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Early pathogenesis of classical swine fever virus (CSFV) strains in Danish pigs
Veterinary Microbiology 159 (2012) 327–336
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Early pathogenesis of classical swine fever virus (CSFV) strains in Danish pigs Louise Lohse *, Jens Nielsen, A˚se Uttenthal National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 February 2012 Received in revised form 19 April 2012 Accepted 20 April 2012
Host–virus interactions play an important role for the clinical outcome of classical swine fever virus (CSFV) infections in pigs. Strain virulence, host characteristics and environment are all factors that markedly influence disease severity. We tested CSFV strains of varying virulence in an experimental set-up, reducing the influence of host and environmental factors. Thus, weaner pigs were inoculated with one of 4 CSFV strains in order to compare the pathogenesis for a 3-week-period after infection. CSFV strains selected were 2 new and 2 previously characterized. None of these strains had been tested in Danish outbred pigs before. Clinical observations grouped the infected pigs into two different categories reflecting either non-specific, mainly gastro-intestinal, problems, or severe disease including high fever within the first week after inoculation. Gross-pathological findings varied between strains, however, lymphoid atrophy and growth retardation represented a consistent finding for all 4 strains. Virus distribution, viral load and in particular virus persistence differed, but supported present practice that recommends lymphoid tissue, most optimal tonsil and lymph nodes, as target material to be applied for early laboratory diagnosis. The present study demonstrated constraints associated with early detection of infections with CSFV strains of low virulence. Since neither clinical symptoms nor pathological lesions observed with these strains constituted characteristic signs of CSF, the risk of neglecting a CSF suspicion is immediate. Therefore, topical information on new outbreaks and continuous enhancement of an efficient surveillance system is of great importance to prevent further spread of CSF within the pig population. ß 2012 Elsevier B.V. All rights reserved.
Keywords: CSFV Strain difference Host–virus interaction Clinical score Pathological score Virus distribution Pathogenesis Pig
1. Introduction Classical swine fever (CSF) is a serious, contagious infection in pigs. It is a listed disease of the World Organization for Animal Health (OIE) causing major damage to the pig population worldwide. The disease is caused by classical swine fever virus (CSFV), a pestivirus belonging to the Flaviviridae family (http://ictvonline.org/ virusTaxonomy.asp?version=2011). The disease mechanisms of CSFV infection are complex, as specific host–virus interactions determine disease
* Corresponding author. Tel.: +45 35887831. E-mail address: [email protected] (L. Lohse). 0378-1135/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2012.04.026
development and clinical outcome. Host factors, such as age, genetic background and immune status, herd sanitary status as well as strain virulence are known to play highly important roles for the course of disease (Depner et al., 1997; Floegel-Niesmann et al., 2003; Blacksell et al., 2006). Recent studies in our laboratory revealed that experimental infection of Danish pigs with the highly virulent CSFV-Eystrup strain (Mittelholzer et al., 2000; Mayer et al., 2003; Durand et al., 2009) produced severe (Uttenthal et al., 2008), moderate (Rasmussen et al., 2007) or even mild (Nielsen et al., 2010) disease in pigs depending on age and sanitary status. These observations drew further attention to a putative situation reflecting country to country variations in the clinical expression of infection with a particular strain of CSFV.
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In many European countries, CSF has been eradicated. However, sporadic as well as massive new outbreaks occur from time to time. Within the last decades, large CSF epidemics have had significant impact on the pig production together with the national economy of several western European countries. Thus, during outbreaks, national as well as international trade of animals and products has been severely hampered for longer periods due to transport restrictions and import/export bans (Elbers et al., 1999; Koenen et al., 1996; Fritzemeyer et al., 2000; Pol et al., 2008). Further, ethical issues concerning animal welfare related to stamping out procedures including not-infected animals from premises located within restrictions zones have been debated. The possibilities of extensive spread of the disease facilitated by modern infrastructure, including intensive transportation and moving/mixing of animals in agriculture, combined with difficulties in establishing an early clinical diagnosis of CSF by the veterinary practitioners, represents a significant problem in outbreak situations. A set of diagnostic tools for laboratory confirmation of a CSFV suspicion is well established at the majority of national reference laboratories within EU. However, a confirmatory laboratory diagnosis needs optimal sample material provided for examination. Therefore, it is of outmost importance that (1) clinical facets of CSF disease become scientifically elucidated and that (2) new knowledge is communicated to the veterinary society to facilitate early detection of an outbreak of CSF by veterinary practitioners. Several experiments have been performed in different countries to characterize virulence and clinical appearance of different CSFV strains in pigs, (Mittelholzer et al., 2000; Floegel-Niesmann et al., 2003, 2009; Kaden et al., 2004; Everett et al., 2010; Nielsen et al., 2010) and scoring systems for objective comparison of individual strains (Mittelholzer et al., 2000; Floegel-Niesmann et al., 2003) have been developed. In order to provide updated information on the putative course of an introduction of CSF in Danish commercial production sites, we compared the performance of different strains in experimentally infected Danish commercial weaner pigs. The overall goal of the present study was to generate information aiming to shorten the time perspective from initial clinical symptoms at an index case locality to a final confirmed diagnosis at the national reference laboratory. The candidate strains for this study included strains of expected low virulence, as such strains are difficult to diagnose at an early time point due to their atypical clinical expression. Strains that are known to circulate in close proximity to Denmark are considered to constitute a current high risk to the Danish pig production. Therefore, we also studied the clinical outcome of 2 strains obtained from recent outbreaks in Eastern Europe and Israel, respectively. 2. Materials and methods 2.1. Animals In total, 60 weaner pigs, 10–16 kg, were obtained from a conventional Danish swine herd with specific pathogen
free (SPF) status, including freedom from enzootic pneumoniae, atrophic rhinitis, swine dysentery, porcine reproductive and respiratory syndrome and most serotypes of Actinobacillus pleuropneumoniae. All pigs were found to be healthy by veterinary inspection at arrival to the Institute, 1 week before experimental start. All pigs were housed in the high containment experimental facilities at the National Veterinary Institute. All pigs were fed a commercial diet for weaning pigs and water was provided ad libitum. Straw was used for bedding. 2.2. Inocula The following strains of CSFV were used for inoculation in the two experiments: CSF0911: Glentorf (genotype 1.1), originally isolated from domestic pigs in Germany, 1968. Characterized to be of low virulence (Meyer et al., 1981), kindly supplied by the Community Reference Laboratory (CRL) for CSF in Hannover. CSF1019: Romania TM/120/07 (genotype 2.3), field isolate obtained from domestic pigs in Romania, 2007. Characterized to be of moderate/high virulence, kindly supplied by the CRL, Hannover, after permission from Dr. Olaru, National Reference Laboratory (NRL), Romania. CSF0906: v.d. Bergen (genotype 2.2), originally isolated from domestic pigs in The Netherlands, 1977. Characterized to be of low virulence (Van Oirschot and Terpstra, 1977), kindly supplied by the CRL, Hannover. CSF1047: Israel (genotype 2.1), field isolate obtained from domestic pigs in Israel, 2009. Characterized to be of high virulence, kindly supplied by the CRL, Hannover, after permission from Dr. Yadin, Kimron Veterinary Institute (KVI), Israel. All experimentally infected pigs were inoculated intranasally with a 50% tissue culture infective dose (TCID50) of 105/pig in 2 ml Eagle’s minimal essential medium (EMEM). 2.3. Design of experiment The study was performed as two subsequentially performed experiments (experiment I and experiment II), with identical experimental designs. For each experiment, 30 pigs were randomly divided into 3 groups, each comprising 10 individuals. On post infection day (PID) 0, the three groups in experiment I were treated as follows: (1) control group, pigs mock-inoculated intranasally with 2 ml EMEM, (2) infected group, pigs inoculated with CSFV-Glentorf, and (3) infected group, pigs inoculated with CSFV-Romania. For experiment II, the inoculation material used for the 2 infected groups was CSFV-Bergen and CSFV-Israel, respectively. Daily, all pigs were clinically monitored, including clinical scoring (Mittelholzer et al., 2000) and recording of body temperature. Blood samples were collected from the jugular vein on PID 0–7, 10/11, 15, 21/22. Three, three and four pigs from each group were killed on PID 5, 10/11, and
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21/22, respectively. Selection of pigs for termination in experiment on individual days was based on clinical score and blood parameters, more specifically, pigs with low total white blood cell count and clear clinical signs were selected before pigs with no affection. In the control groups and groups infected with low virulent virus, pigs were selected at random. Pigs, which displayed clinical symptoms that reached the humane endpoint criteria during the course of experiment, were killed when necessary. These criteria included parameters such as severe affection of general well-being, significant weight loss, dyspnea, convulsions and pain. All pigs were killed by intravenous injection of sodium pentobarbital. All pigs were autopsied. Experimental procedures and animal manage protocols were carried out in accordance with the requirements of the Danish Animal Experimentation Inspectorate, license no. 2003/561-742 and 2008/561-1541.
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2.7. CSFV specific antibodies Serum samples were screened for antibodies to CSFV in a blocking ELISA (Have, 1984). Doubtful or positive samples were further titrated in neutralisation peroxidase-linked assay (NPLA) using CSFV-Alfort 187 in PK-15 cells, previously described by Hyera et al. (1987). 2.8. Statistics Data analysis was performed using GraphPad In Stat version 3.00 (GraphPad Software, San Diego, CA). Unpaired t-test, or alternatively t-test with Welch correction, was used for comparison between means of groups. 3. Results 3.1. Clinical findings
2.4. Clinical monitoring All pigs were weighed at the beginning and termination of the experiment. Daily, clinical examination was performed on the individual pig and a clinical score (CS) was assigned based on a semi-quantitative scoring system developed by Mittelholzer et al. (2000). The individual animal was scored on 10 CSF-relevant parameters (liveliness, body tension, body shape, breathing, walking, skin, eyes/conjunctiva, appetite, defecation, leftovers in feeding trough), using scores from 0 (normal) to 3 (severe CSF symptom), giving a maximum score of 30. One parameter (leftovers in feeding trough) was assigned to each pig as a joint score per box, since the pigs in one box were fed in a common through. Rectal body temperature was recorded on a daily basis. Fever was defined as rectal body temperature 40.0 8C for pigs in this age group. 2.5. Pathological score At autopsy, a pathological score (PS) was calculated for each pig, based on a scoring system developed by FloegelNiesmann et al. (2003). Nine parameters including pathologically important organs for CSF infection (skin, subcutis/ serosa, tonsil, spleen, kidney, lymph nodes, ileum/rectum, brain, respiratory system) were evaluated and scored according to a scale ranging from 0 (no alteration) to 3 (severe CSF lesion), giving a maximum score of 27. 2.6. Virus detection Virus isolation and detection by immunoperoxidase staining was carried out on PK-15 cells and performed on serum samples and selected organs of all pigs in the study using standard diagnostic procedures, as previously described (Uttenthal et al., 2003). Organ materials selected for examination were: Tonsils and lymphoid tissues from spleen, mandibular, ileo-caecal and inguinal lymph nodes together with kidney, lung, and brain tissues. Virus RNA was detected on corresponding samples by silica-based manual extraction of RNA (Uttenthal et al., 2003) followed by quantitative real-time RT-PCR, previously described by Rasmussen et al. (2007).
3.1.1. Body temperature Mean rectal temperatures for the groups included in the 2 experiments are shown in Fig. 1A–D. Pigs inoculated with moderate to highly virulent CSFV-strains developed fever from PID 4 (CSFV-Israel, Fig. 1D) or PID 5 (CSFV-Romania, Fig. 1B), respectively. For both groups, body temperatures continuously remained above the level of control groups, reaching 41 8C or more before termination of the experiment. Pigs inoculated with the CSFV-strains of low virulence, showed body temperatures close to the level of control pigs (CSFV-Glentorf, Fig. 1A, and CSFV-Bergen, Fig. 1C). Significant difference (p < 0.05) in rectal body temperature between inoculated pigs and control pigs was recorded for 5 days (PID 1, 5, 11, 16, 19) for CSFV-Glentorf and 11 days (PID 2, 3, 4, 5, 6, 7, 8, 12, 14, 16, 20) for CSFVBergen. However, the significance was based on two-sided test statistics, so specified time points included deviation from control group at both higher and lower mean rectal temperature level. At one time point only, mean temperature for CSFV-Bergen inoculated pigs exceeded 40.0 8C (PID 7 = 40.1 0.3 8C). 3.1.2. Growth performance Growth retardation was observed in all infected groups. Weight difference from start to termination in experiment was calculated for the individual pig and means for the infected groups compared to the control groups (Table 1). After two weeks of infection, three out of four groups (CSFV-Glentorf, CSFV-Romania and CSFV-Israel) showed a significant lower growth rate than corresponding control pigs (p-values < 0.01). Three weeks after inoculation, mean body weight in the infected groups was still less than the corresponding control group, but only CSFV-Romania showed significant weight difference (p-value < 0.001). 3.1.3. Clinical score A total score for each individual pig per day was calculated and clinical scores (CS) were compared by groups. CS in the control groups of the two experiments did not exceed 2, except for one pig which showed slight depression probably due to minor gastro-intestinal problems and scored 4 on PID 2. Therefore, CS equal to 4 or less
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Fig. 1. Rectal temperature – mean values. Each symbol represents the average SD for all pigs in the group recorded on the specific reading day. Control group = black symbol, CSFV-Glentorf = green symbol (A), CSFV-Romania = red symbol (B), CSFV-Bergen = blue symbol (C) and CSFV-Israel = purple symbol (D). At the x-axis is indicated number of pigs within each group in the specified time interval. * = 1 pig infected with CSFV-Romania was killed at PID 15 for animal welfare reasons, only 3 pigs left in this group from PID 16 and onwards.
than 4 were considered to represent normal background values in this study and as such not reflecting clinical indication of an early CSF infection. It should be noted that in other experimental settings using other strains of CSFV Table 1 Mean difference in body weight per kg from start to termination in experiment for pigs killed in week 2 and 3, respectively. Calculated pvalues describe the difference between inoculated groups and corresponding control group. Group
Weeks of infection
CSFV-Glentorf CSFV-Romania Control exp. I CSFV-Bergen CSFV-Israel Control exp. II CSFV-Glentorf CSFV-Romania Control exp. I CSFV-Bergen CSFV-Israel Control exp. II
2 2 2 2 2 2 3 3 3 3 3 3
Mean body weight gain in kg 1.8 0.8 5.5 0.6 0.4 1.9 4.9 0.7 6.9 3.6 –a 4.7
Number of pigs
p-Value
3 3 3 3 7 3 4 4 4 4 – 4
<0.001 <0.001 – ns <0.01 – ns <0.001 – ns – –
a The experiment was terminated at PID 11 for pigs infected with CSFVIsrael for animal welfare considerations, therefore no data exists for this group in week 3.
or pigs with a different sanitary status, another CS limit may be appropriate. Individual CS for pigs included in the 2 experiments is shown in Fig. 2A–D. Pigs infected with moderate to highly virulent CSFV strains (CSFV-Romania, Fig. 2B, and CSFV-Israel, Fig. 2D) showed CS indicative of a CSF infection from PID 5 and onwards, with clinical signs continuously progressing into a more severe state until the pigs were killed for animal welfare reasons. Initial clinical signs were non-specific and expressed as fever, depression, anorexia and fecal changes. Ataxia developed with progression of infection around PID 8. Furthermore, several of the infected pigs showed a changed visual appearance caused by a bristled coat. Pigs infected with the low virulent strains experienced less clinical signs. Positive CS was observed for CSFV-Bergen (Fig. 2C) on PID 8, 15, 16 and for CSFV-Glentorf (Fig. 2A) on PID 9, predominantly reflecting gastro-intestinal disorders. All pigs belonging to the groups infected with low virulent strains were killed in accordance with the experimental protocol. 3.2. Pathological findings Post mortem (PM) examination was performed on all pigs and a pathological score was designated to each individual pig. General observations for all 4 strains,
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Fig. 2. Clinical score – individual pigs. Control pigs = black symbol, CSFV-Glentorf = green symbol (A), CSFV-Romania = red symbol (B), CSFV-Bergen = blue symbol (C) and CSFV-Israel = purple symbol (D). The dotted line is inserted at CS = 4, lower CS is regarded as background, not representing symptoms indicative of CSFV.
irrespective of strain virulence, represented lymphoid atrophy (Photo 1). This characteristic was recorded for the tonsils, the thymus and the ileal peyer patches already at PM’s performed after one week of infection. Furthermore, hemorrhages visible as blebs in the border of the spleen, petechial bleedings in the kidney together with swellings and petechial bleedings in the lymph nodes were observed for CSFV-Israel and CSFV-Romania. Skin hyperemia and cyanosis was often observed for CSFV-Romania infected pigs from week 2. Lesions included in the pathological score were by far most pronounced in pigs from the CSFVRomania infected group (Fig. 3). However, pigs infected with CSFV-Israel had to be killed before week 3 for animal welfare reasons. 3.3. Virus detection and distribution To elucidate infection kinetics in CSFV infected pigs, the dissemination of virus in the inoculated pigs was followed by virus isolation (VI) and RT-PCR analysis. The examined material was grouped as follows: (1) primary infection and replication of virus: tonsil, (2) viraemia: blood, (3) dissemination of virus to secondary lymphoid target organs: spleen and lymph nodes, (4) dissemination of virus to non-target visceral organs: lung and kidney, and (5) virus crossing the blood–brain barrier: brain. Organ
material was collected every week in continuation of PM examination of killed pigs. A sample was designated positive if the Ct-value in the PCR analysis was less than 40 and/or VI titer was equal to or above 10. In general, RT-PCR analysis was more sensitive in detecting CSFV RNA than VI in detecting live, propagating virus. To provide an overview of virus distribution within the pigs infected with the individual strains, RT-PCR results for material representing the five selected compartments is shown in Table 2. Viral RNA detected in serum from samples collected during the experimental period is shown in Fig. 4A–D. Ct-values in serum from CSFV-Glentorf infected pigs were only 34, which were considerably lower than for pigs infected with CSFV-Romania, CSFV-Bergen and CSFV-Israel. Pigs in these groups reached Ct-values of 26 or less. This difference in Ct-values reflects a 1000 times lower virus load in serum in CSFV-Glentorf infected pigs than in any of the other strains. Further details of interest, including VI results diverging from RT-PCR, for the individual strains are described in the following: CSFV-Glentorf: Virus was detected in serum in the second week of infection. In total, only 2 out of 7 pigs were positive for viral RNA in serum (Fig. 4A). Unexpectedly, VI showed a higher sensitivity than PCR and detected virus in 5 out of 7 pigs (data not shown). Virus persisted in serum only for a short time period (PID 7–15), irrespective of the
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Photo 1. Lymphoid atrophy. Tonsil: A1 = normal tissue from control pig, A2 = atrophied tissue in CSFV-infected pig. Thymus: B1 = normal tissue from control pig, B2 = atrophied tissue in CSFV-infected pig. Focus areas are encircled in blue.
method of analysis used. Examination of organ material revealed that virus persisted for the longest time period (in 3/4 pigs until the end of experiment) and in highest concentrations in tonsils (Ct 30) and in lymph nodes (Ct 32–36). Virus could not be detected in the brain of any of the pigs in this group (Table 2). In 2 out of 10 pigs
Fig. 3. Pathological score – mean values. Each column represents the average SD for all pigs in the group subjected to PM examination within the specified week. Control group = black symbol, CSFV-Glentorf = green symbol, CSFV-Romania = red symbol, CSFV-Bergen = blue symbol and CSFVIsrael = purple symbol. a = There are no CSFV-Israel infected pigs in week 3. All pigs in this group were killed at PID 11 or before.
(killed PID 5 and PID 22, respectively) neither live virus nor viral RNA could be detected at any time during the experiment. CSFV-Romania: In serum, virus was detected from PID 3 and onwards (Fig. 4B). In the second week of infection, seven out of 7 pigs were positive and the virus load gradually increased until termination of experiment. With regard to organ distribution, virus could be detected in tonsils, lymphoid tissue and visceral organs from all examined pigs. Virus could not be detected in brain tissue until 2nd and 3rd week of the experiment. Throughout the experiment, the viral load was highest in tonsils (Ct 26) and in lymph nodes (Ct 25–27) and lowest in brain (Ct 31). CSFV-Bergen: Virus was detected in serum mainly in the second week of infection. In total 4 out of 7 pigs were positive for viral RNA in serum (Fig. 4C), while VI detected 7 positive out of 7 pigs in the same time period. Examination of organ material revealed the highest viral load in tonsils (Ct 31) and in lymph nodes (Ct 32) and lowest viral load in visceral organs (Ct 36) and brain (Ct 35). By termination of the experiment, live virus could not be isolated and only low amounts of viral RNA (Ct 33–37) were detected in tonsils and in lymph nodes of 4/4 and 2/4 pigs, respectively. CSFV-Israel: Virus was detected in serum from PID 3 and all pigs became virus positive in the first week of infection
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Table 2 Virus distribution in pigs inoculated with CSFV. Results of RT-PCR analysis of serum and tissue material collected in connection to PM examination of sequentially killed pigs in a number of 3, 3 and 4 per week, during the 3-week experimental period. CSFV-strain
Virulence
CSFV-Glentorf Tonsil Serum Lymphoid tissuea Visceral organsb Brain CSFV-Romania Tonsil Serum Lymphoid tissue Visceral organs Brain CSFV-Bergen Tonsil Serum Lymphoid tissue Visceral organs Brain CSFV-Israel Tonsil Serum Lymphoid tissue Visceral organs Brain
Low
a b c
Week 1 (PID 0–6) 2/3 0/3 2/3 2/3 0/3
Week 2 (PID 7–13) RT-PCR positive (Ct value <40)/total 3/3 0/3 3/3 3/3 0/3
Week 3 (PID 14–22) 3/4 0/4 3/4 0/4 0/4
Moderate/high 3/3 0/3 3/3 3/3 0/3
3/3 3/3 3/3 3/3 3/3
4/4 4/4 4/4 4/4 4/4
3/3 0/3 3/3 3/3 2/3
3/3 3/3 3/3 2/3 2/3
4/4 0/4 2/4 0/4 0/4
3/3 3/3 3/3 3/3 3/3
7/7 7/7 7/7 7/7 7/7
ndc nd nd nd nd
Low
High
Lymphoid tissue includes spleen and lymph nodes (Ln. mandibularis, Ln. inguinalis and Ln. ileocaecalis). Visceral organs includes lung and kidney. nd = not done – the pigs were dead PID 11.
(Fig. 4D). Viral load in serum increased gradually until PID 11, when the pigs were killed. By examination of organ material, high loads of viral RNA were detected in all organs, the highest levels found in tonsils (Ct 26), and the lowest in brain (Ct 33). 3.4. Development of CSFV specific antibodies Neutralizing antibodies were developed against CSFV low virulent strains and could be detected in serum at PID 22 (last day of the experiment), when 2/4 (CSFV-Glentorf) and 4/4 (CSFV-Bergen) pigs had seroconverted displaying titers ranging between 14 and 80. One of the 2 antibody negative CSFV-Glentorf pigs was never detected virus positive and may therefore be considered as non-infected. Pigs infected with moderate or highly virulent strains did not develop any antibody response at all. However, none of the pigs infected with these strains survived beyond PID 11 (CSFV-Israel) and PID 18 (CSFV-Romania), respectively. 4. Discussion The low specificity of clinical signs in the initial phase of CSFV infection accentuates the difficulties associated with early detection of CSF (Moennig et al., 2003). Some of the fatal consequences related to delayed CSF diagnosis can be illustrated by the progression of the severe epidemics of CSF in The Netherlands in 1997–1998 and in England in 2000, when the time between introduction of CSFV into the country and diagnosis of CSF in the primary outbreak was estimated to approximately 6 weeks (Elbers et al., 1999; Sandvik et al., 2000), thus enabling extensive further spread of the disease among herds. These experiences
underline the urgent need for improvements of the possibilities for early diagnosis of CSF in order to prevent similar epidemic catastrophes. A socio-psychological investigation (Elbers et al., 2010) identified 6 themes limiting early detection of CSF outbreaks and a number of solutions to address this issue were suggested. One of these strategies proposed the establishment of an up-to-date website with information on clinical signs of CSF, thus stressing the importance of attention to the variations in the expression of CSF. Recently, a clinical decision-support system (CDSS) has been developed to supply practitioners and state-veterinary officers with an objective tool to early indentify CSFsuspect situations (Elbers et al., 2011). The successful implementation of these initiatives, however, relies on the availability of currently updated topical information on the course of infection with CSFV in different scenarios. Classical swine fever has not occurred in Denmark for many years and obviously updated knowledge on the clinical and pathological result of CSFV infection in commercial Danish pigs has not been available to the ‘first line’ veterinarians. Since virus–host interactions are known to play important roles for the outcome of infection with CSFV (Depner et al., 1997; Floegel-Niesmann et al., 2003; Blacksell et al., 2006), data from infection studies may not be immediately transferable from country to country or even not from herd to herd due to differences in sanitary status and/or genetic profile. The results of the present study elucidate various aspects of CSF disease in 6week-old Danish pigs experimentally infected with 4 different strains of CSFV. In a previous study with pigs of unique high sanitary status, we demonstrated that an agedifference of 5 weeks did not markedly influence the
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Fig. 4. Viral RNA in serum of individual pigs. Control pigs = black symbol, CSFV-Glentorf = green symbol (A), CSFV-Romania = red symbol (B), CSFVBergen = blue symbol (C) and CSFV-Israel = purple symbol (D).
outcome of infection with CSFV (Nielsen et al., 2010). Generally, however, infection with CSFV is considered to be less virulent with increasing age (Moennig et al., 2003; Kaden et al., 2004), thus indicating a different outcome of the experiment if infection of markedly older pigs had been studied. Clinically, the selected strains could be divided into two groups: One group included the 2 strains characterized by mild symptoms i.e. CSFV-Glentorf and CSFV-Bergen. Previously, these strains have been used in experimental studies for investigation of persistent infection of swine fever in pigs (Van Oirschot and Terpstra, 1977), for vaccine testing (Ahrens et al., 2000) and for laboratory diagnostic optimization (Handel et al., 2004). Although different age groups were studied, general observations including slight depression and intermittent diarrhea were repeated in our study. These coincident observations point out that suspicion of a CSFV infection should be included at a very early time point when new disease problems, even with mild clinical signs, emerge in pig herds. The second group included the 2 CSFV-strains that showed severe disease including high fever, watery diarrhea and locomotory disorders within eight days after infection i.e. CSFVRomania and CSFV-Israel. These strains have not been experimentally characterized before. On basis of field outbreaks, CSFV-Romania has been designated a
moderate/high virulence (personal communication, CRL, Hannover). Further, a case study from the first outbreak in Israel since 60 years (Yadin et al., 2009) reported clinical symptoms such as anorexia, hyperemia of the ears, purple discoloration of the skin, pyrexia and convulsions. The infected farm included pigs of the entire production chain from sow to fatteners. However, it was not specified whether clinical signs differed between age groups. In our study, CSFV-Israel infected pigs experienced a very fast generated viraemia, which resulted in an acute, fatal infection. Hyperemia and discoloration of the skin was not prevalent in these pigs. CSFV-Romania infected pigs demonstrated clinical symptoms as severe as CSFV-Israel, however, disease development was few days protracted and the pigs were kept 4–7 days longer in the experiment. Due to classification following the clinical score system (CS) introduced by Mittelholzer (2000) both the CSFVIsrael and the CSFV-Romania should be designated as highly virulent in Danish production pigs 6 weeks of age. A general consideration for the CSFV-Romania and CSFVIsrael strains is to maintain the awareness of the risk of introduction to new geographic areas. However, due to the more pronounced expression of disease, clinical signs and pathological lesions, the occurrence of these strains are likely to be detected at a relatively early stage, i.e. within a period of 1–2 weeks after infection.
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In our study, lymphoid atrophy and growth retardation reflected consistent findings regardless of the specific strain used for inoculation. Lymphoid tissue alterations are in several studies described as lymphadenopathy, including edema and congestion in lymph nodes (FloegelNiesmann et al., 2003; Handel et al., 2004; Belak et al., 2008; Everett et al., 2010). This finding was also true for the highly virulent CSFV-strains in our study. In addition, however, we observed a macroscopically visible atrophy of tonsil, thymus and ileal peyer patches at PM examinations performed from one week after infection. This observation may be an important early indicator of CSFV. The weight loss in the pigs infected with mild strains seemed to be almost equalized by termination of the experiment 3 weeks after inoculation. This indicates that weight reduction is only temporary for pigs with mild infection. Still, recurrent weighing of the pigs should not be neglected as one of the tools for early warning of CSF. As gross-pathological findings varied between strains from almost no lesions (CSFV-Bergen) to hyperemia and bleedings in internal organs (CSFV-Romania and CSFVIsrael) CSF(V) should not be excluded as a possible differential diagnosis due to lack of CSF(V) typical pathological lesions. The observation that 9 out of 38 infected pigs did not show any typical pathological lesion is in agreement with Elbers et al. (2003) who evaluated pathological findings as a diagnostic tool for detection of CSF outbreak during the CSF-epidemic in The Netherlands. This study concluded that due to a moderate sensitivity as well as specificity, PM examination could only contribute to a limited extent to detection of CSF. As a diagnostic sample material, blood is invaluable for disease surveillance as well as in situations with clinical suspicions due to its easy accessibility and its capability to reflect systemic changes in the immune system of the individual animal. Therefore, establishment of a reliable time window to detect CSFV in blood would be of high importance. In the present study, virus was demonstrated to be present in serum early after inoculation for both CSFV-Romania and CSFV-Israel (both, PID = 3). For these 2 strains, virus remained in blood until termination of the experiment. In contrast, the low virulent strains did not show same consistency. Most optimal time for virus detection in blood from pigs infected with low virulent strains was the second week after infection, however, CSFV-Glentorf detection of virus in this compartment was encumbered with uncertainty, as only 50% of the infected pigs, showed detectable viraemia. More consistent results was found by Handel et al. (2004), who could demonstrate virus for a longer time period (PID 3–21) in 6 out of 6 CSFVGlentorf inoculated pigs. It should be noted that these results were based on RNA extraction on samples of whole blood. For CSFV-Bergen the viral load in serum was rather high in 2 pigs in week two and at the same level as the 2 highly virulent strains. As these pigs were killed at PID 11, it can only be speculated how the viral RNA profile would have developed. Regarding serum samples from pigs infected with low virulent strains, laboratory analyses indicated that VI detected viraemia in more pigs than RTPCR performed on corresponding samples. This observation did neither match the general impression for the total
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sample set nor previous results from our group (Nielsen et al., 2010), but have been recorded from an experimental study in pigs infected with a mild strain, CSFV-Bavaro (Donahue et al., 2012). Altogether, our results discriminate serum and acknowledge lymphoid tissue as superior diagnostic material, for early CSFV diagnosis, as only CSFV-Israel infected pigs proved to be virus positive in serum by the first PM examination 5 days after inoculation. This makes sense, as viraemia appears after the initial multiplication in lymphoid tissue. As such, blood samples do not alone constitute a reliable material for early diagnosis of a CSFV infection in individual pigs. In the field, however, blood samples will provide a valuable tool in confirmation of a clinical CSF suspicion, since herd based sampling differs from individual animal sampling with regard to detectability. Virologic examination on tissue samples revealed considerable differences in virus distribution, load and persistence between pigs infected with different strains. For all strains, however, virus could first be detected in lymphoid tissue, i.e. tonsils and lymph nodes, and in all infected pigs within the first week after infection. In general, viral load and persistence increased with virulence, while virus distribution was almost constant. Only one strain (CSFV-Glentorf) did not cross the blood–brain barrier. After 3 weeks, virus isolation was often not possible from samples of pigs infected with a low virulent strain, but viral RNA could still be detected in target organs i.e. tonsils and eventually lymph nodes. This observation is in accordance with recent observations by Donahue (2012), who isolated the low virulent CSFV-Bavaro in tonsil tissue until 14 days after infection, while CSFV-RNA was demonstrable 70 days after infection. In this study virus persisted for the longest time in the primary target organ (tonsil), regardless of strain type. Three of the strains crossed the blood–brain barrier, however, the presence of virus in brain did not seem to correlate with CNS disorders, which support previous observations from an experimental study with CSFV-Kozlov (Hansen et al., 2011). In conclusion, the demonstrated variations in distribution, load and persistence of virus within the different tissues, confirm that lymphoid tissues, especially tonsil and lymph nodes, remain the target material to be sent for laboratory diagnosis. The development of virus neutralizing antibodies in the pigs infected with CSFV was sparse in pigs infected with clinically low virulent strains and non-existent for pigs infected with clinical moderate/high virulent strains, probably due to impairment and depletion of B-cell function. The demonstrated late or absent occurrence of antibodies during the course of infection supports the previous recognition that antibody measurements do not constitute a useful tool in the early detection of CSF (Koenen et al., 1996). The present study points out problems related to early diagnosis of CSF in index cases where a low virulent strain is the cause of infection. In these situations, only few, non-specific clinical symptoms, short viraemia and lack of virus persistence will be expected. A veterinary system including surveillance and early sampling of target organ material from commercial swine herds with recent clinical
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problems of non-specific nature may benefit the diagnostic preparedness and optimize the time perspective from a clinical outbreak to confirmed diagnosis. It is emphasized that if CSFV should be included as a possible differential diagnosis it is highly important to include tissue material such as tonsil and lymph nodes when animal material is submitted for laboratory confirmation. Finally, our study also provides updated information and experimental characterization of the early pathogenesis of 2 recently circulating strains of highly virulent nature. In the case of an outbreak, both strains will probably lead to rather early detection of CSF as clinical signs pointing in this direction are to be expected within 1– 2 weeks after infection. Acknowledgements The authors thank Bent Eriksen and co-workers for taking care of the animals. Likewise, we thank MajBritt Eicke and Katrine Fog Thomsen for excellent technical assistance. The current study was financially supported by Directorate for Food, Fisheries and Agri Business in Denmark, grant no. 2007-776. References Ahrens, U., Kaden, V., Drexler, C., Visser, N., 2000. Efficacy of the classical swine fever (CSF) marker vaccine Porcilis1 Pesti in pregnant sows§ Vet. Microbiol. 77, 83–97. Belak, K., Koenen, F., Vanderhallen, H., Mittelholzer, C., Feliziani, F., De Mia, G.M., Belak, S., 2008. Comparative studies on the pathogenicity and tissue distribution of three virulence variants of classical swine fever virus, two field isolates and one vaccine strain, with special regard to immunohistochemical investigations. Acta Vet. Scand. 50, 34. Blacksell, S.D., Khounsy, S., Van, A.D., Gleeson, L.J., Westbury, H.A., 2006. Comparative susceptibility of indigenous and improved pig breeds to Classical swine fever virus infection: practical and epidemiological implications in a subsistence-based, developing country setting. Trop. Anim. Health Prod. 38, 467–474. Depner, K.R., Hinrichs, U., Bickhardt, K., Greiser-Wilke, I., Pohlenz, J., Moennig, V., Liess, B., 1997. Influence of breed-related factors on the course of classical swine fever virus infection. Vet. Rec. 140, 506– 507. Donahue, B.C., Petrowski, H.M., Melkonian, K., Ward, G.B., Mayr, G.A., Metwally, S., 2012. Analysis of clinical samples for early detection of classical swine fever during infection with low, moderate, and highly virulent strains in relation to the onset of clinical signs. J. Virol. Methods 179, 108–115. Durand, B., Davila, S., Cariolet, R., Mesple`de, A., Le Potier, M.-F., 2009. Comparison of viraemia- and clinical-based estimates of within- and between-pen transmission of classical swine fever virus from three transmission experiments. Vet. Microbiol. 135, 196–204. Elbers, A.R., Stegeman, A., Moser, H., Ekker, H.M., Smak, J.A., Pluimers, F.H., 1999. The classical swine fever epidemic 1997–1998 in the Netherlands: descriptive epidemiology. Prev. Vet. Med. 42, 157–184. Elbers, A.R.W., Vos, J.H., Bouma, A., van Exsel, A.C.A., Stegeman, A., 2003. Assessment of the use of gross lesions at post-mortem to detect outbreaks of classical swine fever. Vet. Microbiol. 96, 345–356. Elbers, A.R.W., Gorgievski-Duijvesteijn, M.J., van der Velden, P.G., Loeffen, W.L.A., Zarafshani, K., 2010. A socio-psychological investigation into limitations and incentives concerning reporting a clinically suspect situation aimed at improving early detection of classical swine fever outbreaks. Vet. Microbiol. 142, 108–118. Elbers, A., van der Gaag, L., Schmeiser, S., Uttenthal, A˚., Lohse, L., Nielsen, J., Crooke, H., Blome, S., Loeffen, W., 2011. A Bayesian clinical decision support system for early detection of classical swine fever in individual pigs – evaluation of the sensitivity and specificity of the model. In: Proceedings of 8th ESVV Pestivirus Symposium, September 25– 28, 2011, Hannover, Germany.
Everett, H., Salguero, F.J., Graham, S.P., Haines, F., Johns, H., Clifford, D., Nunez, A., La Rocca, S.A., Parchariyanon, S., Steinbach, F., Drew, T., Crooke, H., 2010. Characterization of experimental infections in domestic pigs with genotype 2.1 and 3.3 isolates of classical swine fever virus. Vet. Microbiol. 142, 26–33. Floegel-Niesmann, G., Bunzenthal, C., Fischer, S., Moennig, V., 2003. Virulence of recent and former classical swine fever isolates evaluated by their clinical and pathological signs. J. Vet. Med. B 50, 214– 220. Floegel-Niesmann, G., Blome, S., Gerß-Du¨lmer, H., Bunzenthal, C., Moennig, V., 2009. Virulence of classical swine fever virus isolates from Europe and other areas during 1996 until 2007. Vet. Microbiol. 139, 165–169. Fritzemeyer, J., Teuffert, J., Greiser-Wilke, I., Staubach, C., Schlu¨ter, H., Moennig, V., 2000. Epidemiology of classical swine fever in Germany in the 1990s. Vet. Microbiol. 77, 29–41. Handel, K., Kehler, H., Hills, K., Pasick, J., 2004. Comparison of reverse transcriptase-polymerase chain reaction, virus isolation, and immunoperoxidase assays for detecting pigs infected with low, moderate, and high virulent strains of classical swine fever virus. J. Vet. Diagn. Invest. 16, 132–138. Hansen, M.S., Bille-Hansen, V., Uttenthal, A˚., Lohse, L., Nielsen, J., 2011. Localization of classical swine fever virus (CSFV) in the brain of pigs infected with strain Kozlov. In: Proceedings of 8th ESVV Pestivirus Symposium, September 25–28, 2011, Hannover, Germany. Have, P., 1984. Detection of antibodies against swine fever virus by enzyme-linked immunosorbent assay (ELISA). Acta Vet. Scand. 25, 462–465. Hyera, J.M., Liess, B., Frey, H.R., 1987. A direct neutralizing peroxidaselinked antibody assay for detection and titration of antibodies to bovine viral diarrhea virus. Zbl. Vet. Med. B 34, 227–239. Kaden, V., Lange, E., Polster, U., Klopfleisch, R., Teifke, J.P., 2004. Studies on the virulence of two field isolates of the classical swine fever virus genotype 2.3 Rostock in wild boars of different age groups. J. Vet. Med. 51, 202–208. Koenen, F., Van Caenegem, G., Vermeersch, J.P., Vandenheede, J., Deluyker, H., 1996. Epidemiological characteristics of an outbreak of classical swine fever in an area of high pig density. Vet. Rec. 139, 367–371. Mayer, D., Thayer, T.M., Hofmann, M.A., Tratschin, J.D., 2003. Establishment and characterization of two cDNA-derived strains of classical swine fever virus, one highly virulent and one avirulent. Vet. Res. 98, 105–116. Meyer, H., Liess, B., Frey, H.-R., Hermanns, W., Trautwein, G., 1981. Experimental transplacental transmission of hog cholera virus in pigs. IV. Virological and serological studies in newborn pigs. Zbl. Vet. Med. B 28, 659–668. Mittelholzer, C., Moser, C., Tratschin, J.D., Hofmann, M.A., 2000. Analysis of classical swine fever virus replication kinetics allows differentiation of highly virulent from avirulent strains. Vet. Microbiol. 74, 293– 308. Moennig, V., Floegel-Niesmann, G., Greiser-Wilke, I., 2003. Clinical signs and epidemiology of classical swine fever: a review of new knowledge. Vet. J. 165, 11–20. Nielsen, J., Lohse, L., Rasmussen, T.B., Uttenthal, A˚., 2010. Classical swine fever in 6- and 11-week-old pigs: haematological and immunological parameters are modulated in pigs with mild clinical disease. Vet. Immunol. Immunopathol. 138, 159–173. Pol, F., Rossi, S., Mesple`de, A., Kuntz-Simon, G., Le Potier, M.F., 2008. Two outbreaks of classical swine fever in wild boar in France. Vet. Rec. 162, 811–816. Rasmussen, T.B., Uttenthal, A˚., Reimann, I., Nielsen, J., Depner, K., Beer, M., 2007. Virulence, immunogenicity and vaccine properties of a novel chimeric pestivirus. J. Gen. Virol. 88, 481–486. Sandvik, T., Drew, T., Paton, D., 2000. CSF virus in East Anglia: where from? Vet. Rec. 147, 251. Uttenthal, A., Storgaard, T., Oleksiewicz, M.B., de Stricker, K., 2003. Experimental infection with the Paderborn isolate of classical swine fever virus in 10-week-old pigs: determination of viral replication kinetics by quantitative RT-PCR, virus isolation and antigen ELISA. Vet. Microbiol. 92, 197–212. Uttenthal, A., Rasmussen, T.B., Nielsen, J., 2008. Classical swine fever in one-week-old piglets. In: Proceedings of the 7th ESSV Pestivirus Symposium, Uppsala, Sweden, 16–19 September, p. 125. Van Oirschot, J.T., Terpstra, C., 1977. A congenital persistent swine fever infection. I. Clinical and virological observations. Vet. Microbiol. 2, 121–132. Yadin, H., Deike, S., David, D., Bombarov, V., Fridgout, O., Elad, D., Brener, Y., Edri, N., Hadany, Y., Perl, S., 2009. Case study – occurrence of classical swine fever in Israel. In: Abstract in Report on Annual Meeting of National Swine Fever Laboratories, Madrid, Spain.
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Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Early pathogenesis of classical swine fever virus (CSFV) strains in Danish pigs Louise Lohse *, Jens Nielsen, A˚se Uttenthal National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 February 2012 Received in revised form 19 April 2012 Accepted 20 April 2012
Host–virus interactions play an important role for the clinical outcome of classical swine fever virus (CSFV) infections in pigs. Strain virulence, host characteristics and environment are all factors that markedly influence disease severity. We tested CSFV strains of varying virulence in an experimental set-up, reducing the influence of host and environmental factors. Thus, weaner pigs were inoculated with one of 4 CSFV strains in order to compare the pathogenesis for a 3-week-period after infection. CSFV strains selected were 2 new and 2 previously characterized. None of these strains had been tested in Danish outbred pigs before. Clinical observations grouped the infected pigs into two different categories reflecting either non-specific, mainly gastro-intestinal, problems, or severe disease including high fever within the first week after inoculation. Gross-pathological findings varied between strains, however, lymphoid atrophy and growth retardation represented a consistent finding for all 4 strains. Virus distribution, viral load and in particular virus persistence differed, but supported present practice that recommends lymphoid tissue, most optimal tonsil and lymph nodes, as target material to be applied for early laboratory diagnosis. The present study demonstrated constraints associated with early detection of infections with CSFV strains of low virulence. Since neither clinical symptoms nor pathological lesions observed with these strains constituted characteristic signs of CSF, the risk of neglecting a CSF suspicion is immediate. Therefore, topical information on new outbreaks and continuous enhancement of an efficient surveillance system is of great importance to prevent further spread of CSF within the pig population. ß 2012 Elsevier B.V. All rights reserved.
Keywords: CSFV Strain difference Host–virus interaction Clinical score Pathological score Virus distribution Pathogenesis Pig
1. Introduction Classical swine fever (CSF) is a serious, contagious infection in pigs. It is a listed disease of the World Organization for Animal Health (OIE) causing major damage to the pig population worldwide. The disease is caused by classical swine fever virus (CSFV), a pestivirus belonging to the Flaviviridae family (http://ictvonline.org/ virusTaxonomy.asp?version=2011). The disease mechanisms of CSFV infection are complex, as specific host–virus interactions determine disease
* Corresponding author. Tel.: +45 35887831. E-mail address: [email protected] (L. Lohse). 0378-1135/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2012.04.026
development and clinical outcome. Host factors, such as age, genetic background and immune status, herd sanitary status as well as strain virulence are known to play highly important roles for the course of disease (Depner et al., 1997; Floegel-Niesmann et al., 2003; Blacksell et al., 2006). Recent studies in our laboratory revealed that experimental infection of Danish pigs with the highly virulent CSFV-Eystrup strain (Mittelholzer et al., 2000; Mayer et al., 2003; Durand et al., 2009) produced severe (Uttenthal et al., 2008), moderate (Rasmussen et al., 2007) or even mild (Nielsen et al., 2010) disease in pigs depending on age and sanitary status. These observations drew further attention to a putative situation reflecting country to country variations in the clinical expression of infection with a particular strain of CSFV.
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In many European countries, CSF has been eradicated. However, sporadic as well as massive new outbreaks occur from time to time. Within the last decades, large CSF epidemics have had significant impact on the pig production together with the national economy of several western European countries. Thus, during outbreaks, national as well as international trade of animals and products has been severely hampered for longer periods due to transport restrictions and import/export bans (Elbers et al., 1999; Koenen et al., 1996; Fritzemeyer et al., 2000; Pol et al., 2008). Further, ethical issues concerning animal welfare related to stamping out procedures including not-infected animals from premises located within restrictions zones have been debated. The possibilities of extensive spread of the disease facilitated by modern infrastructure, including intensive transportation and moving/mixing of animals in agriculture, combined with difficulties in establishing an early clinical diagnosis of CSF by the veterinary practitioners, represents a significant problem in outbreak situations. A set of diagnostic tools for laboratory confirmation of a CSFV suspicion is well established at the majority of national reference laboratories within EU. However, a confirmatory laboratory diagnosis needs optimal sample material provided for examination. Therefore, it is of outmost importance that (1) clinical facets of CSF disease become scientifically elucidated and that (2) new knowledge is communicated to the veterinary society to facilitate early detection of an outbreak of CSF by veterinary practitioners. Several experiments have been performed in different countries to characterize virulence and clinical appearance of different CSFV strains in pigs, (Mittelholzer et al., 2000; Floegel-Niesmann et al., 2003, 2009; Kaden et al., 2004; Everett et al., 2010; Nielsen et al., 2010) and scoring systems for objective comparison of individual strains (Mittelholzer et al., 2000; Floegel-Niesmann et al., 2003) have been developed. In order to provide updated information on the putative course of an introduction of CSF in Danish commercial production sites, we compared the performance of different strains in experimentally infected Danish commercial weaner pigs. The overall goal of the present study was to generate information aiming to shorten the time perspective from initial clinical symptoms at an index case locality to a final confirmed diagnosis at the national reference laboratory. The candidate strains for this study included strains of expected low virulence, as such strains are difficult to diagnose at an early time point due to their atypical clinical expression. Strains that are known to circulate in close proximity to Denmark are considered to constitute a current high risk to the Danish pig production. Therefore, we also studied the clinical outcome of 2 strains obtained from recent outbreaks in Eastern Europe and Israel, respectively. 2. Materials and methods 2.1. Animals In total, 60 weaner pigs, 10–16 kg, were obtained from a conventional Danish swine herd with specific pathogen
free (SPF) status, including freedom from enzootic pneumoniae, atrophic rhinitis, swine dysentery, porcine reproductive and respiratory syndrome and most serotypes of Actinobacillus pleuropneumoniae. All pigs were found to be healthy by veterinary inspection at arrival to the Institute, 1 week before experimental start. All pigs were housed in the high containment experimental facilities at the National Veterinary Institute. All pigs were fed a commercial diet for weaning pigs and water was provided ad libitum. Straw was used for bedding. 2.2. Inocula The following strains of CSFV were used for inoculation in the two experiments: CSF0911: Glentorf (genotype 1.1), originally isolated from domestic pigs in Germany, 1968. Characterized to be of low virulence (Meyer et al., 1981), kindly supplied by the Community Reference Laboratory (CRL) for CSF in Hannover. CSF1019: Romania TM/120/07 (genotype 2.3), field isolate obtained from domestic pigs in Romania, 2007. Characterized to be of moderate/high virulence, kindly supplied by the CRL, Hannover, after permission from Dr. Olaru, National Reference Laboratory (NRL), Romania. CSF0906: v.d. Bergen (genotype 2.2), originally isolated from domestic pigs in The Netherlands, 1977. Characterized to be of low virulence (Van Oirschot and Terpstra, 1977), kindly supplied by the CRL, Hannover. CSF1047: Israel (genotype 2.1), field isolate obtained from domestic pigs in Israel, 2009. Characterized to be of high virulence, kindly supplied by the CRL, Hannover, after permission from Dr. Yadin, Kimron Veterinary Institute (KVI), Israel. All experimentally infected pigs were inoculated intranasally with a 50% tissue culture infective dose (TCID50) of 105/pig in 2 ml Eagle’s minimal essential medium (EMEM). 2.3. Design of experiment The study was performed as two subsequentially performed experiments (experiment I and experiment II), with identical experimental designs. For each experiment, 30 pigs were randomly divided into 3 groups, each comprising 10 individuals. On post infection day (PID) 0, the three groups in experiment I were treated as follows: (1) control group, pigs mock-inoculated intranasally with 2 ml EMEM, (2) infected group, pigs inoculated with CSFV-Glentorf, and (3) infected group, pigs inoculated with CSFV-Romania. For experiment II, the inoculation material used for the 2 infected groups was CSFV-Bergen and CSFV-Israel, respectively. Daily, all pigs were clinically monitored, including clinical scoring (Mittelholzer et al., 2000) and recording of body temperature. Blood samples were collected from the jugular vein on PID 0–7, 10/11, 15, 21/22. Three, three and four pigs from each group were killed on PID 5, 10/11, and
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21/22, respectively. Selection of pigs for termination in experiment on individual days was based on clinical score and blood parameters, more specifically, pigs with low total white blood cell count and clear clinical signs were selected before pigs with no affection. In the control groups and groups infected with low virulent virus, pigs were selected at random. Pigs, which displayed clinical symptoms that reached the humane endpoint criteria during the course of experiment, were killed when necessary. These criteria included parameters such as severe affection of general well-being, significant weight loss, dyspnea, convulsions and pain. All pigs were killed by intravenous injection of sodium pentobarbital. All pigs were autopsied. Experimental procedures and animal manage protocols were carried out in accordance with the requirements of the Danish Animal Experimentation Inspectorate, license no. 2003/561-742 and 2008/561-1541.
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2.7. CSFV specific antibodies Serum samples were screened for antibodies to CSFV in a blocking ELISA (Have, 1984). Doubtful or positive samples were further titrated in neutralisation peroxidase-linked assay (NPLA) using CSFV-Alfort 187 in PK-15 cells, previously described by Hyera et al. (1987). 2.8. Statistics Data analysis was performed using GraphPad In Stat version 3.00 (GraphPad Software, San Diego, CA). Unpaired t-test, or alternatively t-test with Welch correction, was used for comparison between means of groups. 3. Results 3.1. Clinical findings
2.4. Clinical monitoring All pigs were weighed at the beginning and termination of the experiment. Daily, clinical examination was performed on the individual pig and a clinical score (CS) was assigned based on a semi-quantitative scoring system developed by Mittelholzer et al. (2000). The individual animal was scored on 10 CSF-relevant parameters (liveliness, body tension, body shape, breathing, walking, skin, eyes/conjunctiva, appetite, defecation, leftovers in feeding trough), using scores from 0 (normal) to 3 (severe CSF symptom), giving a maximum score of 30. One parameter (leftovers in feeding trough) was assigned to each pig as a joint score per box, since the pigs in one box were fed in a common through. Rectal body temperature was recorded on a daily basis. Fever was defined as rectal body temperature 40.0 8C for pigs in this age group. 2.5. Pathological score At autopsy, a pathological score (PS) was calculated for each pig, based on a scoring system developed by FloegelNiesmann et al. (2003). Nine parameters including pathologically important organs for CSF infection (skin, subcutis/ serosa, tonsil, spleen, kidney, lymph nodes, ileum/rectum, brain, respiratory system) were evaluated and scored according to a scale ranging from 0 (no alteration) to 3 (severe CSF lesion), giving a maximum score of 27. 2.6. Virus detection Virus isolation and detection by immunoperoxidase staining was carried out on PK-15 cells and performed on serum samples and selected organs of all pigs in the study using standard diagnostic procedures, as previously described (Uttenthal et al., 2003). Organ materials selected for examination were: Tonsils and lymphoid tissues from spleen, mandibular, ileo-caecal and inguinal lymph nodes together with kidney, lung, and brain tissues. Virus RNA was detected on corresponding samples by silica-based manual extraction of RNA (Uttenthal et al., 2003) followed by quantitative real-time RT-PCR, previously described by Rasmussen et al. (2007).
3.1.1. Body temperature Mean rectal temperatures for the groups included in the 2 experiments are shown in Fig. 1A–D. Pigs inoculated with moderate to highly virulent CSFV-strains developed fever from PID 4 (CSFV-Israel, Fig. 1D) or PID 5 (CSFV-Romania, Fig. 1B), respectively. For both groups, body temperatures continuously remained above the level of control groups, reaching 41 8C or more before termination of the experiment. Pigs inoculated with the CSFV-strains of low virulence, showed body temperatures close to the level of control pigs (CSFV-Glentorf, Fig. 1A, and CSFV-Bergen, Fig. 1C). Significant difference (p < 0.05) in rectal body temperature between inoculated pigs and control pigs was recorded for 5 days (PID 1, 5, 11, 16, 19) for CSFV-Glentorf and 11 days (PID 2, 3, 4, 5, 6, 7, 8, 12, 14, 16, 20) for CSFVBergen. However, the significance was based on two-sided test statistics, so specified time points included deviation from control group at both higher and lower mean rectal temperature level. At one time point only, mean temperature for CSFV-Bergen inoculated pigs exceeded 40.0 8C (PID 7 = 40.1 0.3 8C). 3.1.2. Growth performance Growth retardation was observed in all infected groups. Weight difference from start to termination in experiment was calculated for the individual pig and means for the infected groups compared to the control groups (Table 1). After two weeks of infection, three out of four groups (CSFV-Glentorf, CSFV-Romania and CSFV-Israel) showed a significant lower growth rate than corresponding control pigs (p-values < 0.01). Three weeks after inoculation, mean body weight in the infected groups was still less than the corresponding control group, but only CSFV-Romania showed significant weight difference (p-value < 0.001). 3.1.3. Clinical score A total score for each individual pig per day was calculated and clinical scores (CS) were compared by groups. CS in the control groups of the two experiments did not exceed 2, except for one pig which showed slight depression probably due to minor gastro-intestinal problems and scored 4 on PID 2. Therefore, CS equal to 4 or less
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Fig. 1. Rectal temperature – mean values. Each symbol represents the average SD for all pigs in the group recorded on the specific reading day. Control group = black symbol, CSFV-Glentorf = green symbol (A), CSFV-Romania = red symbol (B), CSFV-Bergen = blue symbol (C) and CSFV-Israel = purple symbol (D). At the x-axis is indicated number of pigs within each group in the specified time interval. * = 1 pig infected with CSFV-Romania was killed at PID 15 for animal welfare reasons, only 3 pigs left in this group from PID 16 and onwards.
than 4 were considered to represent normal background values in this study and as such not reflecting clinical indication of an early CSF infection. It should be noted that in other experimental settings using other strains of CSFV Table 1 Mean difference in body weight per kg from start to termination in experiment for pigs killed in week 2 and 3, respectively. Calculated pvalues describe the difference between inoculated groups and corresponding control group. Group
Weeks of infection
CSFV-Glentorf CSFV-Romania Control exp. I CSFV-Bergen CSFV-Israel Control exp. II CSFV-Glentorf CSFV-Romania Control exp. I CSFV-Bergen CSFV-Israel Control exp. II
2 2 2 2 2 2 3 3 3 3 3 3
Mean body weight gain in kg 1.8 0.8 5.5 0.6 0.4 1.9 4.9 0.7 6.9 3.6 –a 4.7
Number of pigs
p-Value
3 3 3 3 7 3 4 4 4 4 – 4
<0.001 <0.001 – ns <0.01 – ns <0.001 – ns – –
a The experiment was terminated at PID 11 for pigs infected with CSFVIsrael for animal welfare considerations, therefore no data exists for this group in week 3.
or pigs with a different sanitary status, another CS limit may be appropriate. Individual CS for pigs included in the 2 experiments is shown in Fig. 2A–D. Pigs infected with moderate to highly virulent CSFV strains (CSFV-Romania, Fig. 2B, and CSFV-Israel, Fig. 2D) showed CS indicative of a CSF infection from PID 5 and onwards, with clinical signs continuously progressing into a more severe state until the pigs were killed for animal welfare reasons. Initial clinical signs were non-specific and expressed as fever, depression, anorexia and fecal changes. Ataxia developed with progression of infection around PID 8. Furthermore, several of the infected pigs showed a changed visual appearance caused by a bristled coat. Pigs infected with the low virulent strains experienced less clinical signs. Positive CS was observed for CSFV-Bergen (Fig. 2C) on PID 8, 15, 16 and for CSFV-Glentorf (Fig. 2A) on PID 9, predominantly reflecting gastro-intestinal disorders. All pigs belonging to the groups infected with low virulent strains were killed in accordance with the experimental protocol. 3.2. Pathological findings Post mortem (PM) examination was performed on all pigs and a pathological score was designated to each individual pig. General observations for all 4 strains,
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Fig. 2. Clinical score – individual pigs. Control pigs = black symbol, CSFV-Glentorf = green symbol (A), CSFV-Romania = red symbol (B), CSFV-Bergen = blue symbol (C) and CSFV-Israel = purple symbol (D). The dotted line is inserted at CS = 4, lower CS is regarded as background, not representing symptoms indicative of CSFV.
irrespective of strain virulence, represented lymphoid atrophy (Photo 1). This characteristic was recorded for the tonsils, the thymus and the ileal peyer patches already at PM’s performed after one week of infection. Furthermore, hemorrhages visible as blebs in the border of the spleen, petechial bleedings in the kidney together with swellings and petechial bleedings in the lymph nodes were observed for CSFV-Israel and CSFV-Romania. Skin hyperemia and cyanosis was often observed for CSFV-Romania infected pigs from week 2. Lesions included in the pathological score were by far most pronounced in pigs from the CSFVRomania infected group (Fig. 3). However, pigs infected with CSFV-Israel had to be killed before week 3 for animal welfare reasons. 3.3. Virus detection and distribution To elucidate infection kinetics in CSFV infected pigs, the dissemination of virus in the inoculated pigs was followed by virus isolation (VI) and RT-PCR analysis. The examined material was grouped as follows: (1) primary infection and replication of virus: tonsil, (2) viraemia: blood, (3) dissemination of virus to secondary lymphoid target organs: spleen and lymph nodes, (4) dissemination of virus to non-target visceral organs: lung and kidney, and (5) virus crossing the blood–brain barrier: brain. Organ
material was collected every week in continuation of PM examination of killed pigs. A sample was designated positive if the Ct-value in the PCR analysis was less than 40 and/or VI titer was equal to or above 10. In general, RT-PCR analysis was more sensitive in detecting CSFV RNA than VI in detecting live, propagating virus. To provide an overview of virus distribution within the pigs infected with the individual strains, RT-PCR results for material representing the five selected compartments is shown in Table 2. Viral RNA detected in serum from samples collected during the experimental period is shown in Fig. 4A–D. Ct-values in serum from CSFV-Glentorf infected pigs were only 34, which were considerably lower than for pigs infected with CSFV-Romania, CSFV-Bergen and CSFV-Israel. Pigs in these groups reached Ct-values of 26 or less. This difference in Ct-values reflects a 1000 times lower virus load in serum in CSFV-Glentorf infected pigs than in any of the other strains. Further details of interest, including VI results diverging from RT-PCR, for the individual strains are described in the following: CSFV-Glentorf: Virus was detected in serum in the second week of infection. In total, only 2 out of 7 pigs were positive for viral RNA in serum (Fig. 4A). Unexpectedly, VI showed a higher sensitivity than PCR and detected virus in 5 out of 7 pigs (data not shown). Virus persisted in serum only for a short time period (PID 7–15), irrespective of the
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Photo 1. Lymphoid atrophy. Tonsil: A1 = normal tissue from control pig, A2 = atrophied tissue in CSFV-infected pig. Thymus: B1 = normal tissue from control pig, B2 = atrophied tissue in CSFV-infected pig. Focus areas are encircled in blue.
method of analysis used. Examination of organ material revealed that virus persisted for the longest time period (in 3/4 pigs until the end of experiment) and in highest concentrations in tonsils (Ct 30) and in lymph nodes (Ct 32–36). Virus could not be detected in the brain of any of the pigs in this group (Table 2). In 2 out of 10 pigs
Fig. 3. Pathological score – mean values. Each column represents the average SD for all pigs in the group subjected to PM examination within the specified week. Control group = black symbol, CSFV-Glentorf = green symbol, CSFV-Romania = red symbol, CSFV-Bergen = blue symbol and CSFVIsrael = purple symbol. a = There are no CSFV-Israel infected pigs in week 3. All pigs in this group were killed at PID 11 or before.
(killed PID 5 and PID 22, respectively) neither live virus nor viral RNA could be detected at any time during the experiment. CSFV-Romania: In serum, virus was detected from PID 3 and onwards (Fig. 4B). In the second week of infection, seven out of 7 pigs were positive and the virus load gradually increased until termination of experiment. With regard to organ distribution, virus could be detected in tonsils, lymphoid tissue and visceral organs from all examined pigs. Virus could not be detected in brain tissue until 2nd and 3rd week of the experiment. Throughout the experiment, the viral load was highest in tonsils (Ct 26) and in lymph nodes (Ct 25–27) and lowest in brain (Ct 31). CSFV-Bergen: Virus was detected in serum mainly in the second week of infection. In total 4 out of 7 pigs were positive for viral RNA in serum (Fig. 4C), while VI detected 7 positive out of 7 pigs in the same time period. Examination of organ material revealed the highest viral load in tonsils (Ct 31) and in lymph nodes (Ct 32) and lowest viral load in visceral organs (Ct 36) and brain (Ct 35). By termination of the experiment, live virus could not be isolated and only low amounts of viral RNA (Ct 33–37) were detected in tonsils and in lymph nodes of 4/4 and 2/4 pigs, respectively. CSFV-Israel: Virus was detected in serum from PID 3 and all pigs became virus positive in the first week of infection
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Table 2 Virus distribution in pigs inoculated with CSFV. Results of RT-PCR analysis of serum and tissue material collected in connection to PM examination of sequentially killed pigs in a number of 3, 3 and 4 per week, during the 3-week experimental period. CSFV-strain
Virulence
CSFV-Glentorf Tonsil Serum Lymphoid tissuea Visceral organsb Brain CSFV-Romania Tonsil Serum Lymphoid tissue Visceral organs Brain CSFV-Bergen Tonsil Serum Lymphoid tissue Visceral organs Brain CSFV-Israel Tonsil Serum Lymphoid tissue Visceral organs Brain
Low
a b c
Week 1 (PID 0–6) 2/3 0/3 2/3 2/3 0/3
Week 2 (PID 7–13) RT-PCR positive (Ct value <40)/total 3/3 0/3 3/3 3/3 0/3
Week 3 (PID 14–22) 3/4 0/4 3/4 0/4 0/4
Moderate/high 3/3 0/3 3/3 3/3 0/3
3/3 3/3 3/3 3/3 3/3
4/4 4/4 4/4 4/4 4/4
3/3 0/3 3/3 3/3 2/3
3/3 3/3 3/3 2/3 2/3
4/4 0/4 2/4 0/4 0/4
3/3 3/3 3/3 3/3 3/3
7/7 7/7 7/7 7/7 7/7
ndc nd nd nd nd
Low
High
Lymphoid tissue includes spleen and lymph nodes (Ln. mandibularis, Ln. inguinalis and Ln. ileocaecalis). Visceral organs includes lung and kidney. nd = not done – the pigs were dead PID 11.
(Fig. 4D). Viral load in serum increased gradually until PID 11, when the pigs were killed. By examination of organ material, high loads of viral RNA were detected in all organs, the highest levels found in tonsils (Ct 26), and the lowest in brain (Ct 33). 3.4. Development of CSFV specific antibodies Neutralizing antibodies were developed against CSFV low virulent strains and could be detected in serum at PID 22 (last day of the experiment), when 2/4 (CSFV-Glentorf) and 4/4 (CSFV-Bergen) pigs had seroconverted displaying titers ranging between 14 and 80. One of the 2 antibody negative CSFV-Glentorf pigs was never detected virus positive and may therefore be considered as non-infected. Pigs infected with moderate or highly virulent strains did not develop any antibody response at all. However, none of the pigs infected with these strains survived beyond PID 11 (CSFV-Israel) and PID 18 (CSFV-Romania), respectively. 4. Discussion The low specificity of clinical signs in the initial phase of CSFV infection accentuates the difficulties associated with early detection of CSF (Moennig et al., 2003). Some of the fatal consequences related to delayed CSF diagnosis can be illustrated by the progression of the severe epidemics of CSF in The Netherlands in 1997–1998 and in England in 2000, when the time between introduction of CSFV into the country and diagnosis of CSF in the primary outbreak was estimated to approximately 6 weeks (Elbers et al., 1999; Sandvik et al., 2000), thus enabling extensive further spread of the disease among herds. These experiences
underline the urgent need for improvements of the possibilities for early diagnosis of CSF in order to prevent similar epidemic catastrophes. A socio-psychological investigation (Elbers et al., 2010) identified 6 themes limiting early detection of CSF outbreaks and a number of solutions to address this issue were suggested. One of these strategies proposed the establishment of an up-to-date website with information on clinical signs of CSF, thus stressing the importance of attention to the variations in the expression of CSF. Recently, a clinical decision-support system (CDSS) has been developed to supply practitioners and state-veterinary officers with an objective tool to early indentify CSFsuspect situations (Elbers et al., 2011). The successful implementation of these initiatives, however, relies on the availability of currently updated topical information on the course of infection with CSFV in different scenarios. Classical swine fever has not occurred in Denmark for many years and obviously updated knowledge on the clinical and pathological result of CSFV infection in commercial Danish pigs has not been available to the ‘first line’ veterinarians. Since virus–host interactions are known to play important roles for the outcome of infection with CSFV (Depner et al., 1997; Floegel-Niesmann et al., 2003; Blacksell et al., 2006), data from infection studies may not be immediately transferable from country to country or even not from herd to herd due to differences in sanitary status and/or genetic profile. The results of the present study elucidate various aspects of CSF disease in 6week-old Danish pigs experimentally infected with 4 different strains of CSFV. In a previous study with pigs of unique high sanitary status, we demonstrated that an agedifference of 5 weeks did not markedly influence the
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Fig. 4. Viral RNA in serum of individual pigs. Control pigs = black symbol, CSFV-Glentorf = green symbol (A), CSFV-Romania = red symbol (B), CSFVBergen = blue symbol (C) and CSFV-Israel = purple symbol (D).
outcome of infection with CSFV (Nielsen et al., 2010). Generally, however, infection with CSFV is considered to be less virulent with increasing age (Moennig et al., 2003; Kaden et al., 2004), thus indicating a different outcome of the experiment if infection of markedly older pigs had been studied. Clinically, the selected strains could be divided into two groups: One group included the 2 strains characterized by mild symptoms i.e. CSFV-Glentorf and CSFV-Bergen. Previously, these strains have been used in experimental studies for investigation of persistent infection of swine fever in pigs (Van Oirschot and Terpstra, 1977), for vaccine testing (Ahrens et al., 2000) and for laboratory diagnostic optimization (Handel et al., 2004). Although different age groups were studied, general observations including slight depression and intermittent diarrhea were repeated in our study. These coincident observations point out that suspicion of a CSFV infection should be included at a very early time point when new disease problems, even with mild clinical signs, emerge in pig herds. The second group included the 2 CSFV-strains that showed severe disease including high fever, watery diarrhea and locomotory disorders within eight days after infection i.e. CSFVRomania and CSFV-Israel. These strains have not been experimentally characterized before. On basis of field outbreaks, CSFV-Romania has been designated a
moderate/high virulence (personal communication, CRL, Hannover). Further, a case study from the first outbreak in Israel since 60 years (Yadin et al., 2009) reported clinical symptoms such as anorexia, hyperemia of the ears, purple discoloration of the skin, pyrexia and convulsions. The infected farm included pigs of the entire production chain from sow to fatteners. However, it was not specified whether clinical signs differed between age groups. In our study, CSFV-Israel infected pigs experienced a very fast generated viraemia, which resulted in an acute, fatal infection. Hyperemia and discoloration of the skin was not prevalent in these pigs. CSFV-Romania infected pigs demonstrated clinical symptoms as severe as CSFV-Israel, however, disease development was few days protracted and the pigs were kept 4–7 days longer in the experiment. Due to classification following the clinical score system (CS) introduced by Mittelholzer (2000) both the CSFVIsrael and the CSFV-Romania should be designated as highly virulent in Danish production pigs 6 weeks of age. A general consideration for the CSFV-Romania and CSFVIsrael strains is to maintain the awareness of the risk of introduction to new geographic areas. However, due to the more pronounced expression of disease, clinical signs and pathological lesions, the occurrence of these strains are likely to be detected at a relatively early stage, i.e. within a period of 1–2 weeks after infection.
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In our study, lymphoid atrophy and growth retardation reflected consistent findings regardless of the specific strain used for inoculation. Lymphoid tissue alterations are in several studies described as lymphadenopathy, including edema and congestion in lymph nodes (FloegelNiesmann et al., 2003; Handel et al., 2004; Belak et al., 2008; Everett et al., 2010). This finding was also true for the highly virulent CSFV-strains in our study. In addition, however, we observed a macroscopically visible atrophy of tonsil, thymus and ileal peyer patches at PM examinations performed from one week after infection. This observation may be an important early indicator of CSFV. The weight loss in the pigs infected with mild strains seemed to be almost equalized by termination of the experiment 3 weeks after inoculation. This indicates that weight reduction is only temporary for pigs with mild infection. Still, recurrent weighing of the pigs should not be neglected as one of the tools for early warning of CSF. As gross-pathological findings varied between strains from almost no lesions (CSFV-Bergen) to hyperemia and bleedings in internal organs (CSFV-Romania and CSFVIsrael) CSF(V) should not be excluded as a possible differential diagnosis due to lack of CSF(V) typical pathological lesions. The observation that 9 out of 38 infected pigs did not show any typical pathological lesion is in agreement with Elbers et al. (2003) who evaluated pathological findings as a diagnostic tool for detection of CSF outbreak during the CSF-epidemic in The Netherlands. This study concluded that due to a moderate sensitivity as well as specificity, PM examination could only contribute to a limited extent to detection of CSF. As a diagnostic sample material, blood is invaluable for disease surveillance as well as in situations with clinical suspicions due to its easy accessibility and its capability to reflect systemic changes in the immune system of the individual animal. Therefore, establishment of a reliable time window to detect CSFV in blood would be of high importance. In the present study, virus was demonstrated to be present in serum early after inoculation for both CSFV-Romania and CSFV-Israel (both, PID = 3). For these 2 strains, virus remained in blood until termination of the experiment. In contrast, the low virulent strains did not show same consistency. Most optimal time for virus detection in blood from pigs infected with low virulent strains was the second week after infection, however, CSFV-Glentorf detection of virus in this compartment was encumbered with uncertainty, as only 50% of the infected pigs, showed detectable viraemia. More consistent results was found by Handel et al. (2004), who could demonstrate virus for a longer time period (PID 3–21) in 6 out of 6 CSFVGlentorf inoculated pigs. It should be noted that these results were based on RNA extraction on samples of whole blood. For CSFV-Bergen the viral load in serum was rather high in 2 pigs in week two and at the same level as the 2 highly virulent strains. As these pigs were killed at PID 11, it can only be speculated how the viral RNA profile would have developed. Regarding serum samples from pigs infected with low virulent strains, laboratory analyses indicated that VI detected viraemia in more pigs than RTPCR performed on corresponding samples. This observation did neither match the general impression for the total
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sample set nor previous results from our group (Nielsen et al., 2010), but have been recorded from an experimental study in pigs infected with a mild strain, CSFV-Bavaro (Donahue et al., 2012). Altogether, our results discriminate serum and acknowledge lymphoid tissue as superior diagnostic material, for early CSFV diagnosis, as only CSFV-Israel infected pigs proved to be virus positive in serum by the first PM examination 5 days after inoculation. This makes sense, as viraemia appears after the initial multiplication in lymphoid tissue. As such, blood samples do not alone constitute a reliable material for early diagnosis of a CSFV infection in individual pigs. In the field, however, blood samples will provide a valuable tool in confirmation of a clinical CSF suspicion, since herd based sampling differs from individual animal sampling with regard to detectability. Virologic examination on tissue samples revealed considerable differences in virus distribution, load and persistence between pigs infected with different strains. For all strains, however, virus could first be detected in lymphoid tissue, i.e. tonsils and lymph nodes, and in all infected pigs within the first week after infection. In general, viral load and persistence increased with virulence, while virus distribution was almost constant. Only one strain (CSFV-Glentorf) did not cross the blood–brain barrier. After 3 weeks, virus isolation was often not possible from samples of pigs infected with a low virulent strain, but viral RNA could still be detected in target organs i.e. tonsils and eventually lymph nodes. This observation is in accordance with recent observations by Donahue (2012), who isolated the low virulent CSFV-Bavaro in tonsil tissue until 14 days after infection, while CSFV-RNA was demonstrable 70 days after infection. In this study virus persisted for the longest time in the primary target organ (tonsil), regardless of strain type. Three of the strains crossed the blood–brain barrier, however, the presence of virus in brain did not seem to correlate with CNS disorders, which support previous observations from an experimental study with CSFV-Kozlov (Hansen et al., 2011). In conclusion, the demonstrated variations in distribution, load and persistence of virus within the different tissues, confirm that lymphoid tissues, especially tonsil and lymph nodes, remain the target material to be sent for laboratory diagnosis. The development of virus neutralizing antibodies in the pigs infected with CSFV was sparse in pigs infected with clinically low virulent strains and non-existent for pigs infected with clinical moderate/high virulent strains, probably due to impairment and depletion of B-cell function. The demonstrated late or absent occurrence of antibodies during the course of infection supports the previous recognition that antibody measurements do not constitute a useful tool in the early detection of CSF (Koenen et al., 1996). The present study points out problems related to early diagnosis of CSF in index cases where a low virulent strain is the cause of infection. In these situations, only few, non-specific clinical symptoms, short viraemia and lack of virus persistence will be expected. A veterinary system including surveillance and early sampling of target organ material from commercial swine herds with recent clinical
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problems of non-specific nature may benefit the diagnostic preparedness and optimize the time perspective from a clinical outbreak to confirmed diagnosis. It is emphasized that if CSFV should be included as a possible differential diagnosis it is highly important to include tissue material such as tonsil and lymph nodes when animal material is submitted for laboratory confirmation. Finally, our study also provides updated information and experimental characterization of the early pathogenesis of 2 recently circulating strains of highly virulent nature. In the case of an outbreak, both strains will probably lead to rather early detection of CSF as clinical signs pointing in this direction are to be expected within 1– 2 weeks after infection. Acknowledgements The authors thank Bent Eriksen and co-workers for taking care of the animals. Likewise, we thank MajBritt Eicke and Katrine Fog Thomsen for excellent technical assistance. The current study was financially supported by Directorate for Food, Fisheries and Agri Business in Denmark, grant no. 2007-776. References Ahrens, U., Kaden, V., Drexler, C., Visser, N., 2000. Efficacy of the classical swine fever (CSF) marker vaccine Porcilis1 Pesti in pregnant sows§ Vet. Microbiol. 77, 83–97. Belak, K., Koenen, F., Vanderhallen, H., Mittelholzer, C., Feliziani, F., De Mia, G.M., Belak, S., 2008. Comparative studies on the pathogenicity and tissue distribution of three virulence variants of classical swine fever virus, two field isolates and one vaccine strain, with special regard to immunohistochemical investigations. Acta Vet. Scand. 50, 34. Blacksell, S.D., Khounsy, S., Van, A.D., Gleeson, L.J., Westbury, H.A., 2006. Comparative susceptibility of indigenous and improved pig breeds to Classical swine fever virus infection: practical and epidemiological implications in a subsistence-based, developing country setting. Trop. Anim. Health Prod. 38, 467–474. Depner, K.R., Hinrichs, U., Bickhardt, K., Greiser-Wilke, I., Pohlenz, J., Moennig, V., Liess, B., 1997. Influence of breed-related factors on the course of classical swine fever virus infection. Vet. Rec. 140, 506– 507. Donahue, B.C., Petrowski, H.M., Melkonian, K., Ward, G.B., Mayr, G.A., Metwally, S., 2012. Analysis of clinical samples for early detection of classical swine fever during infection with low, moderate, and highly virulent strains in relation to the onset of clinical signs. J. Virol. Methods 179, 108–115. Durand, B., Davila, S., Cariolet, R., Mesple`de, A., Le Potier, M.-F., 2009. Comparison of viraemia- and clinical-based estimates of within- and between-pen transmission of classical swine fever virus from three transmission experiments. Vet. Microbiol. 135, 196–204. Elbers, A.R., Stegeman, A., Moser, H., Ekker, H.M., Smak, J.A., Pluimers, F.H., 1999. The classical swine fever epidemic 1997–1998 in the Netherlands: descriptive epidemiology. Prev. Vet. Med. 42, 157–184. Elbers, A.R.W., Vos, J.H., Bouma, A., van Exsel, A.C.A., Stegeman, A., 2003. Assessment of the use of gross lesions at post-mortem to detect outbreaks of classical swine fever. Vet. Microbiol. 96, 345–356. Elbers, A.R.W., Gorgievski-Duijvesteijn, M.J., van der Velden, P.G., Loeffen, W.L.A., Zarafshani, K., 2010. A socio-psychological investigation into limitations and incentives concerning reporting a clinically suspect situation aimed at improving early detection of classical swine fever outbreaks. Vet. Microbiol. 142, 108–118. Elbers, A., van der Gaag, L., Schmeiser, S., Uttenthal, A˚., Lohse, L., Nielsen, J., Crooke, H., Blome, S., Loeffen, W., 2011. A Bayesian clinical decision support system for early detection of classical swine fever in individual pigs – evaluation of the sensitivity and specificity of the model. In: Proceedings of 8th ESVV Pestivirus Symposium, September 25– 28, 2011, Hannover, Germany.
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