Robert B. Morrison

Isolation of swine infertility and respiratory syndrome virus (isolate ATCC VR-2332) in North America and experimental reproduction of the disease in gnotobiotic pigs

A recent epizootic of swine infertility and respiratory syndrome (SIRS) in a Minnesota swine herd was investigated. Examination of a sow, neonatal piglets, and stillborn fetuses obtained during the epizootic from the affected herd revealed interstitial pneumonitis, lymphomononuclear encephalitis, and lymphomononuclear myocarditis in the piglets and focal vasculitis in the brain of the sow. Fetuses did not have microscopic lesions. No cause for the infertility and respiratory syndrome was determined. Therefore, attempts were made to experimentally reproduce the disease. Eleven 3-day-old gnotobiotic piglets exposed intranasally to tissue homogenates of piglets from the epizootic became inappetent and febrile by 2–4 days postexposure and had interstitial pneumonitis and encephalitis similar to that seen in the field outbreak. After 2 blind passages in gnotobiotic piglets, tissue homogenates were cultured on continuous cell line CL2621, and a cytopathic virus (ATCC VR-2332), provisionally named SIRS virus, was isolated. Gnotobiotic piglets exposed intranasally to the SIRS virus developed clinical signs and microscopic lesions that were the same as those in piglets exposed to the tissue homogenates, and the virus was reisolated from their lungs. This is the first isolate of SIRS virus in the United States that fulfills Kochs postulates in producing the respiratory form of the disease in gnotobiotic piglets and the first report of isolation and propagation of the virus on a continuous cell line (CL2621). The virus is designated as American Type Culture Collection VR-2332.

Characterization of Swine Infertility and Respiratory Syndrome (SIRS) Virus (Isolate ATCC VR-2332)

The characterization of an isolate of swine infertility and respiratory syndrome (SIRS) virus (ATCC VR-2332) is reported. A commercial cell line (CL262 1) was used for the propagation of the virus for all assays. Laboratory studies indicate that this isolate is a fastidious, nonhemagglutinating, enveloped RNA virus. Cesium chloride-purified virions visualized by electron microscopy were spherical particles with an average diameter of 62 nm (range: 48–83 nm) and a 25–30 nm core surrounded by an envelope. Virus replication was restricted to the cytoplasm, as demonstrated by immunofluorescence. The virus did not react serologically with antisera to several common porcine viruses or with antisera to known viruses in the alphavirus, rubivirus, pestivirus, and ungrouped lactic dehydrogenase virus genera of the Togaviridae. However, convalescent sow sera and rabbit hyperimmune sera neutralized the SIRS virus at titers of 1:256 and 1:512, respectively. The virus was stable at 4 and −70 C, but was labile at 37 and 56 C. The properties of this isolate of SIRS virus resemble those of the family Togaviridae but do not match the described genera.

Experimental porcine reproductive and respiratory syndrome virus infection in one-, four-, and 10-week-old pigs.

One-, 4-, and lo-week-old pigs were exposed to porcine reproductive and respiratory syndrome virus (PRRSV) to determine the effect of age on clinical signs, hematologic alterations, the onset and duration of viremia, routes of virus shedding, antibody production, and microscopic lesions produced by PRRSV isolate ATCC VR-2332. The response to PRRSV infection was similar among age groups. Fever, usually prolonged, and a marked dyspnea with cutaneous erythema when restrained for sample collection were the most consistent clinical signs. Prolonged periocular edema was unique to the 1-week-old pigs. The white blood cell count was decreased on day 4 postexposure (PE) due to decreases in neutrophils and lymphocytes. The virus was isolated from buffy coats at day 1 PE and was isolated from serum, buffy coat, or plasma at each sample collection period through the end of the trial (day 28 PE). Virus was most consistently isolated from lung, lymph node, spleen, and tonsil on day 7 PE and exclusively from lymph node, spleen, and tonsil on day 28 PE. Virus was infrequently isolated from urine and fecal and nasal swabs. Consistent microscopic changes in all age groups included interstitial pneumonia and lymph node hypertrophy and hyperplasia on days 7 and 28 PE, lymph node necrosis on day 7 PE, and subacute mononuclear myocarditis on day 28 PE. Findings presented here indicate that interstitial pneumonia, lymphoid necrosis, and mononuclear myocarditis are characteristic lesions of PRRSV isolate ATCC VR-2332 infection in 1-, 4-, and lo-week-old pigs.

An indirect fluorescent antibody test for the detection of antibody to swine infertility and respiratory syndrome virus in swine sera

An indirect fluorescent antibody (IFA) test was developed and standardized to detect and quantitate antibody for swine infertility and respiratory syndrome (SIRS) virus in swine sera. Test results were evaluated using sera of pigs infected both experimentally and naturally with SIRS virus. The IFA test used swine alveolar macrophage (SAM) monolayers prepared in 96-well microplates and infected with SIRS virus. The monolayers were incubated with test sera, washed, and stained with fluorescein isothiocyanate-labeled rabbit anti-swine IgG. After another wash step, the monolayers were examined under a fluorescent microscope. A noninfected SAM control well was included for each sample. The antibody titers for each serum sample were recorded as the highest serum dilutions with specific cytoplasmic fluorescence but no fluorescence in the control wells. To evaluate the test, sera of 4 6-week-old pigs that had been infected with SIRS virus, 2 contact pigs, and 13 experimentally infected sows were used. In the experimentally infected pigs, antibody was first detected at 7 days postexposure (PE) and peaked (1:256–1,024) between 11 and 21 days PE. All 13 sow sera were negative at time of infection but were positive (1:64-> 1: 1,024) at 14–26 days PE. Seven hundred twenty sera collected from 25 different swine farms with or without a history of SIRS were also tested. Of 344 sera from 15 swine farms with a clinical history of SIRS, 257 (74.7%) sera had IFA titers ≥ 1:4, whereas 371 (98.7%) of 376 sera from herds with no history of SIRS were negative. The present results indicate that the IFA is a useful test for the detection and quantitation of SIRS virus antibody in swine sera.

Control and elimination of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSv) can have a significant economic impact on swine herds due to reproductive failure, preweaning mortality and reduced performance in growing pigs. Control at the farm level is pursued through different management procedures (e.g. pig flow, gilt acclimation, vaccination). PRRSv is commonly eliminated from sow herds by a procedure called herd closure whereby the herd is closed to new introductions for a period of time during which resident virus dies out. However, despite thorough application of biosecurity procedures, many herds become re-infected from virus that is present in the area. Consequently, some producers and veterinarians are considering a voluntary regional program to involve all herds present within an area. Such a program was initiated in Stevens County in west central Minnesota in 2004. PRRSv has been eliminated from most sites within the region and the area involved has expanded to include adjacent counties. The program has been relatively successful and reflects local leadership, a cooperative spirit, and a will to eliminate virus from the region.

Evidence of infectivity of airborne porcine epidemic diarrhea virus and detection of airborne viral RNA at long distances from infected herds

Porcine epidemic diarrhea virus (PEDV) spread rapidly after being diagnosed in the USA in April 2013. In this study we assessed whether PEDV could become airborne and if so, whether the virus was infectious. Air samples were collected both from a room containing experimentally infected pigs and at various distances from the outside of swine farms experiencing acute PEDV outbreaks. Results indicated presence of infectious PEDV in the air from experimentally infected pigs and genetic material of PEDV was detected up to 10 miles downwind from naturally infected farms. Airborne transmission should be considered as a potential route for PEDV dissemination.

Relationships between tail biting in pigs and disease lesions and condemnations at slaughter

Two matched case-control studies were performed at an abattoir with a capacity of 780 pigs per hour, each study using the approximately 7000 pigs slaughtered on one day. In the first study, the severity of tail biting and pneumonia were recorded in pigs with bitten or intact tails. In the second study, the tail score, sex, and the presence of pleuritis, externally visible abscesses and trimming were recorded in pigs with bitten or intact tails. In study 1, there was no significant association between the tail score and the percentage of lung tissue affected by lesions typical of enzootic pneumonia, but there was a significant association between the severity of tail biting and the prevalence of lungs with abscesses and/or pleuritic lesions (P<0·0001). In study 2, there were significant associations between the severity of tail biting, and the prevalence of external carcase abscesses and carcase trimming; the carcases of castrated males had evidence of tail biting more frequently than the carcases of females (P<0·05).

Serologic evidence incriminating a recently isolated virus (ATCC VR-2332) as the cause of swine infertility and respiratory syndrome (SIRS).

and swine infertility and respiratory syndrome (SIRS). tality ranged from 37% to 83% and was accompanied by late term (>day 100) abortion in all but 1 herd. The absence of anorexia in 2 herds and absence of dyspnea in 1 herd may reflect a failure of detection rather than absence of these clinical signs (Table 2). Each case herd was matched with a herd located within 10 miles, of similar size and type of production, and having no history of undiagnosed reproductive failure or respiratory disease in the last 3 years. Sera were collected from approximately 30 representative sows at each of these 16 herds.

Previous infection of sows with a “mild” strain of porcine epidemic diarrhea virus confers protection against infection with a “severe” strain

Porcine epidemic diarrhea virus (PEDv) infected approximately 50% of the US swine breeding herds from July 2013 to July 2014 as estimated by the Swine Health Monitoring Project. In the absence of effective vaccines or standard control protocols, there is an urgent need for evidence of cross-protective immune countermeasures. Here, we evaluated the response of 3-day-old piglets born to sows exposed seven months earlier to a mild strain of PEDv to challenge with a virulent PEDv isolate. Piglet survival to one week of age was 100% compared to 67% in piglets born to sows not previously exposed, and morbidity was 43% compared to 100%, respectively. At necropsy at 7 days of age, the PEDv Ct value was 23.6 (range 16.6-30.6) in intestinal contents, compared to 17.2 (range 15.9-18.5) (p<0.06) in litters from sows with no previous exposure to PEDv. The findings indicated that durable lactogenic immunity was present in sows previously exposed to a mild strain of PEDv and this immunity induced cross-protection to representative virulent PEDv. Thus, a naturally attenuated form of PEDv provided significant passive immune protection for seven months against piglet challenge with virulent PEDv.

Evaluation of probiotics as a substitute for antibiotics in a large pig nursery

ANTIMICROBIALS, when used as growth promoters, are claimed to improve daily weight gain by 3 to 9 per cent and feed utilisation by 2 to 7 per cent, with fewer scour problems (Visk 1978, Hedde 1984, Doyle 2001). For this reason they are customarily used in pigs even on high-health status farms. They appear to act by reducing the pathogenic bacteria and modifying the microflora in the gut, providing more nutrient availability for the animal itself and less substrate for the bacterial organisms to use for their own growth (Visk 1978, Hedde 1984). Dritz and others (2002) showed that antibiotics are justified for use only in nursery pigs but not in growers and finishers. Despite their beneficial effects, recent concerns regarding the presence of drug residues in edible animal products and the potential transfer of antibiotic resistance to human pathogens has directed research towards alternative solutions such as probiotics. Legislation to phase out the routine feeding to food animals of eight classes of antibiotics as feed additives (penicillin, tetracycline, bacitracin, macrolides, lincomycin, streptogramin, aminoglycoside and sulphonamides) judged to be directly or indirectly of human medical importance has been recently introduced in the USA and has already been applied in the EU. Thus, alternative solutions should always be sought. Probiotics are live cultures of harmless bacteria or yeast species (for example, Lactobacillus, Streptococcus and Saccharomyces species) that equilibrate intestinal microflora, to the benefit of the animal (Fuller 1989, Ferencik and others 2000). They may promote growth by competing with harmful gut flora, and by stimulating the immune system of the animal and therefore increasing its resistance to infectious agents (Tannock 1980, Fuller 1989, Khajarern and Khajarern 1994). Several studies in various animal species have indicated a beneficial role of probiotics against diseases; for example, they may control some Escherichia coli-induced diseases (Khajarern and Khajarern 1994, Zani and others 1998, Kyriakis and others 1999, Genovese and others 2000). Other studies including untreated, antibiotic-free control pigs have shown that the average daily weight gain (ADG) and feed conversion ratio (FCR) are significantly improved after probiotic treatment (Kyriakis and others 1999, 2003, Kritas and others 2000). This short communication describes a study to examine the ability of probiotics to act as a substitute for growthpromoting antibiotics on the health and productivity of weaned pigs on one farm with high-health status. The study was carried out from October 2002 to October 2003, on a commercial, three-site production farm of 1600 sows in Minnesota, USA. All breeding animals were of the same breed and they were vaccinated against Aujeszky’s disease, porcine reproductive and respiratory syndrome, parvovirus infection, erysipelas, leptospirosis and E coli. The piglets on the farm were weaned in weekly batches of (mean [sd]) 520 (50) animals at the age of 20 (2) days and, after separation by sex and bodyweight, were transferred to two adjacent off-site nurseries, where they remained until the age of 64 (3) days. Each nursery contained four small rooms of 16 pens and two large rooms of 32 pens, with approximately 22 pigs per pen. The nursery rooms were filled sequentially one after another, and each small room contained pigs of the same week of age, or of two sequential weeks of age in the large rooms. A common outer corridor connected the rooms in each nursery, and footbaths in the corridor outside each room were used before personnel entered. A separate feed bin supplied each room. The pigs were fed ad libitum rations containing low doses of antibiotics: 400 ppm neomycin (Neomix; Phibro Animal Health) for the first five to seven days postweaning; 100 ppm neomycin plus 100 ppm oxytetracycline (Neo-Terramycin; Phibro Animal Health) for the next seven days, and thereafter 20 ppm tylosin (Tylan 10; Elanco Animal Health) up to the age of 70 days. The programme had been started five years previously to prevent E coli postweaning diarrhoea. Postweaning mortality in the nurseries was historically low (<2 per cent). To test the effects of probiotics compared with subtherapeutic doses of antibiotics, the product BioPlus 2B (Chr Hansen) containing 1012 colony-forming units total Bacillus content (Bacillus licheniformis and Bacillus subtilis) per 0·45 kg of product was used instead of the antibiotics. The US Food and Drug Administration has approved this product for use in feeds, and the Bacillus species strains included do not produce any antibiotic (Anon 2005). The product was included in the usual rations of the nursery period at the standard dose of 0·45 kg/tonne of complete feed. Forty-two nursery rooms were used chronologically but in an alternate mode; for example, the pigs of the first room received feed with probiotics, those of the second room feed with antibiotics, those of the third room feed with probiotics, and so on. At the end of the trial, the two treatment groups, that is, pigs receiving the usual feed that contained antibiotics (21 rooms) and pigs receiving the same type of feed but containing probiotics instead of antibiotics for the same period (21 rooms), were compared. As shown in Table 1, the average age and bodyweight of the pigs between the groups did not differ significantly at the beginning of the trial. Mortality and reasons of death were recorded by the farmer for each group of pigs up to the end of the nursery period. At that time, the pigs were weighed again. The feed consumption per group was also recorded during this period, and the ADG, the average daily feed intake (ADFI) and the FCR were calculated. At least 20 treated and 20 control rooms were needed to detect a 1·50 per cent difference in mortality (α=0·05, power 80 per cent, sd 1·5 per cent). Each parameter was recorded and analysed with ‘room’ as the experimental unit by one-way analysis of variance using the SPSS for Windows statistical package (release 11.0.1; SPSS). The results shown in Table 1 were derived from a total of 10,955 pigs in 21 probiotic rooms and 10,800 pigs in 21 antibiotic rooms. In both treatment groups, the nursery periods had the same average length. The bodyweight of pigs at the end of the nursery period, the ADG, ADFI and FCR were similar for both groups. The cost of feed per pig and per kg of bodyweight was also similar. There was no difference in morVeterinary Record (2005) 156, 447-448