host. Instead, they may act as spillover hosts in situations when infection pressure is high and barriers between rabbits and hares are limited, resulting in occasional infections causing EBHS‐like lesions. The serological survey of stocked hare sera taken from Italian and Spanish hare populations provided an understanding of naturally occurring RHDV2 infection in the field confirming its sporadic occurrence in EBH. Our findings increase the knowledge on distribution, host range and epidemiology of RHDV2.

Experimental infection with chamois border disease virus causes long-lasting viraemia and disease in Pyrenean chamois (Rupicapra pyrenaica)

Since 2001, severe outbreaks of disease associated with border disease virus (BDV) infection have been reported in Pyrenean chamois. The disease is characterized by variable degrees of cachexia, alopecia and neurological manifestations prior to death. The aim of this study was to investigate this disease under experimental conditions. To assess viral virulence, humoral immune response, dissemination and probable routes of transmission, seven chamois (five seronegative and two seropositive for BDV) were inoculated with a BDV isolated from a naturally infected chamois. A group of three chamois were maintained as uninfected controls. The five seronegative chamois became viraemic from day 2 post-inoculation (p.i.) until their death (three animals) or the end of the experiment (on day 34 p.i.) and developed neutralizing antibodies from day 18 p.i. until the end of the study. Continuous shedding of the virus was detected by RT-PCR in oral, nasal and rectal swabs in viraemic chamois from day 5 p.i. Despite none of the viraemic chamois showing obvious neurological signs, all of them had a non-suppurative meningoencephalitis as seen in naturally infected chamois. The two inoculated BDV-seropositive chamois did not become viraemic. This study confirms that BDV is the primary agent of the disease that has been affecting chamois populations in recent years in the Pyrenees and that previously acquired humoral immunity is protective.

Long-term assessment of wild boar harvesting and cattle removal for bovine tuberculosis control in free ranging populations

Wild boar is a recognized reservoir of bovine tuberculosis (TB) in the Mediterranean ecosystems, but information is scarce outside of hotspots in southern Spain. We describe the first high-prevalence focus of TB in a non-managed wild boar population in northern Spain and the result of eight years of TB management. Measures implemented for disease control included the control of the local wild boar population through culling and stamping out of a sympatric infected cattle herd. Post-mortem inspection for detection of tuberculosis-like lesions as well as cultures from selected head and cervical lymph nodes was done in 745 wild boar, 355 Iberian ibexes and five cattle between 2004 and 2012. The seasonal prevalence of TB reached 70% amongst adult wild boar and ten different spoligotypes and 13 MIRU-VNTR profiles were detected, although more than half of the isolates were included in the same clonal complex. Only 11% of infected boars had generalized lesions. None of the ibexes were affected, supporting their irrelevance in the epidemiology of TB. An infected cattle herd grazed the zone where 168 of the 197 infected boars were harvested. Cattle removal and wild boar culling together contributed to a decrease in TB prevalence. The need for holistic, sustained over time, intensive and adapted TB control strategies taking into account the multi-host nature of the disease is highlighted. The potential risk for tuberculosis emergence in wildlife scenarios where the risk is assumed to be low should be addressed.

Severity of bovine tuberculosis is associated with co-infection with common pathogens in wild boar

Co-infections with parasites or viruses drive tuberculosis dynamics in humans, but little is known about their effects in other non-human hosts. This work aims to investigate the relationship between Mycobacterium bovis infection and other pathogens in wild boar (Sus scrofa), a recognized reservoir of bovine tuberculosis (bTB) in Mediterranean ecosystems. For this purpose, it has been assessed whether contacts with common concomitant pathogens are associated with the development of severe bTB lesions in 165 wild boar from mid-western Spain. The presence of bTB lesions affecting only one anatomic location (cervical lymph nodes), or more severe patterns affecting more than one location (mainly cervical lymph nodes and lungs), was assessed in infected animals. In addition, the existence of contacts with other pathogens such as porcine circovirus type 2 (PCV2), Aujeszkys disease virus (ADV), swine influenza virus, porcine reproductive and respiratory syndrome virus, Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Haemophilus parasuis and Metastrongylus spp, was evaluated by means of serological, microbiological and parasitological techniques. The existence of contacts with a structured community of pathogens in wild boar infected by M. bovis was statistically investigated by null models. Association between this community of pathogens and bTB severity was examined using a Partial Least Squares regression approach. Results showed that adult wild boar infected by M. bovis had contacted with some specific, non-random pathogen combinations. Contact with PCV2, ADV and infection by Metastrongylus spp, was positively correlated to tuberculosis severity. Therefore, measures against these concomitant pathogens such as vaccination or deworming, might be useful in tuberculosis control programmes in the wild boar. However, given the unexpected consequences of altering any community of organisms, further research should evaluate the impact of such measures under controlled conditions. Furthermore, more research including other important pathogens, such as gastro-intestinal nematodes, will be necessary to complete this picture.

Abwesenheit von Haematozoa bei einer Brutpopulation des SeeregenpfeifersCharadrius alexandrinus in Nordspanien

A low presence of haematophagus parasites has been reported in arctic waders. Three hypotheses try to explain these low levels: higher inherent resistance to parasites in the Charadrii, lower presence of parasites in the breeding grounds of waders in the arctic, and influence of the vegetation structure in the habitat of these birds. To test these hypotheses, we took blood samples from 87 alive-trapped Kentish PloversCharadrius alexandrinus nesting in Northeast Spain. None of the samples had haematophagus parasites. Therefore, very low infection rates occur also in a non-arctic species. These data support the hypotheses that Charadrii have a higher resistance or a lower probability of being infected by blood parasites than other taxa, independently of their breeding distribution.

Border Disease Virus: An Exceptional Driver of Chamois Populations Among Other Threats

Though it is accepted that emerging infectious diseases are a threat to planet biodiversity, little information exists about their role as drivers of species extinction. Populations are also affected by natural catastrophes and other pathogens, making it difficult to estimate the particular impact of emerging infectious diseases. Border disease virus genogroup 4 (BDV-4) caused a previously unreported decrease in populations of Pyrenean chamois (Rupicapra pyrenaica pyrenaica) in Spain. Using a population viability analysis, we compared probabilities of extinction of a virtual chamois population affected by winter conditions, density dependence, keratoconjunctivitis, sarcoptic mange, and BD outbreaks. BD-affected populations showed double risk of becoming extinct in 50 years, confirming the exceptional ability of this virus to drive chamois populations.

Presumptive Babesia ovis infection in a spanish ibex (Capra pyrenaica).

On December 29 1995, a 13-year old, male Spanish ibex was easily captured by hand, with depression, weakness and severe tick infestation, mainly in the periocular and auricular regions. Blood and serum samples were collected and haematological analysis and serum iron levels were determined. Red blood cell count, haematocrit, haemoglobin concentration and mean corpuscular haemoglobin concentration (MCHC) were decreased and mean corpuscular volume (MCV) increased (macrocytic-hypochromic anemia). Serum iron and transferrin saturation were decreased and total and unbound iron-binding capacity were increased. Piroplasms were observed within parasitized erythrocytes and presumptively identified as Babesia spp. Ticks were identified exclusively as Ripicephalus bursa. The animal was treated with imidocarb but died after 15 days of capture. Histopathological examination revealed congestion of pulmonary capillaries and spleen, glomerulonephritis, hemoglobinuric nephrosis and generalized hemosiderosis. An indirect fluorescent antibody test was performed using a Babesia ovis isolate of ovine origin as antigen and the animal was positive with a titre of 1:640.

Uses and limitations of faecal egg count for assessing worm burden in wild boars.

The most widely used technique to assess helminth infection in both domestic and wild mammals is the faecal egg count (FEC). Most efforts to test the reliability of FEC as a proxy for parasite load are in small ruminant studies and limited work has evaluated the use of FEC in pigs. The aim of this study was to explore whether FEC is a reliable indicator of helminth load, and to evaluate the effects of sample storage on FEC accuracy in 59 wild boars. Though FEC was useful for assessing most helminth infections (e.g., Metastrongylus spp., Ascaris suum, Trichuris suis), stomach nematodes were often missed. The accuracy of FEC decreased over time, and thus it is recommended that samples be processed within 5 days of collection.