【病毒外文文献】1996 Dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respira

veterinary microbiology ELSEVIER Veterinary Microbiology 48 1996 325 335 Dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus a clinical and virological study Kristien Van Reeth Hans Nauwynck Maurice Pensaert Laborator of Veterinary Virology Faculty of Veterinary Medicine University of Gem Salisburylaan 133 B 9820 Merelbeke Belgium Received 13 July 1994 accepted 9 June 1995 Abstract Dual infections of pigs with porcine reproductive and respiratory syndrome virus PRRSV fol lowed by a second common respiratory virus either porcine respiratory coronavirus PRCV or swine influenza virus SW were studied The aim was to determine if dual infections as compared to single virus infections result in enhanced clinical manifestations It was also examined if PRRSV replication affects replication of PRCV or SIV in the respiratory tract Groups of conventional 10 week old pigs were inoculated with PRRSV only 3 pigs PRCV only 4 pigs or SIV only 4 pigs Dual inoculations with PRRSV PRCV 4 pigs and PRRSV SIV 3 groups of 4 4 and 5 pigs were performed at a 3 day interval A group of uninoculated control pigs 8 pigs was included The infection with PRRSV only induced a transient fever 40 2 C at 2 DPI but no respiratory signs The PRCV only infection remained subclinical The SIV only infection resulted in a one day fever 40 1 C with moderate tachypnoea and dyspnoea Mean weight gain in the virus inoculated groups was retarded compared with the control group The PRRSV PRCV infection induced a 9 day lasting fever peak 40 9 C with tachypnoea dyspnoea and productive coughing The PRRSV SIV infection resulted in fever and respiratory signs in all 3 groups Clinical signs however were more pronounced in group 1 than in groups 2 and 3 Pigs of group 1 showed fever during 10 days peak 41 4 Z tachypnoea marked dyspnoea with abdominal breathing and a productive cough Pigs of groups 2 and 3 had fever for 5 and 3 days peaks 40 6 and 40 3 C respectively and mild respiratory disorders Mean weight gain during 14 DPI of the 2nd virus was 5 9 kg in the PRRSV PRCV group and 4 0 6 8 and 6 7 kg in PRRSV SIV University of Gent Faculty of Veterinary Medicine Salisburylaan 133 982O Merelbeke Belgium Tel 32 9 264 7366 Fax 32 9 264 7495 0378 l 135 96 15 00 0 1996 Elsevier Science B V All rights reserved SSD10378 1135 95 00145 x 326 K Van Reeth et al Veterinary Microbiology 48 1996 325 335 groups 1 2 and 3 respectively Mean weight gain during the corresponding period in the PRRSV only group was 8 6 kg It was concluded that dual infections with viruses causes more severe disease and growth retardation than single PRRSV infection PRCV excretion curves were similar in single and dual virus inoculated groups Excretion of SIV was delayed by 2 days in the dual inoculated pigs Thus replication of the second virus is not PRCV or only slightly SIV affected by a prior infection with PRRSV Keywords Porcine reproductive and respiratory syndrome virus Porcine respiratory coronavirns Swine influenza virus Dual infections Pigs 1 Introduction Porcine reproductive and respiratory syndrome virus PRRSV appeared for the first time in Europe in the northwestern part of Germany in November 1990 Ohlinger et al 199 1 During the next months the virus spread rapidly through the major pig producing areas of Western Europe The virus has become enzootic in Germany The Netherlands Belgium Spain United Kingdom France and Denmark since 1992 Ohlinger et al 1991 Wensvoort et al 1991 Vynckier and Pensaert 1993 Plana Duran et al 1992a Edwards et al 1992 Albina et al 1992 and Boetner et al 1993 Since that time an increase in respiratory disease in weaners growing and fattening pigs has been widely reported The clinical picture is characterized by respiratory disorders increased mortality and suboptimal performance The PRRSV is often incriminated as the causative agent but the signs observed in the field have never been reproduced experimentally The pattern of infection with PRRSV on Belgian swine farms has been studied by Houben et al 1995 Most pigs are born from immune sows and thus have maternal antibodies during their first weeks of life On breeding farms where the virus persists pigs may or may not become infected with PRRSV before the age of 10 weeks When infection occurs maternal immunity is replaced by active immunity In the absence of infection maternal PRRSV antibodies do not persist beyond 10 l 1 weeks of age Thus pigs which are to be transported to industrial fattening farms and which have not previously been infected with PRRSV on the breeding farm are fully susceptible and have a high risk to contract the infection shortly after they are grouped together In the study mentioned it was shown that on the average 50 of the pigs entering intensive fattening units were devoid of PRRSV antibodies and that all these pigs had seroconverted to PRRSV 1 month later Houben et al 1995 Around that age the pigs are confronted with several respiratory infectious agents Indeed earlier studies have shown that infections with porcine respiratory corona virus PRCV and swine influenza viruses SIV are extremely common shortly after grouping together of pigs in intensive fattening units Van Reeth and Pensaert 1994b Combined infections with the latter viruses and with PRRSV are therefore likely Upon a respiratory infection with PRRSV primary virus replication takes place in the lungs and there seems to be a predilection for alveolar lung macrophages Pol et al 1991 Some researchers suggest that lung defence mechanisms may be temporarily impaired as a result of virus replication in alveolar macrophages A synergism between PRRSV and other viruses possibly leading to disease has often been proposed Groschup et al 1993 but K Van Reeth et al Veterinary Microbiology 48 1996 325 335 321 clinical experiments to document this hypothesis have not been done It was the purpose of the present study to examine the clinical effects of dual infections with PRRSV followed either by PRCV or by SIV In a previous study on dual infections with PRCV followed by SIV the authors demon strated that PRCV infection highly interferes with SIV replication Van Reeth and Pensaert 1994a Replication of HlNl influenza virus in the lungs of pigs was reduced by a factor of 99 Thus it was also the purpose of the present study to find out if PRRSV is able to interfere with replication of PRCV or SIV 2 Materials and methods 2 1 Viruses The Lelystad virus strain of PRRSV Wensvoort et al 1991 was used in this study It was kindly provided by G Wensvoort Institute for Animal Science and Health ID DLO Lelystad The Netherlands Virus used for inoculation was at the 5th passage in alveolar macrophages The PRCV strain 9 1 V44 Van Reeth and Pensaert 1994a was isolated from the lungs of a feeder pig in February 1991 It had been passaged twice in swine testicle ST cells The A Sw Belg l 83 isolate of HlNl influenza virus was obtained from an outbreak of swine influenza on a breeding farm in January 1983 and used at the third passage in embryonated eggs 2 2 Pigs and inoculation Thirty six conventional feeder pigs free of antibodies against the respective virus es with which they were to be inoculated were used at the age of 10 weeks The pigs were allocated to 8 groups each of which was housed in separate isolation facilities Eight pigs served as uninoculated controls control group Three pigs were inoculated with PRRSV only PRRSV only group 4 with PRCV only PRCV only group and 4 with SIV only SIV only group Four pigs were inoculated with PRRSV first and 3 days later with PRCV PRRSV PRCV group and in 3 separate experiments 4 4 and 5 pigs were inoculated with PRRSV first and 3 days later with SIV PRRSV SIV group 1 2 and 3 All inoculations were performed individually by aerosol during 35 min with 8 ml phosphate buffered saline PBS containing either 104 9 median tissue culture infective doses TCID of PRRSV 10 TCIDS of PRCV or 1O7 5 median egg infective doses EID50 of SIV using the Wright nebulizer particle size 45 min dyspnoea and coughing from 6 days before the first inoculation until 9 control PRCV only and SIV only groups or 14 PRRSV only PRRSV PRCV 328 K Van Reeth et al Veterinary Microbiology 48 19 325 335 and PRRSV SIV groups days after the last inoculation The pigs were weighed daily throughout the observation period 2 4 Virological studies Nasal swabs were taken before and 3 days post inoculation DPI with PRRSV in the respective groups to make sure that PRRSV infection was successful Nasal swabs were collected daily for titration of PRCV or SIV during 10 consecutive days PI to follow the excretion curves in the respective groups The swabs from the individual animals in each group were pooled daily for virus titration Cotton swabs were weighed before and after collection to determine virus titers per 100 mg nasal secretions Swabs from both nostrils were suspended in 1 ml of PBS supplemented with penicillin 100 IU ml and streptomycin 0 1 pug ml and mixed vigorously for 1 h The medium was collected clarified by centrifugation and used for virus isolation titration Isolation of PRRSV was performed in porcine alveolar macrophages seeded in 96 well microtiter plates and reading occurred at 60 h PI using an immunoperoxidase monolayer assay IPMA Wensvoort et al 1991 Titration of PRCV and SIV was performed in ST cells Van Reeth and Pensaert 1994a and 10 day old embryonated chicken eggs respec tively Palmer et al 1975 Two passages in embryonated eggs were made 2 5 Serology At the end of the experiments sera from all pigs were tested for antibodies against PRRSV by the IPMA Wensvoort et al 1991 against PRCV by virus neutralization Voets et al 1980 and against HlNl influenzavirus by haemagglutination inhibition Palmer et al 1975 2 6 Statistical analysis Multiple regression analysis was used to compare groups on the basis of weight gain Pair wise comparisons were made between each of the dual virus inoculated groups and a the control group b the PRRSV only group c the group inoculated with the 2nd virus only 3 Results All the pigs were clinically healthy before virus inoculation At the time of the first inoculation nasal swabs were virus negative and sera were free of antibodies against the respective viruses Three DPI with PRRSV virus excretion was detected in every group inoculated These results indicate that PRRSV was replicating in the respiratory tract of pigs at the time of inoculation with the second virus At the end of the experiments all pigs had seroconverted against the viruses with which they had been inoculated K Van Reeth et al Veterinary Microbiology 48 1996 325 335 329 Table 1 Summary of body temperatures and weight gain in control and single PRRSV PRCV and SIV infected groups Group Days of fever DPI MWG Control 0 6 3 PRRSV only 1 2 4 4 PRCV only 0 3 9 SIV only 1 5 5 3 Mean weight gain during the 9 day post inoculation period in kg 3 1 Control group and single PRRSV PRCV and SIV infections Body temperatures and weight gain in the uninoculated control and single PRRSV PRCV and SIV infected groups are summarized in Table 1 The control group was clinically healthy and mean body temperatures were between 39 4 and 39 9 C Mean weight gain during the 9 day observation period was 6 3 kg The infection with PRRSV only induced an increase in body temperature 40 2 Z at 2 DPI From the 3th DPI on body temperatures returned to normal and the further course of the infection was subclinical Except for a slight nasal discharge respiratory signs were not observed Growth arrest was recorded the first 2 DPI only and mean weight gain between 0 and 9 DPI was 4 4 kg Between 3 and 17 DPI mean weight gain was 8 6 kg as shown in Fig 2 Clinical responses were not observed in the PRCV only group Growth was retarded between 0 and 3 DPI but thereafter daily weight gain parallelly increased with that of the control group data not shown Mean weight gain was 3 9 kg Pigs of the SIV only group showed fever 4O l C at 5 DPI Respiration rates were increased peak 65 breaths min 4 5 and 6 DPI Coughing was observed occasionally particularly when the pigs were forced to move Mean weight gain was 5 3 kg 3 2 Dual PRRSV PRCV and PRRSV SIV infections Body temperatures and weight evolution of the dual virus infected groups are compared with those of the PRRSV only group in Fig 1 and Fig 2 respectively The time of inocu lation with PRRSV is represented as day 3 the time of inoculation with the 2nd virus as day 0 In the PRRSV PRCV group pigs developed fever and respiratory disease A transient rise in body temperature 2 DPI with PRRSV was followed by a 9 day lasting fever after inoculation with PRCV The peak of fever 40 9 C was accompanied by increased respi ration rates peak 55 breaths min All 4 pigs showed respiratory distress with abdominal respiration and a productive cough between 8 and 11 DPI They huddled together while showing signs of chilling listlessness and inappetence Mean weight gain 5 9 kg was statistically different P 0 01 from that of the control and PRRSV only groups but not from that of the PRCV only group Pigs of PRRSV SIV group 1 had severe and long lasting febrile responses with fever for 10 days peaking at 41 4 C They were dull and made no effort to rise when disturbed There 330 K Van Iteeth et al Veterinary Microbiology 48 I 996 325 335 41 5 0 41 o L E E 40 5 B E 3 6 40 o CG 39 s 39 0 38 5 3 0 5 10 DAYS A A PRRSV 2nd virus inoculation PRRSV PRCV PRRSV SW group 1 x PRRSV SW group 2 mUV U PRRSV SD group 3 Fig I Body temperatures in single PRRSV and dual PRRSV PRCV and PRRSV SIV infected groups was a marked anorexia All 4 pigs exhibited prominent respiratory disease between 4 and 10 DPI There was tachypnoea peak 90 breaths min labored jerking abdominal breathing and severe productive coughing Mean weight gain at the end of the experiment was 4 0 kg Because of the marked clinical disturbances in PRRSV SIV group 1 the experimental PRRSV SIV infection was repeated twice PRRSV SIV groups 2 and 3 In these 2 addi tional experiments pigs exhibited fever for 5 group 2 peak 40 6 C and 3 days group 3 peak 40 3 C Respiratory disorders were noted between 5 and 8 DPI These groups were less severely affected than PRRSV SIV group 1 Pigs were only slightly less alert than before virus inoculations and respiratory rates were moderately increased peak 69 breaths min in group 2 62 breaths mm in group 3 Sneezing and nasal discharge were recorded in all pigs superficial respiration and coughing were recorded occasionally Mean weight gains were 6 8 group 2 and 6 7 kg group 3 In each of the 3 PRRSV SIV groups mean weight gains were statistically different P 0 01 from that of the control PRRSV only and SIV only groups 3 3 Excretion of PRCV or SIV afer a prior inoculation with PRRSV Data on excretion of PRCV and SIV in single and dual virus inoculated groups are presented in Table 2 and Table 3 respectively K Van Reeth et al Veterinar Microbiology 48 1996 325 335 331 10 9 8 7 2 6 5 5 4 g 3 3 2 1 0 1 2 PRRSV PRCV PRRSV SIV group 1 PRRSV 2nd virus inoculation Fig 2 Weight evolution in single PRRSV and dual PRRSV PRCV and PRRSV SIV infected groups Table 2 PRCV excretion in PRCV only and PRRSV PRCV groups Group Virus titer log TCIDS 100 mg nasal secrete at days after inoculation with PRCV 0 I 2 3 4 5 6 7 8 9 IO PRCV only n 4 ncg 2 5 3 3 5 0 5 2 5 2 5 2 3 6 2 6 neg neg PRRSV PRCV neg 2 1 3 4 5 6 5 9 5 6 5 0 3 5 2 4 2 7 neg n 4 virus titer in pooled nasal swabs TCID median tissue culture infective doses Table 3 SIV excretion in XV only group and in PRRSV SIV group 1 Group Virus titer log EID J 100 mg nasal secrete at days after inoculation with SIV 0 1 2 3 4 5 6 7 8 9 10 SIV only n 4 neg 2 9 5 2 6 2 5 6 3 8 PRRSV SIV 1 n 4 neg neg neg 2 6 4 1 6 4 virus titer in pooled nasal swabs EID median egg infective doses 4 1 5 2 neg 4 3 neg neg neg neg neg eg 332 K Van Reeth et al Veterinary Microbiology 48 1996 32 5 335 PRCV was detected in nasal swabs from 1 to 8 DPI in the PRCV only group and from 1 to 9 DPI in the PRRSV PRCV group Table 2 Virus excretion curves were similar and highest amounts of virus log 5 0 5 9 TCID lOO mg nasal secretions were found between 3 and 6 DPI in both groups The excretion of SIV in the PRRSV SIV group was delayed by 2 days not only with regard to the presence of virus but also with regard to the peak amounts Table 3 4 Discussion This study confirms the hypothesis that the clinical effects of a PRRSV infection may be exacerbated when a concurrent infection with common respiratory viruses occurs As in previous experimental studies Plana Duran et al 1992b Ramos et al 1992 it was found that the uncomplicated PRRSV infection has no clinical importance in feeder pigs A short transient fever and inappetence were the only clinical signs observed in the present and other studies Combined PRRSV PRCV and PRRSV SIV infections on the other hand consistently resulted in fever respiratory illness and growth retardation Growth retardation over a 14 day period was 2 7 4 6 1 8 and 1 9 kg in PRRSV PRCV and PRRSV SIV groups 1 2 and 3 respectively We cannot explain why growth retardation also occurred following inoculation with PRCV only because pigs of this group were clinically normal and even lively throughout the observation period Also experimental PRCV infections have been performed frequently by the authors Van Reeth and Pensaert 1992 and generally had a mild or subclinical course In the authors opinion secondary infection with PRCV or SIV is one of the factors that may determine whether or not disease results from an infection with PRRSV There is no doubt that other factors will also play a role The variable clinical outcome in the pigs inoculated with PRRSV SIV in the present experiment shows that the effect of the dual infections is not reproducible to the same level in every group Three separate groups of pigs were inoculated with PRRSV SIV Respiratory disease fever and growth retardation were much more severe in PRRSV SIV group 1 than in groups 2 and 3 Still there were no indications of concurrent infections with mycoplasma or other respiratory viruses in any of the groups All the sera collected 1 month after the virus inoculations scored negative in a blocking ELISA test Dako Mycoplusma hyopneumoniue ELISA Feld et al 1992 for detection of Mycoplasma hyopneumoniae antibodies There was no serological evidence of an accidental infection with other viruses such as PRCV Aujeszky s disease virus or H3N2 influenza virus Certainly the variation in disease observed between experimentally inoc ulated pigs in the present study must also occur under field conditions and may be a reflection of hitherto undefined physiological parameters The viruses used for the experimental dual infections were purposely selected on the basis of a serologic study earlier performed in intensive fattening units in Belgium Van Reeth and Pensaert 1994b This study showed that infections with PRCV and HlNl influenzavirus are very common when 10 week old feeder pigs derived from several breeding farms are grouped together in intensive fattening units Also it was shown that infections with PRRSV systematically occur in such pigs Houben et al 1995 Thus combined infections with PRRSV and or PRCV and or HlNl influenzavirus are likely to K Van Reeth et al Veterinary Microbiology 48 I 996 325 335 333 occur within a short time interval Experiments in the authors laboratory have shown that PRRSV can be isolated from the lungs of experimentally infected pigs until at least 1 month PI Consequently a primary infection with PRRSV can be easily followed by infection with other respiratory viruses even when they occur with several weeks intervals Definite proof is however still lacking to show that combined PRRSV PRSV or PRRSV SIV infections are the cause of increased respiratory problems in the field There is an urgent need to study the true economical signi