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1 Central Veterinary Research Laboratory, PO Box 597, Dubai, United Arab Emirates
2 Institute for Animal Health (IAH), Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK
Correspondence
Nick J. Knowles
nick.knowles{at}bbsrc.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this paper are EF204767–EF204772.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). ERAV was first identified in 1962 in the UK (Plummer, 1962) and subsequently found in horses in many countries around the world (Ditchfield & Macpherson, 1965; Hofer et al., 1973; Holmes et al., 1978; Studdert & Gleeson, 1977). ERAV infection of horses may result in acute febrile respiratory disease accompanied by viraemia and persistent virus shedding in urine and faeces (Plummer & Kerry, 1962; McCollum & Timoney, 1991). Little is known about the epidemiology and pathogenesis of ERAV, but it is thought to occur worldwide and has been responsible for relatively large outbreaks of acute respiratory illness in adult horse populations. Many ERAVs are not cytopathic in cell culture and may be missed in disease investigations that rely on virus isolation alone (Li et al., 1997). Despite being primarily an infectious agent of horses, ERAV is pathogenic for a broad range of other animal species, including man (Plummer, 1962, 1963). The genome sequences of seven ERAV isolates have been determined so far, as well as the complete P1 capsid-coding regions of eight additional virus isolates (Li et al., 1996; Wutz et al., 1996; Varrasso et al., 2001; A. Aminev and A. C. Palmenberg, unpublished data), allowing molecular diagnostic tests to be developed (Dynon et al., 2001). This paper describes the isolation of ERAV from fetuses during an abortion outbreak in a dromedary (Camelus dromedarius) herd in Dubai and the results of a subsequent experimental infection of two pregnant dromedaries with the isolated virus.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Several weeks after the onset of these abortions, the 57 resident horses from the retirement home were inspected for signs of disease. Blood samples were subsequently taken for serological assessment from the 258 camels in the study herd, 200 randomly sampled camels from other premises in Dubai and the 57 horses in the retirement home.
Isolation and titration of virus.
Tissues from four of the eight aborted fetuses, including placentas, were collected for bacteriological, mycological, virological and histopathological investigation. Tissue samples (liver, lung, spleen and placenta) taken from each fetus were homogenized and then pooled for virological assay.
One millilitre of each clarified tissue suspension, diluted 1 : 10 in minimal essential medium (MEM) without fetal calf serum (FCS), was inoculated onto separate monolayer cultures of Vero and fetal camel kidney (Caki) cells. Inoculated cultures were incubated at 37 °C and examined microscopically for 7 days. Virus was harvested from the infected cell cultures when they showed 100 % cytopathic effect (CPE). Samples that did not show any visible CPE were blind-passaged on two further occasions.
Serum neutralization test.
Sera decanted from the clotted blood were tested for antibodies against ERAV in 96-well microtitre plates using a serum neutralization test (SNT). Briefly, a triplicate twofold dilution series of each serum, diluted from 1 : 2 to 1 : 128 in MEM medium without FCS, was prepared on the plates. An equal volume of the reference ERAV strain NM-11/67, containing 100 TCID50, was added to each of the serum wells. A virus control titration was included in each test to confirm the virus dose used. Serum/virus mixtures were incubated for 1 h at 37 °C. Vero cells at a concentration of 2.0x106 cells ml–1 were added to all of the wells. Plates were sealed and incubated for 3 days at 37 °C and read microscopically on days 2 and 3. Titres were calculated as the reciprocal of the last dilution of serum in the serum/virus mixtures showing CPE at the 50 % end point (Finney, 1964). The sera were also tested against the virus isolated from one of the aborted fetuses as described above.
Experimental infection.
Two 7-months-pregnant dromedaries were housed at the Central Veterinary Research Laboratory (CVRL), Dubai, in separate roofed pens. Tarpaulin sheets were secured on three sides of the pens. The dromedaries were restrained in a sitting position. One was sprayed in both nostrils with 3 ml infectious tissue culture supernatant fluid containing the camel virus isolate (designated D1305-03; pooled homogenate of lung, liver, spleen and placenta collected from one of the aborted fetuses and passaged three times in Vero cells) at a titre of 104.0 TCID50 ml–1 using a nebulizer (Omron MicroAir U22) according to the manufacturer's recommendation. The particle size was approximately 4.9 µm and the nebulization rate was 0.25 ml min–1. The procedure lasted for 7 min in each nostril. The second animal was infected intratracheally with 5 ml of the same virus suspension. Both dromedaries were examined daily, and rectal temperature and clinical signs were recorded. Blood samples were collected before infection and at intervals thereafter for 3 weeks. Tissue samples (bone marrow, brain, intestine, liver, lung, placenta, skin, spleen, thymus, tonsil and trachea) were collected from the aborted fetuses of the intranasally and intratracheally infected dromedaries at 12 and 82 days post-inoculation (p.i.), respectively. Each tissue was homogenized separately and assayed for virus as described above.
RT-PCR.
Total RNA was extracted from 460 µl virus-infected cell culture supernatant using an RNeasy kit (Qiagen) following the manufacturer's protocol and resuspended in 50 µl nuclease-free water. This RNA (5 µl) was used as template in a one-step RT-PCR (Ready-To-Go RT-PCR beads; Amersham Pharmacia Biotech) following the manufacturer's instructions. Forward and reverse primer amounts were 20 and 40 pmol, respectively. Two different primer combinations were used to obtain amplicons containing either the complete ERAV VP1 gene (primers ERAV-1C616F and ERAV-2A22R; amplicon of 851 bp) or a partial VP1 product (primers ERAV-1D7F and ERAV-2A22R; amplicon of 782 bp) (Table 1). The mixes were subjected to the following thermal profile: one cycle of 30 min at 42 °C and 5 min at 94 °C, followed by 45 cycles of 1 min at 94 °C, 1 min at 45 °C and 1.5 min at 72 °C, with a final extension of 5 min at 72 °C, and then held at 4 °C. PCR amplicons were analysed on a 1.5 % agarose/TBE gel containing 0.5 µg ethidium bromide ml–1. DNA molecular mass markers (GeneRuler 100 bp DNA Ladder Plus, Ready-To-Use; MBI Fermentas) were run alongside the samples to facilitate product identification and quantification. Post-PCR removal of dNTPs and primers was achieved enzymically using ExoSAP-IT (GE Healthcare) according to the manufacturer's instructions.
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) and NM-11/67 (RK-13 passage 9, LLC-MK2 passage 3) (Burrows, 1968), respectively. The viruses were propagated on African green monkey kidney cells (either Vero or LLC-MK2). Both Vero and Caki cells were used for isolation of viruses from dromedaries.
DNA sequencing.
PCR amplicons were sequenced with the four primers used for PCR shown in Table 1
using a DTS Quick Start kit (Beckman Coulter) according to the manufacturer's instructions. The sequencing reactions were run on a CEQ800 Automated Sequencer (Beckman Coulter) according to the manufacturer's instructions. The complete VP1 nucleotide sequences of the two reference ERAV strains NM-11/67 and SWI/V1722/70 were also determined.
Phylogenetic analysis.
Sequences were compiled and edited using the EpiSeq 2.0 package (N. J. Knowles, unpublished data). Phylogenetic analysis was conducted using MEGA version 3.1 (Kumar et al., 2004). A neighbour-joining tree was constructed using a difference matrix based on the Kimura two-parameter model of nucleotide substitution. Confidence levels on branches were estimated by bootstrap resampling (1000 pseudoreplicates). The following publicly available ERAV VP1 sequences were also used in the analysis: PERV/UK/62 (AF347671
[GenBank]
; X96870
[GenBank]
; DQ272578
[GenBank]
), AUS/393/76 (AF347668
[GenBank]
), AUS/967/90 (AF347669
[GenBank]
), SWI/V1722/70 (AF347675
[GenBank]
), SWI/P200/75 (AF347676
[GenBank]
), SWI/P346/75 (AF347677
[GenBank]
), SWI/P1316/92 (AF347678
[GenBank]
), USA/360007 (AF347672
[GenBank]
), USA/4066/79 (AF347674
[GenBank]
), USA/544/82 (AF347673
[GenBank]
), T3 (USA) (DQ268580
[GenBank]
), Plowright (USA) (DQ272127
[GenBank]
), U188 (USA) (DQ272128
[GenBank]
) and T10 (USA) (DQ272577
[GenBank]
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The dromedaries experimentally infected with the camel isolate via the intranasal and the intratracheal routes both developed mild, transient pyrexia on day 6 (Table 2). Between days 9 and 12, both experimental animals recorded a slight drop in temperature to below 37 °C. Other clinical signs observed in the intranasally infected dromedary, from day 6 until the day of abortion, included lethargy, inappetence and an oedematous udder. A clear vaginal discharge was also observed on day 11, 24 h before abortion. The second camel aborted spontaneously 82 days after the intratracheal infection but in the absence of any obvious clinical signs.
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). The chorial epithelium observed in field and experimental cases had a greyish floury appearance with some petechial and ecchymotic haemorrhages. Severe subcutaneous, generalized oedema was seen in all aborted fetuses, most prominently in the dorsal regions from the head to the hump area, and less so in the legs (Fig. 1b). In all cases, the abdominal and thoracic cavities were completely filled with dark haemolytic fluid but without coagulation (Fig. 1c). Similar fluids were also noted in the stomach of all fetuses. No other lesions were observed.
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). The heart showed severe interstitial oedema, which had caused degeneration of single fibres. Numerous eosinophilic round globules of different sizes were seen between the fibres and attached to the endocardium, the larger ones containing round crystalline structures (Fig. 2b). Similar eosinophilic round structures, which stained positively with Perl's stain (for haemosiderin pigment), were also found throughout the extensive subcutaneous oedema. Grocott, periodic acid–Schiff (PAS) and Guinea stains revealed no evidence of fungal spores in these red globules. These structures were similar to the ‘myospherules’ described by Rosai (1978) in cystic lesions of skeletal muscles, which have been classified as ‘altered erythrocytes’. These formations are simply erythrocytes that have been altered and clumped by haemostatic conditions. Marked intramural oedema with numerous small haemorrhages was seen in the trachea. The lung was still in the tubular development stage and showed moderate interstitial oedema. No inflammation and no inclusion bodies were seen in any organ.
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).
Isolation and identification of the virus
A cytopathic virus was isolated from the pooled, homogenized organs of two of the four aborted fetuses. No other cytopathic viruses were found. The virus isolate, designated D1305-03 (isolated from one of the fetal organ pools), was identified as ERAV using a pan-picornavirus RT-PCR (primer set ICYGDD/3'-RACE-A; N. J. Knowles, unpublished data) followed by DNA sequencing with the forward primer, ICYGDD (Table 1). The sequence of 441 nt at the 3' end of the D1305-03 genome was determined and included 328 nt of the 3D polymerase gene and the complete 3' UTR (113 nt including the stop codon). Additionally, the sequence of the 3' 438 nt was determined for ERAV PERV/UK/62 and this was identical to the published sequence (Fig. 3; Wutz et al., 1996). This diagnosis was confirmed using an ERAV-specific RT-PCR and sequencing of the VP1 gene. The length of the VP1 gene was 738 nt, as it was in all of the horse ERAV isolates. The relationship of D1305-03 to ERAVs isolated from horses is shown in Fig. 4.
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). This virus was later confirmed by RT-PCR and sequencing to be identical to the original camel virus strain (D1305-03; data not shown). Although the second dromedary, which was infected intratracheally, did not abort until 82 days p.i., both dromedaries seroconverted 7 days after experimental inoculation (Table 2). An identical virus was isolated from the same organs taken at 82 days p.i. from the aborted fetus of the intratracheally infected dromedary (Table 3).
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. The results were identical for both the field and the reference ERAV strains. Most of the dromedary (>85 %) and horse (>75 %) sera recorded antibody titres greater than 1 : 128. The sera from the 200 randomly selected racing camels were found to be negative by SNT. Table 2 shows temperature responses and SNT results for the two dromedaries experimentally infected with the camel strain of ERAV.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The virus is highly contagious and widespread in equids, being transmitted by the respiratory route (Plummer & Kerry, 1962). Experimental infection of man, monkeys, rabbits and guinea pigs has resulted in viraemia, and virus has been recovered from various tissues and an antibody response has been reported in all species studied. However, there is no evidence of virus spreading horizontally in any of these species (Plummer, 1963). Intranasal inoculation of a human volunteer with ERAV resulted in severe pharyngitis, lymphadenitis, fever and viraemia (Plummer, 1963). Plummer (1962) also found high neutralization titres against ERAV in the sera of 3/12 stable workers, whilst no ERAV antibodies could be detected in a control group of 159 non-stable workers. Kriegshauser et al. (2005) found only a low prevalence (2.7 %) of weakly neutralizing antibodies to ERAV in human sera obtained from 137 mixed-practice veterinarians, whilst 90 % of 200 horses sampled were positive, many with high titres. A previous serological survey carried out on 439 equine sera from the seven emirates of the United Arab Emirates in 1998 showed that more than 50 % of the horses tested had antibodies to ERAV (Wernery et al., 1999). Similarly, in this study, at least 80 % of the 57 retired horses tested were seropositive for ERAV. Together, these results suggest that ERAV is circulating in the United Arab Emirates equine population. The isolation of ERAV from aborted dromedary fetuses, the extremely high prevalence of antibody (>90 %) against ERAV demonstrated in the adult dromedary herd and the subsequent experimental field trials, although small in number, clearly demonstrate that ERAV not only infects dromedaries but can also cause abortion. Although not proven at this time, it is highly likely that the dromedaries, which had easy access to the horse retirement premises, received their infection from these horses. No respiratory disease was or has ever been observed or reported in the dromedary herd and no such symptoms were observed in the pregnant dromedaries experimentally infected with the camel isolate of ERAV. To check for a possible viraemia, 19 sera collected from each of the experimentally infected dromedaries between days 0 and 22 p.i. were examined for the presence ERAV RNA using a real-time RT-PCR (K. Ebert, S. M. Reid, N. J. Knowles & D. P. King, unpublished data); however, none was detected. Placentation in Camelidae is diffuse epitheliochorial, similar to that of the equine species, and this anatomical particularity may, in part, explain the susceptibility of dromedaries to an equine virus.
The absence of detectable antibody against ERAV in the racing camels is interesting but might only be a reflection of the lack of direct contact between the camels and infectious horses. Further studies are ongoing to study the epidemiology of ERAV in dromedaries.
Two picornaviruses have been isolated previously from camelids, one from a dromedary in an American zoological collection and one from llamas, also in the USA. The virus isolated from heart tissue of the adult dromedary was identified as encephalomyocarditis virus (genus Cardiovirus) and the disease was characterized by pale foci within the myocardium and epicardial haemorrhages associated with excessive pericardial fluid (Wells et al., 1989). The second virus was isolated from two aborted llama fetuses but was only identified as ERAV following the report by Stehman et al. (1997) of a picornavirus infection in llamas that caused abortion in 15 llamas over a 3.5 month period and at a mean gestation period of 220 days (7–8 months). Concurrent diabetes mellitus was also observed in the dams.
In Kenya, serological evidence of the exposure of free-ranging and captive zebra to ERAV has been presented (Kimber et al., 2002). The free-range animals had a higher seroprevalence than captive zebra and the authors speculated on the possible transmission of ERAV between the two groups.
To our knowledge, no reports of abortion in horses have been published, although two viruses (360007 and P1316/92), isolated from a fetus and placenta, respectively (Varrasso et al., 2001), and for which the complete capsid sequences have been determined, were said to have come from cases of equine abortion. This might suggest that ERAV-seronegative mares could be at risk if they become infected during pregnancy, although further studies are required to quantify this. In light of the potential risk, it would also be of benefit to ascertain the serological status of mares before and during pregnancy.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Stehman, S. M., Morris, L. I., Wiesensel, L., Freeman, W., Del Piero, F., Zylich, N. & Dubovi, E. J. (1997). Case report: picornavirus infection associated with abortion and adult onset diabetes mellitus in a herd of llamas. In Proceedings of the American Association of Veterinary Laboratory Diagnosticians Annual Conference, p. 43. Louisville, USA, 18–24 October 1997.
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Received 18 May 2006;
accepted 19 November 2007.
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