Jorge Ramón López-Olvera

EFFECTS OF ACEPROMAZINE ON CAPTURE STRESS IN ROE DEER (CAPREOLUS CAPREOLUS)

The aim of this study was to evaluate effect of a short-acting neuroleptic (acepromazine) on capture stress response in roe deer (Capreolus capreolus). Sixteen roe deer were captured by drive-nets in the winters of 1998, 1999, and 2001. Roe deer were divided into two groups: animals in the treatment group received an intramuscular injection of acepromazine (0.093 mg/kg±0.003 SEM; n=8) while animals in the control group (n=8) did not receive tranquilizer. Heart rate and body temperature, as well as hematologic and biochemical indicators of stress, were used to evaluate effect of the neuroleptic over 3 hr. Heart rate decreased over time after capture in both groups (P<0.05), but stabilized sooner in the treated roe deer (75 min after capture) than in the controls (105 min after capture). Body temperature decreased over 45 min and then stabilized in both groups (P<0.05). Comparisons of blood parameters revealed significantly lower red blood cell count (RBC), lymphocyte count, hemoglobin concentration, packed cell volume (PCV), and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), and lactate dehydrogenase (LDH) activities in tranquilized animals compared with controls (at least P<0.05). A reduction in PCV, lymphocyte count, and serum cortisol concentrations (at least P<0.05) and an increase in serum creatinine levels (P<0.05) were recorded over time in control animals, while a reduction in RBC and hemoglobin concentration (at least P<0.05) and an increase in serum urea concentrations (P<0.05) over time were observed in the treated group. Finally, a decrease in serum lactate and potassium levels and an increase in CK, AST, ALT, and LDH activities were recorded over time in both groups. Results obtained showed the suitability of using acepromazine in capture operations in order to reduce stress response and prevent its adverse effects in roe deer. The beneficial effect was not only due to the sedative effect of acepromazine, but also to peripheral vasodilatation.

EFFECT OF VENIPUNCTURE SITE ON HEMATOLOGIC AND SERUM BIOCHEMICAL PARAMETERS IN MARGINATED TORTOISE (TESTUDO MARGINATA)

Blood samples were obtained from the dorsal coccygeal vein and the brachial vein of five adult (four females and one male) and two subadult males of marginated tortoise (Testudo marginata) and hematologic and biochemical parameters were compared. Significant differences were found for red blood cell count, hematocrit, hemoglobin concentration, total proteins, uric acid, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, calcium, and phosphorus, which were greater in the brachial vein samples. Hemodilution due to lymph was observed when collecting blood from the dorsal coccygeal vein, and it is thought to be the cause of the differences found. This research documented that the brachial vein is a more reliable and consistent venipuncture site than dorsal coccygeal vein in marginated tortoise.

Molecular Identification of a New Pestivirus Associated with Increased Mortality in the Pyrenean Chamois (Rupicapra pyrenaica pyrenaica) in Spain

Pestivirus infection was identified in 16 of 17 chamois during an outbreak of a previously unreported disease in Pyrenean chamois (Rupicapra pyrenaica pyrenaica) in northeastern Spain in 2001–02. By analysis of the 5′ noncoding regions of the virus, we assigned it to the border disease virus cluster with pairwise similarity values ranging from 82.1% to 88.1%. It will be important to investigate the association of this pestivirus with disease in Pyrenean chamois.

Experimental infection of European red deer (Cervus elaphus) with bluetongue virus serotypes 1 and 8

Bluetongue (BT) is a climate change-related emerging infectious disease in Europe. Outbreaks of serotypes 1, 2, 4, 6, 8, 9, 11, and 16 are challenging Central and Western Europe since 1998. Measures to control or eradicate bluetongue virus (BTV) from Europe have been implemented, including movement restrictions and vaccination of domestic BTV-susceptible ruminants. However, these measures are difficult to apply in wild free-ranging hosts of the virus, like red deer (Cervus elaphus), which could play a role in the still unclear epidemiology of BT in Europe. We show for the first time that BTV RNA can be detected in European red deer blood for long periods, comparable to those of domestic ruminants, after experimental infection with BTV-1 and BTV-8. BTV RNA was detected in experimentally infected red deer blood up to the end of the study (98-112 dpi). BTV-specific antibodies were found in serum both by enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VNT) from 8 to 12 dpi to the end of the study, peaking at 17-28 dpi. Our results indicate that red deer can be infected with BTV and maintain BTV RNA for long periods, remaining essentially asymptomatic. Thus, unvaccinated red deer populations have the potential to be a BT reservoir in Europe, and could threaten the success of the European BTV control strategy. Therefore, wild and farmed red deer should be taken into account for BTV surveillance, and movement restrictions and vaccination schemes applied to domestic animals should be adapted to include farmed or translocated red deer.

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.

Serologic and virologic investigations into pestivirus infection in wild and domestic ruminants in the Pyrenees (NE Spain).

An outbreak of disease associated to a border disease virus was described in the Southern chamois (Rupicapra pyrenaica) in Spain in 2002. Sera and/or spleen samples from 57 mouflon, 15 red deer, 21 roe deer, 3 fallow deer, 55 sheep, 32 cattle, and 68 goats sharing the chamois habitat were studied. An antibody ELISA test yielded an inconclusive result in 2 mouflon and positive results in 5 goat sera. Comparative virus neutralization tests were performed on the 2 inconclusive mouflons, 3 of the 5 seropositive goats, 55 sheep and 32 cattle, using 6 pestivirus strains. Positive results were obtained in 1 mouflon, 2 goats, 69% of sheep and 78% of cattle. Virological investigations performed with an antigen ELISA test yielded negative results in 21 goats and 39 mouflons, the result in 1 mouflon being inconclusive. PCR performed on 12 goats and the inconclusive mouflon gave negative results. These results suggested that it is unlikely that chamois BDV is infecting wild and domestic ruminants.

Comparative evaluation of effort, capture and handling effects of drive nets to capture roe deer (Capreolus capreolus), Southern chamois (Rupicapra pyrenaica) and Spanish ibex (Capra pyrenaica)

The objective of this study is to assess the usefulness of drive nets to capture roe deer (Capreolus capreolus), Southern chamois (Rupicapra pyrenaica) and Spanish ibex (Capra pyrenaica), comparing the results obtained with other capture methods and amongst the three species. Sixty-five drive net capture operations using beaters were conducted from January 1998 to September 2004. A total of 161 wild ungulates (31 roe deer, 95 Southern chamois and 35 Spanish ibexes) were captured. The average number of animals captured per operation was 1.07 for roe deer, 3.96 for Southern chamois and 2.92 for Spanish ibex. The average number of person–days per captured animal was 21.5, 7.1 and 10.6 for roe deer, Southern chamois and Spanish ibex, respectively. Specificity was 100% for Southern chamois and Spanish ibex (only the target species captured) and 77.5% for roe deer. Risk for the animals (mortality plus injuries) was 3.23% for roe deer, 5.27% for Southern chamois and 0% for Spanish ibex, whereas injuries to the operators occurred with 3.1% of the handled animals. Sex ratio was skewed towards females in roe deer, towards males in Southern chamois and balanced in Southern chamois. Drive nets showed good performance, although many operators were required. Safety for the animals and specificity were higher than traditionally attributed to this capture method. It is concluded that drive nets are an efficient and safe method to capture many ungulate species.

Prevalence of antibodies against selected agents shared between Cantabrian chamois (Rupicapra pyrenaica parva) and domestic goats.

Southern chamois (Rupicapra pyrenaica) share the habitat with domestic ungulates, and may, therefore, play a role in the epidemiology of shared agents. The objective of this study was to determine the seroprevalence for Brucella spp., Mycobacterium avium ssp. paratuberculosis (MAP), pestivirus, and Sarcoptes scabiei in Cantabrian chamois (Rupicapra pyrenaica parva) and compare these data with those of sympatric domestic goats (Capra hircus). From 2005 to 2008, blood samples were obtained from 236 adult Cantabrian chamois in two different populations, the western one and the eastern one. Seroprevalence for Brucella spp. and pestivirus was assessed using commercial ELISA kits, whereas specifically designed ELISA tests were used for MAP and S. scabiei. No antibodies against Brucella spp. were detected. Conversely, antibodies against MAP, pestivirus (chamois 3.8%; goat 2.3%), and S. scabiei (chamois 11.9%; goat 12.8%) were detected in both species. Seroprevalence for MAP was significantly higher for domestic goats (26%) than for chamois (9.7%). In chamois, seroprevalence for pestivirus was higher in the west (6.5%) than in the east (range 0–1.8%), whereas seroprevalence for S. scabiei followed the opposite trend (west 4.6%; east 16.7–21.4%). We suggest that certain diseases could circulate between Cantabrian chamois and domestic goat populations, and domestic livestock may suppose a threat for the health status of sympatric Cantabrian chamois.

Effects of parasitic helminths and ivermectin treatment on clinical parameters in the European wild boar (Sus scrofa)

Limited information exists on serum biochemistry and haematology of the European wild boar, and few correlations have been found between parasitic burden and clinical parameters in this species. Naturally infected wild boars were experimentally treated to study the effect of nematode parasites and ivermectin treatment on phytohaemagglutinin (PHA) skin reaction and haematological and serum biochemical parameters. White blood cells decreased in untreated wild boars, whereas triglyceride, lactate and creatinine concentrations increased in ivermectin-treated wild boars, and total protein and aspartate aminotransferase activity increased in both groups. Band neutrophils variation was positively correlated with the number of total adult parasites. Band neutrophils, creatinine and total protein variations were negatively correlated with both total egg counts and Metastrongylus eggs per gram variations. Alkaline phosphatase activity showed a negative correlation with Ascaris sp. eggs. The PHA skin reaction was positively correlated with the number of total adult parasites in untreated wild boars and with Metastrongylus sp. eggs of all wild boars at time 0. Two models including leukocytic and serum biochemical parameters were also highly correlated with the variation of Metastrongylus sp. eggs. Clinical parameters were thus in our study affected by helminth parasitism in the European wild boar, particularly those related to nutrients uptake, physical condition and immune response. Therefore, they could be useful in studies on subclinical effects of parasites, and parasitic burden must be considered when assessing the physical condition of European wild boars through haematological and serum biochemical parameters.

Evaluation of the efficacy of commercial vaccines against bluetongue virus serotypes 1 and 8 in experimentally infected red deer (Cervus elaphus)

Red deer (Cervus elaphus) is a widespread and abundant species susceptible to bluetongue virus (BTV) infection. Inclusion of red deer vaccination among BTV control measures should be considered. Four out of twelve BTV antibody negative deer were vaccinated against serotype 1 (BTV-1), and four against serotype 8 (BTV-8). The remaining four deer acted as unvaccinated controls. Forty-two days after vaccination (dpv), all deer were inoculated with a low cell passage of the corresponding BTV strains. Serological and virological responses were analyzed from vaccination until 28 days after inoculation (dpi). The vaccinated deer reached statistically significant (P<0.05) higher specific antibody levels than the non vaccinated deer from 34 (BTV-8) and 42 (BTV-1) dpv, maintaining stable neutralizing antibodies until 28 dpi. The non vaccinated deer remained seronegative until challenge, showing neutralizing antibodies from 7 dpi. BTV RNA was detected in the blood of the non vaccinated deer from 2 to 28 dpi, whereas no BTV RNA was found in the vaccinated deer. BTV was isolated from the blood of non vaccinated deer from 7 to 28 dpi (BTV-1) and from 9 to 11 dpi (BTV-8). BTV RNA could be identified by RT-PCR at 28 dpi in spleen and lymph nodes, but BTV could not be isolated from these samples. BT-compatible clinical signs were inapparent and no gross lesions were found at necropsy. The results obtained in the present study confirm that monovalent BTV-1 and BTV-8 vaccines are safe and effective to prevent BTV infection in red deer. This finding indicates that vaccination programs on farmed or translocated red deer could be a useful tool to control BTV.