Exposure of Asian Elephants and Other Exotic Ungulates to Schmallenberg Virus

Schmallenberg virus (SBV) is an emerging Orthobunyavirus, first described in 2011 in cattle in Germany and subsequently spread throughout Europe, affecting mainly ruminant livestock through the induction of foetal malformations. To gain a better understanding of the spectrum of susceptible species and to assess the value of current SBV serological assays, screening of serum samples from exotic artiodactyls and perissodactyls collected at the Living Collections from the Zoological Society of London (Whipsnade and London Zoos) and Chester Zoo was carried out. There was compelling evidence of SBV infection in both zoological collections. The competitive ELISA has proved to be applicable for the detection of SBV in exotic Bovidae, Cervidae, Suidae, Giraffidae and most notably in endangered Asian elephants (Elephas maximus), but unreliable for the screening of Camelidae, for which the plaque reduction neutralisation test was considered the assay of choice.

Despite the detection of SBV-induced antibodies in wild and captive ruminant species mentioned above, there is an under-appreciation of the spectrum of susceptible species, due to the very short viraemia and a lack of clinical symptoms in the acute phase of the infection. This study was therefore aimed to address this by examining archived sera from exotic ungulates collected from the Zoological Society of London (ZSL) Whipsnade and London Zoos and Chester Zoo. In addition, the value of current SBV serological assays, a competitive ELISA (cELISA) and a plaque reduction neutralisation test (PRNT), for screening serum samples from artiodactyls and perissodactyls was also assessed.

Ethical statement
All samples used for this study were surplus archived serum samples, initially collected for clinical purposes under the Veterinary Surgeons Act 1966, United Kingdom.

Archived serum sample selection
Frozen serum samples stored at -20°C from Artiodactyla, Perissodactyla and Proboscidea collected at ZSL Whipsnade Zoo (Collection A) between January 1, 2011 and February 1, 2014 (211 samples from 136 individuals of 39 species) and at Chester Zoo (Collection B) between August 1, 2012 and December 1, 2013 (28 samples from 28 individuals of 16 exotic ungulate species) were used for this study. Two further samples from two camelid species from ZSL London Zoo (Collection C) were also included. In total, 241 samples from 165 individuals of 48 different exotic ungulate species were tested using the cELISA and 35 samples from 34 individuals of 20 different species were further verified using PRNT (Table 1).

ELISA
The cELISA 'ID Screen Schmallenberg virus Competition Multi-species' (ID.vet) was used for the initial serological screening, according to manufacturer's instructions. The ratio of the OD of the sample (S) to the OD of the negative control (N) was subsequently calculated to obtain the S/N %. The serum was considered positive for SBV antibodies when the S/N % was smaller than or equal to 40 and negative when the S/N % was more than 50. Test results between these measures were considered inconclusive.

PRNT
To validate the results obtained from the cELISA and to quantify the levels of SBV neutralising antibodies, samples with an S/N % of 40-60 and a random selection of positives and negatives from a variety of species were tested using PRNT [17]. For this test, a 2-fold dilution series of the serum was mixed with a standard quantity of SBV and incubated on a monolayer of cells. Non-neutralised virus leads to cytopathic effect (cpe) in the infected cells. The resultant plaques were visualised following fixation and staining of the monolayer. The number of plaques is inversely proportional to the level of antibodies present in the tested sera.
Briefly, the assay was carried out using Vero cells (kidney epithelial cells from Chlorocebus sp. monkeys, ECACC, No: 85020206) in Eagle's minimum essential medium (EMEM, Life Technologies) in a 24-well plate. A 70 μl volume of each selected serum was first placed in a 56°C water bath for 30 minutes to inactivate the complement. To prepare the serial dilutions, 25 μl of each sample was mixed with 175 μl EMEM for a 1/8 dilution. Subsequently, 100 μl of this dilution was mixed with 100 μl EMEM for a serial two-fold dilution. The serial dilutions were mixed with 100 μl of virus suspension (40-80 plaque forming units) and incubated at Table 1. List of serum samples used in this study, detailing number of individual species and samples tested with cELISA and PRNT. The two tests were carried out on archived sera collected from exotic wildlife species in the Bovidae, Cervidae, Suidae, Giraffidae, Camelidae and Elephantidae families. The sera were from one to several individual animals of each species, All but one were tested with cELISA, and a random selection of cELISA positives and negatives were further investigated using PRNT. 37°C for 1 hour before being added to the cells. The plates were incubated at 37°C for 1 hour before a carboxymethylcellulose (Sigma-Aldrich) overlay was added to each well. After incubation, cells were fixed with buffered formalin, stained with Crystal Violet (Sigma-Aldrich) and the plates were placed on a light box to count the plaques and determine the PRNT. Samples were considered SBV positive if the 1/8 and 1/16 dilutions of the serum had plaque reductions of 95% (PRNT 95 ) and 80% (PRNT 80 ) respectively.

Results
The Serial sampling at Collection A confirmed sero-conversion in 21 individuals from 11 different species: one greater kudu, two scimitar-horned oryx (Oryx dammah), three yaks, one gemsbok (Oryx gazelle gazella), one reticulated giraffe (Giraffa camelopardalis reticulata), one llama   PRNT was carried out on 35 samples from 17 individuals representing 18 different species from Collection A and on 2 samples from one species from Collection B. There was complete concordance between the PRNT and cELISA results for deer (hog deer Axis porcinus, reindeer Rangifer tardinus), antelope (greater kudu, blackbuck Antelope cervicapra), bovids (yak, gaur Bos gaurus), giraffes and red river hogs (Potamochoerus porcus). There was also concordance between the positive and negative outcomes of the cELISA and PRNT for Asian elephants. One inconclusive cELISA result from an Asian elephant (S/N% = 42.7) was negative on PRNT.
The cELISA and PRNT results did not correlate for the serum samples tested from camelids and perissodactyls. Of the three positive PRNT results in camelids (an alpaca, a llama and a Bactrian camel Camelus bactrianus), only the alpaca, with the highest PRNT titre of these three camelids, produced a positive cELISA result. Forty-two perissodactyl serum samples from 27 individuals of 9 species were screened. One onager (Equus hemionus) and a Grevy's zebra (Equus grevyi) were positive on cELISA but not on PRNT, although two out of four individual onagers screened showed some plaque reduction at the initial serum dilution. One further cELISA inconclusive result (a male white rhinoceros, Ceratotherium simum) was also PRNT negative with an insufficient plaque reduction (<90%) at the 1/8 dilution.

Discussion
To our knowledge this is the first systematic evaluation of previous SBV infection in exotic ungulates in Europe. The study provides compelling evidence of SBV infection in exotic Bovidae, Cervidae, Suidae, Giraffidae and most notably in Asian elephants (Elephas maximus)-one of the endangered flagship ungulate species [18]. Serial serum samples of several individuals suggest that ungulates at Collections A, B and C were first exposed to SBV in the late summer of 2012, the second year of SBV circulation in the UK. Whilst this is generally in line with the spread of this virus to livestock holdings across the UK [19], both ZSL collections were in geographical areas where the first incursion of SBV occurred in 2011. Notably, some animals, including one of the Asian elephants, sero-converted only in 2013, the third SBV season in the UK, demonstrating the continuous spread of SBV in an affected area of the UK (South East) for three consecutive years. Interestingly, in this context and in relation to the biology of the Culicoides vector midges, all the positive cases at Collection A were animals housed near buildings or which preferred to shelter under bushes and/or trees. Most species in the 'Drive Through', an exposed wide open space with limited housing and vegetation, were negative for SBV. Examples are Chinese water deer (Hydropotis inermis), hog deer and Pere David's deer (Elaphurus davidianus). Other studies have also shown that landscape elements and shelter can influence the spatial distribution of Culicoides-borne diseases [20][21], likely through a variation of exposure time to the midges. A previous study comparing an indirect ELISA (iELISA) with serum neutralisation test (SNT) on a large number of red deer sera confirmed the reliability of using iELISA for this species [22]. Similar conclusion could be drawn on the use of the cELISA for the majority of species screened in this study, with the exclusion of camelids and perissodactyls. However, it has to be taken into consideration that only a small number of individuals of each species were tested in this study.
SBV exposure of camelids has previously been established using an iELISA [23]. However, in this study the cELISA proved unsuitable for the screening of camelids, resulting in false negatives in both a llama and a Bactrian camel. This was unexpected as SBV cELISA performance in other ruminants, including wild deer and bison, is considered to be optimal [22]. However, this study only examined a handful of camelid samples and hence cELISA appliance in Inconclusive. c The cELISA result was also confirmed by PRNT.
camelids requires further validation. In the meantime, virus neutralisation assays and iELISA should be considered as the preferred tests for the screening of camelids.
As far as perissodactyls are concerned, a fair number of sera returned inconclusive (white rhinoceros) or positive (onager, Grevy's zebra) results on the cELISA, which could not be verified by the PRNT. This variability and, at times, marked plaque reduction in the initial dilution potentially points towards an SBV-related virus, against which the antibodies cross-react in the cELISA. Similar observation has also been made testing horse sera for SBV antibodies (unpublished data). Further research is therefore required to resolve these inconsistencies.
Following the first serologically positive yak at Collection A in August 2012, almost two thirds (59%) of individual artiodactyls at Collection A tested positive for SBV antibodies. This is similar to in-herd sero-prevalence findings in livestock following initial infection [24]. Screening of the large number of serial serum samples from the Asian elephant herd at Collection A showed that the majority of animals within an exposed herd sero-converted within a similar time frame, and that, after initial exposure, antibody levels remained high. Similar trends were also seen in serial serum samples from other species, including a greater kudu and a scimitar-horned oryx, in which the antibody level remained high for at least 6 to 12 months, respectively. This is consistent with persistent antibody levels seen in cattle [24].
As yet, clinical evidence of the presence of SBV has not been seen at Collections A, B or C. Transient fever, impaired general condition and diarrhoea, as seen in cattle [25], may have been missed in exotic ungulates due to the nature of their hands-off management. However, as yet an increase in congenital deformities and/or abortions has not been observed at any of the three sites studied. Routine tissue testing at Collection A of several aborted foetuses by PCR has also yielded negative results (unpublished data). As SBV antibodies are considered protective [26][27] disease is only expected in naïve animals or those that have not sero-converted [28]. Therefore both clinical and economical impacts at zoos can be considered negligible even for naïve populations. In line with the notion that previous SBV infection (documented through the presence of antibodies) protects from re-infection for a considerable length of time, no clinical signs consistent with SBV infection have been observed in any of the animals studied at these zoological collections.
Further research is currently taking place on the vector presence, infection rate and seasonality at Collection A, in an attempt to gain insights into potential correlation between seasonality of virus load in vectors and sero-prevalence. Based on such further findings, conclusions may potentially be drawn as to the value of vaccination of exotic ungulates against SBV.