Accepted Manuscript Title: Experimental infection of sheep and goats with a recent isolate of peste des petits ruminants virus from Kurdistan Author: Kerstin Wernike Michael Eschbaumer Angele Breithaupt Julia Maltzan Henning Wiesner Martin Beer Bernd Hoffmann PII: DOI: Reference:
S0378-1135(14)00246-6 http://dx.doi.org/doi:10.1016/j.vetmic.2014.05.010 VETMIC 6616
To appear in:
VETMIC
Received date: Revised date: Accepted date:
16-9-2013 15-4-2014 3-5-2014
Please cite this article as: Wernike, K., Eschbaumer, M., Breithaupt, A., Maltzan, J., Wiesner, H., Beer, M., Hoffmann, B.,Experimental infection of sheep and goats with a recent isolate of peste des petits ruminants virus from Kurdistan, Veterinary Microbiology (2014), http://dx.doi.org/10.1016/j.vetmic.2014.05.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Experimental infection of sheep and goats with a recent isolate of peste des petits ruminants virus
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from Kurdistan
3 Kerstin Wernike1, +, Michael Eschbaumer1,4, +, Angele Breithaupt2,5, Julia Maltzan3, Henning Wiesner3,
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Martin Beer1,*, Bernd Hoffmann1
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Insel Riems, Germany;
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Institute of Diagnostic Virology, Friedrich-Loeffler-Institut (FLI), Suedufer 10, 17493 Greifswald–
Department of Experimental Animal Facilities and Biorisk Management, Friedrich-Loeffler-Institut
(FLI), Suedufer 10, 17493 Greifswald–Insel Riems, Germany
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+ Both authors contributed equally.
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Academy for the Protection of Zoo and Wild Animals, Munich, Germany
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* Corresponding author. Phone: +49 38351 71200, Fax: +49 38351 71226, E-mail:
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[email protected]
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Center, P.O. Box 848, Greenport, NY, 11944, USA. E-mail: [email protected]
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Universität Berlin, Berlin, Germany. E-mail: [email protected]
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Present affiliation: Foreign Animal Disease Research Unit, USDA/ARS Plum Island Animal Disease
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Present affiliation: Department of Veterinary Pathology, Faculty of Veterinary Medicine, Freie
Short title: PPRV in European sheep and goats
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Highlights
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Experimental infection of both sheep and goats with a recent isolate of peste des petits ruminants virus (PPRV from Kurdistan) PPRV RNA was detected in blood as well as nasal, oral and fecal swabs and tissues The 2011 Kurdish strain of PPRV is highly virulent in European goats and spreads easily to in contact animals Disease severity and contagiosity is lower in sheep
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34 Abstract
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Peste des petits ruminants (PPR) is a contagious viral disease of sheep and goats common in Africa
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and Asia. Its high morbidity and mortality has a devastating impact on agriculture in developing
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countries. As an example, an Asian lineage IV strain of PPRV was responsible for mass fatalities
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among wild goats in Kurdistan in 2010/11.
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In separate experiments, three sheep and three goats of German domestic breeds were
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subcutaneously inoculated with the Kurdish virus isolate; three uninfected sheep and goats were
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housed together with the inoculated animals. All inoculated animals, all in-contact goats and two
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in-contact sheep developed high fever (up to 41.7 °C), depression, severe diarrhea, ocular and nasal
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discharge as well as ulcerative stomatitis and pharyngitis. Infected animals seroconverted within a
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few days of the first detection of viral genome. Clinical signs were more pronounced in goats; four
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out of six goats had to be euthanized. Necropsy revealed characteristic lesions in the alimentary
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tract. Peste des petits ruminants virus (PPRV) RNA was detected in blood as well as nasal, oral and
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fecal swabs and tissues.
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The 2011 Kurdish strain of PPRV is highly virulent in European goats and spreads easily to in-contact
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animals, while disease severity and contagiosity in sheep are slightly lower. PPRV strains like the
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tested recent isolate can have a high impact on small ruminants in the European Union, and
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therefore, both early detection methods and intervention strategies have to be improved and
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updated regularly.
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54 Keywords: Peste des petits ruminants; peste des petits ruminants virus; PPRV; goat plague; ovine
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rinderpest; experimental infection; transmission
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Introduction
59 Peste des petits ruminants (PPR) is a highly contagious viral disease of small ruminants. Like the
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closely related rinderpest virus and the distemper and measles viruses, PPR virus (PPRV) is a
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morbillivirus of the Paramyxoviridae family. Besides sheep and goats, PPR occasionally affects other
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small ruminants in the wild (Munir, 2013). Disease severity depends on various factors such as
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species, breed or virus strain; in severe outbreaks, mortality can reach 100% (Banyard et al., 2010;
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Couacy-Hymann et al., 2007; Diop et al., 2005). PPR is characterized by high fever, thick discharge of
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eyes and nostrils, necrotizing, erosive stomatitis, and enteritis with diarrhea. In fatal infections, death
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is caused by primary viral bronchopneumonia or severe dehydration due to acute diarrhoea (Banyard
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et al., 2010; Couacy-Hymann et al., 2007).
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PPRV was first identified in West Africa in 1942. The virus is currently present all over West and
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Central Africa, the Middle East and Central to South-East Asia in four genetically distinct lineages
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(Banyard et al., 2010; Dhar et al., 2002). Small ruminants are an essential part of the livelihood of
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many subsistence farmers and pastoralists, and PPR is considered the main constraint to increased
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animal production in endemic areas (Diallo, 2006). It has never been reported in the European Union,
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but is regarded as a credible threat to domestic agriculture (Libeau et al., 2011).
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A lineage IV strain of PPRV was presumed responsible for mass fatalities among wild goats in
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Kurdistan in 2010/11 (Hoffmann et al., 2012), and the present study intended to confirm the
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virulence of the Kurdish strain in an animal experiment. Furthermore, the potential impact of an
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introduction of this strain into the European Union was investigated by using European breeds of
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sheep and goats for the experimental infection.
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Materials and Methods
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Virus
84 PPRV was isolated from a swab sample of a Kurdish goat that had died during the 2010/11 outbreak.
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After initial isolation on grivet kidney (CV1) cells expressing the caprine signalling lymphocyte
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activation molecule (SLAM) (Adombi et al., 2011), the virus was passaged once on Vero/dog-SLAM
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cells (von Messling et al., 2003) for use in the animal experiment. The isolate has been designated
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PPRV Kurdistan/2011, and partial nucleocapsid gene sequence information is available online
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(GenBank accession no. JF969755).
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91 Animals and experimental design
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An infection experiment with sheep and goats of German domestic breeds was carried out in the
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BSL3-ag containment facility of the Friedrich-Loeffler-Institut, Insel Riems. Three sheep (S1-S3) and
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three goats (G1-G3) were subcutaneously inoculated with 1x10^4 TCID50 of virus. Sheep and goats
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were kept in separate rooms. Three uninfected sheep (S4-S6) and goats (G4-G6), respectively, were
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housed together with the inoculated animals. After inoculation, clinical signs and rectal body
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temperature were recorded daily. Whole blood, serum, nasal, oral and fecal swabs were taken every
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other day beginning on day 2. Seven days after inoculation one sheep and one goat (S3/G3) were
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euthanized and necropsied. The remaining goats were euthanized on days 12 (G1/G2), 17 (G4), 18
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(G5) and 20 (G6), the sheep 20 days after infection. A diverse panel of tissues was taken at necropsy
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(14 samples from the alimentary canal, 3 from the respiratory tract, 7 lymphatic tissues, as well as
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liver, brain, kidney, heart and reproductive organ samples).
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The experimental protocol was reviewed by an independent ethics commission pursuant to §15 of
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the German Animal Welfare Act, and it has been approved by the competent authority (State Office
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for Agriculture, Food Safety and Fisheries of Mecklenburg-Vorpommern, Rostock, Germany; ref.
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LALLF M-V/TSD/7221.3-2.5-004/11).
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Clinical observations were recorded and scored with a rubric similar to the one used by El Harrak et
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al. (2012). Depending on the severity of clinical signs, up to three points were awarded in the
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categories “general attitude”, “congestion and edema”, “oculonasal discharge”, “mucosal lesions”,
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“diarrhea” and “rectal temperature”. Daily scores and a total cumulative score for the entire
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experiment were calculated for each animal.
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114 Real-time RT-PCR, virus isolation and serology
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Total RNA from bloods, swabs and tissue samples was extracted using the NucleoSpin® 96 RNA Kit
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(Macherey-Nagel, Düren, Germany) on a MICROLAB® STAR liquid handling workstation (Hamilton,
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Bonaduz, Switzerland) according to the manufacturer’s recommendations. PPRV genome load in the
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samples was determined by an N gene-specific RT-qPCR as described previously (Kwiatek et al., 2010)
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with an external RNA standard. The tissue samples were homogenized and tested for the presence of
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PPRV RNA by real-time RT-PCR as well.
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Virus isolation from one nasal swab per animal (day 8 for inoculated animals, day 16 for in-contact
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animals) was attempted on CV1/goat-SLAM cells. G3 and S3 were not tested. Five days after culture
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inoculation, the cells were screened for cytopathic effects (cell death, syncytia) and stained with a
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monoclonal antibody against the PPRV N protein (ID Vet, Montpellier, France). N-protein-positive
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cells were considered infected even in the absence of a visible cytopathic effect.
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All serum samples were tested with a commercially available, competitive PPR-N-protein-antibody
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ELISA (ID Vet, Montpellier, France) using the recommended cut-off of 60% relative optical density
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compared to the negative control. Serum samples taken before infection and on the last sampling
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day before euthanasia (day 6 for S3 and G3, day 12 for G1 and G2, day 16 for G4, day 18 for G5 and
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day 20 for the remaining animals) were further analysed in a standard micro-neutralization test (NT)
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on Vero/dog-SLAM cells against the homologous virus isolate.
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Clinical and virological data collected in the experiment were fitted to a linear mixed-effects model
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using R (R Development Core Team, 2009). Differences between groups were examined by Tukey’s
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all-pair comparisons using the Bonferroni method for multiplicity adjustment of p values. Adjusted p
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values < 0.01 were considered significant.
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Results
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Clinical scores: More severe disease in goats
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The first clear clinical signs appeared within 4 to 5 days post inoculation. Eventually, all inoculated
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animals, all in-contact goats and two out of three in-contact sheep developed high fever (up to
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41.7 °C, figure 1) and depression, severe diarrhea, mucopurulent ocular and nasal discharge as well
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as ulcerations in the oral and pharyngeal mucosa. An overview of the clinical scores in the
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experiment is provided by Figure 2. In general, signs of disease were more severe in goats and lasted
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longer than in sheep; four out of six goats had to be euthanized for humane reasons. The average
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total cumulative score over the course of the experiment in sheep was 22, compared to 41 in goats.
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No in-contact sheep ever exceeded a daily clinical score of 4, whereas all three in-contact goats did
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(maximum scores of 15, 8 and 5, respectively).
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For the statistical analysis, needle-inoculated and in-contact animals were examined separately. Over
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the course of the experiment, the difference in clinical scores between goats and sheep was found to
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be significant, independent of the route of exposure.
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158 Gross pathology: Moderate to severe lesions in multiple organs
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At autopsy, acute, diffuse, catarrhal to catarrhal-purulent rhinitis and sinusitis were found. The oral
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cavity exhibited an acute fibrinosuppurative, partially erosive to ulcerative stomatitis and pharyngitis
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(e.g. figure 3a). Frequently, the lung parenchyma showed multifocal to coalescing areas of
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consolidation affecting 5-15% of lung lobes with adjacent moderate to severe, multifocal to diffuse,
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acute, alveolar emphysema and a mild to moderate, acute, diffuse alveolar edema. Further, an acute,
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focal, extending erosive to ulcerative ruminitis was evident (figure 3b).
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There were moderate, multifocal petechiae, ecchymoses and erosions within the intestinal mucosa,
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predominantly affecting the ileum, cecum and colon. Several animals had an acute, focally extending
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fibrino-necrotizing and hemorrhagic enteritis (i. e. typhlocolitis, also affecting the ileocecal valve).
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Two animals (G3, G6) showed an acute intussusception of the caecum with varying degrees of
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hemorrhagic infarction (figure 3c-d). The mesenteric lymph nodes were moderately to severely
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enlarged. Besides these main findings, intestinal and pulmonary nematodes and associated lesions
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were found in several animals.
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PCR and virus isolation: Abundant viral RNA in whole blood, swabs and tissues
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In whole blood samples of the inoculated animals, viral RNA was first detected on day 4. It was most
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abundant between 4 and 6 days after inoculation, but remained detectable until the end of the
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study. In the two in-contact goats, PPRV RNA could be found for the first time on day 10, in the third 8
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goat on day 12. Two in-contact sheep were positive from days 12 and 16, respectively. In whole
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blood samples of the last sheep (S6), PPRV RNA was not detected at any time (figure 4). In general,
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PPRV RNA was more readily detected in whole blood than in serum samples. Among 107 sample
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pairs tested, 53 whole blood samples were positive, compared to only 26 sera. In pairs where whole
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blood and serum were both positive, detection in serum was delayed by 8 PCR cycles on average
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(data not shown).
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Viral RNA was also present in nasal, oral and fecal swabs of all animals whose blood was PCR-positive
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(figure 5). The viral RNA loads in blood samples or swabs, however, were not significantly different
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between the species. In the inoculated animals, viral RNA detection in blood preceded the detection
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in swabs, while the opposite was true for the in-contact animals. Virus was successfully isolated from
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nasal swabs from all tested animals except S6 and G4.
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See figure S1 for an overview of PPRV genome loads in tissue samples. In goats, viral RNA was most
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abundantly found in the alimentary canal, whereas sheep had the highest loads in the lymphatic
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tissues.
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Serology: Early detection of antibodies by ELISA and strong neutralising antibody response in viremic
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animals
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All inoculated sheep and goat G3 were positive in the ELISA from day 6. Goats G1 and G2 had
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seroconverted by day 8 after inoculation. The in-contact animals became positive between days 14
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and 18, only sheep S6 was still negative at the end of the study (figure 7).
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All animals were negative in the NT before infection. The two animals euthanized on day 6 had no
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detectable neutralising antibodies. In the last available serum sample, sheep S4 had a neutralising
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titre of 1/96, and the titres of all other animals were at least 1/512. Sheep S6 remained negative in
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the NT until the end of the study. 9
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Discussion
207 Experimental infection with the Kurdish PPRV isolate exactly reproduced the spectrum of clinical
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signs and pathological lesions that had been observed during the original outbreak (Hoffmann et al.,
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2011) and in PPR-affected small ruminant populations thousands of miles and ten years apart
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(Roeder et al., 1994; Singh et al., 2004). The clinical signs and pathomorphological findings –
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particularly the lesions observed in the alimentary tract – match previous reports of experimental
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infections with other strains of the virus (Brown et al., 1991; Bundza et al., 1988). Our animal
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experiment confirmed the high epizootic potential of the recent Kurdish strain of PPRV (Hoffmann et
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al., 2012). The strain is highly virulent in goats and spreads easily to in-contact animals. At the same
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time, clinical disease was milder in sheep, and one out of three in-contact sheep did not become
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infected. This animal, sheep S6, remained negative in both serological tests, and PPRV RNA was never
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detected in its blood. Swabs (particularly fecal swabs) and tissue samples (from the alimentary tract
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and reproductive organs) were weak positive in the RT-qPCR, but most likely this is the consequence
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of environmental contamination in the animal room. Hammouchi et al. (2012) have also observed
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severe clinical disease after experimental inoculation of a European goat breed (Alpine goats in their
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case) with a lineage IV strain of PPRV. Even though that virus had been isolated in Morocco, it is
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suspected that the strain originated in Asia (Hammouchi et al., 2012), as in the present experiment.
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In another recent experiment, El Harrak et al. (2012) observed a marked difference in disease
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severity depending on the route of inoculation of PPRV. The clinical outcome in animals that had
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been injected subcutaneously was much milder than when the same amount of virus was given
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intranasally. Assuming an intranasal or oral route of infection for the in-contact animals, no such
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difference was observed in the present experiment. However, the total infectious dose that in-
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contact animals were exposed to cannot be determined, and a direct comparison is not possible.
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For all diagnostic procedures, the choice of the samples has a significant impact on the performance
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of the test. The comparison of the real-time RT-PCR results for whole blood and serum confirms that
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the former is much more suitable for PPRV detection. Other studies consistently report good results
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for buffy coat samples (Luka et al., 2012). Presumably, this is due to PPRV replication in lymphocytes,
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which are obviously only included in whole blood preparations and not in sera.
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Goats are generally considered to be more susceptible to PPRV than sheep (Bundza et al., 1988; Delil
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et al., 2012; Singh et al., 2004). While disease severity and contagiosity in sheep appear lower, in a
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European outbreak scenario this could be set off by the greater overall number of heads and the
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higher stocking density in sheep production1. Severe PPR epizootics with thousands of dead sheep
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have been described before (Taylor et al., 2002).
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PPRV is a prominent transboundary disease with a devastating influence on agriculture in Africa and
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Asia. Historically, the international trade risk associated with PPRV was seen as low, because there is
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very little export of animals and animal products from endemic areas (Diallo, 2006). Therefore, from
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a European perspective, its presence in the Levant is particularly troubling (Banyard et al., 2010), and
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our study provides evidence for the high potential impact of the local strains on small ruminant
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production in the European Union.
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Acknowledgements
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Patrick Zitzow, Christian Korthase and Anja Landmesser provided excellent technical assistance.
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Susanne Röhrs’s help with the animal experiment and the dedicated animal care by the staff of the
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isolation unit of the FLI is gratefully acknowledged. Recombinant cell lines used for virus isolation and
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propagation were kindly provided by Carrie Batten and Geoff Pero of the Pirbright Institute and
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Adama Diallo of the International Atomic Energy Agency. This study was financially supported by the
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Eurostat, http://epp.eurostat.ec.europa.eu/portal/page/portal/agriculture/introduction
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ANIHWA project “Improved understanding of the epidemiology of peste-des-petits ruminants”
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(IUEPPR).
256 Conflict of interest statement
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The authors have no financial, personal, or professional interests that inappropriately influenced this
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paper.
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Figure legends
322 Figure 1: Rectal temperature of inoculated and in-contact animals.
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Figure 2: Clinical score of inoculated and in-contact animals.
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Figure 3: Gross pathology. 3a) Goat no. 4, 17 days post inoculation (dpi): Pharynx and tongue
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showing a moderate, acute, diffuse, fibrinosuppurative pharyngitis. 3b) Goat no. 6, 21 dpi: Rumen,
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with a moderate, acute, focally extending ulcerative ruminitis. 3c) Goat no. 6, 21 dpi: Intestine,
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exhibiting an acute intussusception of the cecum. 3d) Goat no. 6, 21 dpi: Intestine, with an acute
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intussusception of the cecum (after removal and opening) showing mild, acute, multifocal,
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hemorrhagic and fibrino-necrotic typhlitis and intramural edema.
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Figure 4: Detection of PPRV RNA in whole blood samples.
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Figure 5: Detection of PRRV RNA in swab samples.
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Figure 6: Competitive PPRV-N-protein-antibody ELISA.
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Figure S1: Detection of PRRV RNA in tissue samples taken at necropsy.
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S0378-1135(14)00246-6 http://dx.doi.org/doi:10.1016/j.vetmic.2014.05.010 VETMIC 6616
To appear in:
VETMIC
Received date: Revised date: Accepted date:
16-9-2013 15-4-2014 3-5-2014
Please cite this article as: Wernike, K., Eschbaumer, M., Breithaupt, A., Maltzan, J., Wiesner, H., Beer, M., Hoffmann, B.,Experimental infection of sheep and goats with a recent isolate of peste des petits ruminants virus from Kurdistan, Veterinary Microbiology (2014), http://dx.doi.org/10.1016/j.vetmic.2014.05.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Experimental infection of sheep and goats with a recent isolate of peste des petits ruminants virus
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from Kurdistan
3 Kerstin Wernike1, +, Michael Eschbaumer1,4, +, Angele Breithaupt2,5, Julia Maltzan3, Henning Wiesner3,
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Martin Beer1,*, Bernd Hoffmann1
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Insel Riems, Germany;
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Institute of Diagnostic Virology, Friedrich-Loeffler-Institut (FLI), Suedufer 10, 17493 Greifswald–
Department of Experimental Animal Facilities and Biorisk Management, Friedrich-Loeffler-Institut
(FLI), Suedufer 10, 17493 Greifswald–Insel Riems, Germany
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+ Both authors contributed equally.
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Academy for the Protection of Zoo and Wild Animals, Munich, Germany
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* Corresponding author. Phone: +49 38351 71200, Fax: +49 38351 71226, E-mail:
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[email protected]
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Center, P.O. Box 848, Greenport, NY, 11944, USA. E-mail: [email protected]
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Universität Berlin, Berlin, Germany. E-mail: [email protected]
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Present affiliation: Foreign Animal Disease Research Unit, USDA/ARS Plum Island Animal Disease
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Present affiliation: Department of Veterinary Pathology, Faculty of Veterinary Medicine, Freie
Short title: PPRV in European sheep and goats
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Highlights
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Experimental infection of both sheep and goats with a recent isolate of peste des petits ruminants virus (PPRV from Kurdistan) PPRV RNA was detected in blood as well as nasal, oral and fecal swabs and tissues The 2011 Kurdish strain of PPRV is highly virulent in European goats and spreads easily to in contact animals Disease severity and contagiosity is lower in sheep
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34 Abstract
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Peste des petits ruminants (PPR) is a contagious viral disease of sheep and goats common in Africa
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and Asia. Its high morbidity and mortality has a devastating impact on agriculture in developing
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countries. As an example, an Asian lineage IV strain of PPRV was responsible for mass fatalities
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among wild goats in Kurdistan in 2010/11.
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In separate experiments, three sheep and three goats of German domestic breeds were
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subcutaneously inoculated with the Kurdish virus isolate; three uninfected sheep and goats were
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housed together with the inoculated animals. All inoculated animals, all in-contact goats and two
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in-contact sheep developed high fever (up to 41.7 °C), depression, severe diarrhea, ocular and nasal
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discharge as well as ulcerative stomatitis and pharyngitis. Infected animals seroconverted within a
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few days of the first detection of viral genome. Clinical signs were more pronounced in goats; four
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out of six goats had to be euthanized. Necropsy revealed characteristic lesions in the alimentary
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tract. Peste des petits ruminants virus (PPRV) RNA was detected in blood as well as nasal, oral and
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fecal swabs and tissues.
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The 2011 Kurdish strain of PPRV is highly virulent in European goats and spreads easily to in-contact
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animals, while disease severity and contagiosity in sheep are slightly lower. PPRV strains like the
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tested recent isolate can have a high impact on small ruminants in the European Union, and
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therefore, both early detection methods and intervention strategies have to be improved and
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updated regularly.
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54 Keywords: Peste des petits ruminants; peste des petits ruminants virus; PPRV; goat plague; ovine
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rinderpest; experimental infection; transmission
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Introduction
59 Peste des petits ruminants (PPR) is a highly contagious viral disease of small ruminants. Like the
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closely related rinderpest virus and the distemper and measles viruses, PPR virus (PPRV) is a
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morbillivirus of the Paramyxoviridae family. Besides sheep and goats, PPR occasionally affects other
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small ruminants in the wild (Munir, 2013). Disease severity depends on various factors such as
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species, breed or virus strain; in severe outbreaks, mortality can reach 100% (Banyard et al., 2010;
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Couacy-Hymann et al., 2007; Diop et al., 2005). PPR is characterized by high fever, thick discharge of
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eyes and nostrils, necrotizing, erosive stomatitis, and enteritis with diarrhea. In fatal infections, death
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is caused by primary viral bronchopneumonia or severe dehydration due to acute diarrhoea (Banyard
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et al., 2010; Couacy-Hymann et al., 2007).
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PPRV was first identified in West Africa in 1942. The virus is currently present all over West and
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Central Africa, the Middle East and Central to South-East Asia in four genetically distinct lineages
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(Banyard et al., 2010; Dhar et al., 2002). Small ruminants are an essential part of the livelihood of
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many subsistence farmers and pastoralists, and PPR is considered the main constraint to increased
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animal production in endemic areas (Diallo, 2006). It has never been reported in the European Union,
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but is regarded as a credible threat to domestic agriculture (Libeau et al., 2011).
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A lineage IV strain of PPRV was presumed responsible for mass fatalities among wild goats in
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Kurdistan in 2010/11 (Hoffmann et al., 2012), and the present study intended to confirm the
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virulence of the Kurdish strain in an animal experiment. Furthermore, the potential impact of an
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introduction of this strain into the European Union was investigated by using European breeds of
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sheep and goats for the experimental infection.
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Materials and Methods
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Virus
84 PPRV was isolated from a swab sample of a Kurdish goat that had died during the 2010/11 outbreak.
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After initial isolation on grivet kidney (CV1) cells expressing the caprine signalling lymphocyte
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activation molecule (SLAM) (Adombi et al., 2011), the virus was passaged once on Vero/dog-SLAM
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cells (von Messling et al., 2003) for use in the animal experiment. The isolate has been designated
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PPRV Kurdistan/2011, and partial nucleocapsid gene sequence information is available online
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(GenBank accession no. JF969755).
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91 Animals and experimental design
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An infection experiment with sheep and goats of German domestic breeds was carried out in the
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BSL3-ag containment facility of the Friedrich-Loeffler-Institut, Insel Riems. Three sheep (S1-S3) and
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three goats (G1-G3) were subcutaneously inoculated with 1x10^4 TCID50 of virus. Sheep and goats
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were kept in separate rooms. Three uninfected sheep (S4-S6) and goats (G4-G6), respectively, were
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housed together with the inoculated animals. After inoculation, clinical signs and rectal body
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temperature were recorded daily. Whole blood, serum, nasal, oral and fecal swabs were taken every
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other day beginning on day 2. Seven days after inoculation one sheep and one goat (S3/G3) were
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euthanized and necropsied. The remaining goats were euthanized on days 12 (G1/G2), 17 (G4), 18
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(G5) and 20 (G6), the sheep 20 days after infection. A diverse panel of tissues was taken at necropsy
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(14 samples from the alimentary canal, 3 from the respiratory tract, 7 lymphatic tissues, as well as
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liver, brain, kidney, heart and reproductive organ samples).
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The experimental protocol was reviewed by an independent ethics commission pursuant to §15 of
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the German Animal Welfare Act, and it has been approved by the competent authority (State Office
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for Agriculture, Food Safety and Fisheries of Mecklenburg-Vorpommern, Rostock, Germany; ref.
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LALLF M-V/TSD/7221.3-2.5-004/11).
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Clinical observations were recorded and scored with a rubric similar to the one used by El Harrak et
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al. (2012). Depending on the severity of clinical signs, up to three points were awarded in the
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categories “general attitude”, “congestion and edema”, “oculonasal discharge”, “mucosal lesions”,
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“diarrhea” and “rectal temperature”. Daily scores and a total cumulative score for the entire
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experiment were calculated for each animal.
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Total RNA from bloods, swabs and tissue samples was extracted using the NucleoSpin® 96 RNA Kit
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(Macherey-Nagel, Düren, Germany) on a MICROLAB® STAR liquid handling workstation (Hamilton,
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Bonaduz, Switzerland) according to the manufacturer’s recommendations. PPRV genome load in the
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samples was determined by an N gene-specific RT-qPCR as described previously (Kwiatek et al., 2010)
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with an external RNA standard. The tissue samples were homogenized and tested for the presence of
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PPRV RNA by real-time RT-PCR as well.
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Virus isolation from one nasal swab per animal (day 8 for inoculated animals, day 16 for in-contact
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animals) was attempted on CV1/goat-SLAM cells. G3 and S3 were not tested. Five days after culture
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inoculation, the cells were screened for cytopathic effects (cell death, syncytia) and stained with a
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monoclonal antibody against the PPRV N protein (ID Vet, Montpellier, France). N-protein-positive
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cells were considered infected even in the absence of a visible cytopathic effect.
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All serum samples were tested with a commercially available, competitive PPR-N-protein-antibody
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ELISA (ID Vet, Montpellier, France) using the recommended cut-off of 60% relative optical density
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compared to the negative control. Serum samples taken before infection and on the last sampling
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day before euthanasia (day 6 for S3 and G3, day 12 for G1 and G2, day 16 for G4, day 18 for G5 and
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day 20 for the remaining animals) were further analysed in a standard micro-neutralization test (NT)
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on Vero/dog-SLAM cells against the homologous virus isolate.
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Clinical and virological data collected in the experiment were fitted to a linear mixed-effects model
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using R (R Development Core Team, 2009). Differences between groups were examined by Tukey’s
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all-pair comparisons using the Bonferroni method for multiplicity adjustment of p values. Adjusted p
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values < 0.01 were considered significant.
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Results
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Clinical scores: More severe disease in goats
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The first clear clinical signs appeared within 4 to 5 days post inoculation. Eventually, all inoculated
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animals, all in-contact goats and two out of three in-contact sheep developed high fever (up to
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41.7 °C, figure 1) and depression, severe diarrhea, mucopurulent ocular and nasal discharge as well
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as ulcerations in the oral and pharyngeal mucosa. An overview of the clinical scores in the
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experiment is provided by Figure 2. In general, signs of disease were more severe in goats and lasted
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longer than in sheep; four out of six goats had to be euthanized for humane reasons. The average
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total cumulative score over the course of the experiment in sheep was 22, compared to 41 in goats.
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No in-contact sheep ever exceeded a daily clinical score of 4, whereas all three in-contact goats did
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(maximum scores of 15, 8 and 5, respectively).
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For the statistical analysis, needle-inoculated and in-contact animals were examined separately. Over
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the course of the experiment, the difference in clinical scores between goats and sheep was found to
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be significant, independent of the route of exposure.
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158 Gross pathology: Moderate to severe lesions in multiple organs
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At autopsy, acute, diffuse, catarrhal to catarrhal-purulent rhinitis and sinusitis were found. The oral
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cavity exhibited an acute fibrinosuppurative, partially erosive to ulcerative stomatitis and pharyngitis
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(e.g. figure 3a). Frequently, the lung parenchyma showed multifocal to coalescing areas of
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consolidation affecting 5-15% of lung lobes with adjacent moderate to severe, multifocal to diffuse,
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acute, alveolar emphysema and a mild to moderate, acute, diffuse alveolar edema. Further, an acute,
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focal, extending erosive to ulcerative ruminitis was evident (figure 3b).
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There were moderate, multifocal petechiae, ecchymoses and erosions within the intestinal mucosa,
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predominantly affecting the ileum, cecum and colon. Several animals had an acute, focally extending
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fibrino-necrotizing and hemorrhagic enteritis (i. e. typhlocolitis, also affecting the ileocecal valve).
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Two animals (G3, G6) showed an acute intussusception of the caecum with varying degrees of
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hemorrhagic infarction (figure 3c-d). The mesenteric lymph nodes were moderately to severely
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enlarged. Besides these main findings, intestinal and pulmonary nematodes and associated lesions
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were found in several animals.
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PCR and virus isolation: Abundant viral RNA in whole blood, swabs and tissues
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In whole blood samples of the inoculated animals, viral RNA was first detected on day 4. It was most
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abundant between 4 and 6 days after inoculation, but remained detectable until the end of the
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study. In the two in-contact goats, PPRV RNA could be found for the first time on day 10, in the third 8
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goat on day 12. Two in-contact sheep were positive from days 12 and 16, respectively. In whole
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blood samples of the last sheep (S6), PPRV RNA was not detected at any time (figure 4). In general,
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PPRV RNA was more readily detected in whole blood than in serum samples. Among 107 sample
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pairs tested, 53 whole blood samples were positive, compared to only 26 sera. In pairs where whole
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blood and serum were both positive, detection in serum was delayed by 8 PCR cycles on average
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(data not shown).
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Viral RNA was also present in nasal, oral and fecal swabs of all animals whose blood was PCR-positive
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(figure 5). The viral RNA loads in blood samples or swabs, however, were not significantly different
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between the species. In the inoculated animals, viral RNA detection in blood preceded the detection
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in swabs, while the opposite was true for the in-contact animals. Virus was successfully isolated from
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nasal swabs from all tested animals except S6 and G4.
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See figure S1 for an overview of PPRV genome loads in tissue samples. In goats, viral RNA was most
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abundantly found in the alimentary canal, whereas sheep had the highest loads in the lymphatic
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tissues.
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Serology: Early detection of antibodies by ELISA and strong neutralising antibody response in viremic
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animals
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All inoculated sheep and goat G3 were positive in the ELISA from day 6. Goats G1 and G2 had
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seroconverted by day 8 after inoculation. The in-contact animals became positive between days 14
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and 18, only sheep S6 was still negative at the end of the study (figure 7).
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All animals were negative in the NT before infection. The two animals euthanized on day 6 had no
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detectable neutralising antibodies. In the last available serum sample, sheep S4 had a neutralising
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titre of 1/96, and the titres of all other animals were at least 1/512. Sheep S6 remained negative in
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the NT until the end of the study. 9
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Discussion
207 Experimental infection with the Kurdish PPRV isolate exactly reproduced the spectrum of clinical
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signs and pathological lesions that had been observed during the original outbreak (Hoffmann et al.,
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2011) and in PPR-affected small ruminant populations thousands of miles and ten years apart
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(Roeder et al., 1994; Singh et al., 2004). The clinical signs and pathomorphological findings –
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particularly the lesions observed in the alimentary tract – match previous reports of experimental
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infections with other strains of the virus (Brown et al., 1991; Bundza et al., 1988). Our animal
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experiment confirmed the high epizootic potential of the recent Kurdish strain of PPRV (Hoffmann et
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al., 2012). The strain is highly virulent in goats and spreads easily to in-contact animals. At the same
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time, clinical disease was milder in sheep, and one out of three in-contact sheep did not become
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infected. This animal, sheep S6, remained negative in both serological tests, and PPRV RNA was never
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detected in its blood. Swabs (particularly fecal swabs) and tissue samples (from the alimentary tract
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and reproductive organs) were weak positive in the RT-qPCR, but most likely this is the consequence
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of environmental contamination in the animal room. Hammouchi et al. (2012) have also observed
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severe clinical disease after experimental inoculation of a European goat breed (Alpine goats in their
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case) with a lineage IV strain of PPRV. Even though that virus had been isolated in Morocco, it is
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suspected that the strain originated in Asia (Hammouchi et al., 2012), as in the present experiment.
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In another recent experiment, El Harrak et al. (2012) observed a marked difference in disease
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severity depending on the route of inoculation of PPRV. The clinical outcome in animals that had
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been injected subcutaneously was much milder than when the same amount of virus was given
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intranasally. Assuming an intranasal or oral route of infection for the in-contact animals, no such
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difference was observed in the present experiment. However, the total infectious dose that in-
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contact animals were exposed to cannot be determined, and a direct comparison is not possible.
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For all diagnostic procedures, the choice of the samples has a significant impact on the performance
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of the test. The comparison of the real-time RT-PCR results for whole blood and serum confirms that
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the former is much more suitable for PPRV detection. Other studies consistently report good results
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for buffy coat samples (Luka et al., 2012). Presumably, this is due to PPRV replication in lymphocytes,
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which are obviously only included in whole blood preparations and not in sera.
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Goats are generally considered to be more susceptible to PPRV than sheep (Bundza et al., 1988; Delil
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et al., 2012; Singh et al., 2004). While disease severity and contagiosity in sheep appear lower, in a
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European outbreak scenario this could be set off by the greater overall number of heads and the
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higher stocking density in sheep production1. Severe PPR epizootics with thousands of dead sheep
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have been described before (Taylor et al., 2002).
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PPRV is a prominent transboundary disease with a devastating influence on agriculture in Africa and
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Asia. Historically, the international trade risk associated with PPRV was seen as low, because there is
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very little export of animals and animal products from endemic areas (Diallo, 2006). Therefore, from
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a European perspective, its presence in the Levant is particularly troubling (Banyard et al., 2010), and
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our study provides evidence for the high potential impact of the local strains on small ruminant
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production in the European Union.
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Acknowledgements
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Patrick Zitzow, Christian Korthase and Anja Landmesser provided excellent technical assistance.
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Susanne Röhrs’s help with the animal experiment and the dedicated animal care by the staff of the
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isolation unit of the FLI is gratefully acknowledged. Recombinant cell lines used for virus isolation and
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propagation were kindly provided by Carrie Batten and Geoff Pero of the Pirbright Institute and
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Adama Diallo of the International Atomic Energy Agency. This study was financially supported by the
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Eurostat, http://epp.eurostat.ec.europa.eu/portal/page/portal/agriculture/introduction
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ANIHWA project “Improved understanding of the epidemiology of peste-des-petits ruminants”
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(IUEPPR).
256 Conflict of interest statement
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The authors have no financial, personal, or professional interests that inappropriately influenced this
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paper.
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Figure legends
322 Figure 1: Rectal temperature of inoculated and in-contact animals.
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Figure 2: Clinical score of inoculated and in-contact animals.
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Figure 3: Gross pathology. 3a) Goat no. 4, 17 days post inoculation (dpi): Pharynx and tongue
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showing a moderate, acute, diffuse, fibrinosuppurative pharyngitis. 3b) Goat no. 6, 21 dpi: Rumen,
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with a moderate, acute, focally extending ulcerative ruminitis. 3c) Goat no. 6, 21 dpi: Intestine,
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exhibiting an acute intussusception of the cecum. 3d) Goat no. 6, 21 dpi: Intestine, with an acute
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intussusception of the cecum (after removal and opening) showing mild, acute, multifocal,
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hemorrhagic and fibrino-necrotic typhlitis and intramural edema.
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Figure 4: Detection of PPRV RNA in whole blood samples.
332
Figure 5: Detection of PRRV RNA in swab samples.
333
Figure 6: Competitive PPRV-N-protein-antibody ELISA.
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Figure S1: Detection of PRRV RNA in tissue samples taken at necropsy.
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