Correlation of Lawsonia intracellularis positivity in quantitative PCR and herd factors in European pig herds.

BACKGROUND: Lawsonia intracellularis is causing diarrhea, poor growth and sudden death in pigs. It can be found in most pig populations leading to large economic losses worldwide. Many potential risk factors for the occurrence of disease or seropositivity have been described. The current study therefore focused on herd characteristics in European countries associated with direct detection of the pathogen determined by quantitative polymerase chain reaction. RESULTS: A median number of less than 30 nursery pigs per pen was correlated to less positive nursery pigs (p < 0.01) and generally less samples positive per herd (p < 0.05) as well as a lower median of genome equivalents determined per herd (p < 0.05). Routine use of zinc oxide at/ around weaning, which was mentioned by 41.0% of all farmers, was correlated to higher number of positive nursery pigs (p < 0.01) as well as higher median genome equivalents determined per herd (p < 0.05). Slatted flooring of more than 78.0% of the surface in nursery units was correlated to lower number of positive animals (p < 0.05) and a lower median of genome equivalents per herd (p < 0.05). A weight of more than 7.8 kg at weaning was correlated to a higher number of positive growing pigs (p < 0.05) as well as general higher number of positive samples/ herd (p < 0.01). CONCLUSIONS: Weaning and subsequent accommodation of nursery pigs seem to be of particular importance in prevention of infection with Lawsonia intracellularis and the spread of the pathogen within the herd.

Prevalence of Lawsonia intracellularis in pig herds in different European countries.

BACKGROUND: Lawsonia intracellularis causes large economic losses in the pig industry worldwide. Pigs suffer from reduced daily weight gain, poor feed conversion ratio and increased mortality. The number of affected animals and herds in Europe remains unknown. This study will provide an overview of the prevalence of Lawsonia intracellularis in herds with a history of diarrhoea in different European countries and thereby identify country specific differences. RESULTS: Out of the 144 herds sampled in Germany, Denmark, Spain, the Netherlands and the United Kingdom, 90.3% (79.2-100.0%) contained at least one positive faecal sample on quantitative polymerase chain reaction (qPCR). Of the 6450 nursery, growing and finishing pigs of the previously mentioned herds, 26.2% (15.9-41.5%) of the animals were tested positive in faecal samples. Enzyme linked immunosorbent assay (ELISA) results of 60 herds were 91.7% (70-100%) positive. The percentage of positive samples in these 1791 blood samples was 31.6% (20.3-51.0%). Herd prevalence did not differ significantly by qPCR or ELISA. Significant differences between the countries were found regarding: Within-herd prevalence- qPCR: Samples from Denmark were more often positive than samples of Spain or the United Kingdom. Within-herd prevalence- ELISA: Samples from Denmark were more often positive than samples from Spain and the Netherlands. Affected age category- qPCR: Nursery pigs in Denmark were more often positive and shed more genome equivalents than nursery pigs in the other countries. Concentration of detected genome equivalents- qPCR: The concentration of genome equivalents from Lawsonia intracellularis in herds in Denmark was higher compared to all other countries. CONCLUSION: A widespread of Lawsonia intracellularis in the six European countries was confirmed, whereby a large part of the positive animals only excreted small amounts of genome equivalents. Country specific differences were found with Denmark in particular diagnosing more Lawsonia intracellularis then the other countries. Herd data collected in this study needs to be analysed to get more information about possible reasons for the differences found between the countries.

Field evaluation of a ready-to-use combined Porcine circovirus type 2 and Mycoplasma hyopneumoniae vaccine in Denmark - a historical comparison of productivity parameters in 20 nursery and 23 finishing herds.

BACKGROUND: In practice, field evaluation of vaccine efficacy in individual herds is often based on a historical comparison of productivity data following initiation of vaccination. Being biased by time, this study design highly contrasts the more controlled, parallel-group design used for most initial vaccine efficacy studies but offers the possibility of including a larger number of animals and herds. As an important add-on to previous findings in controlled studies, the objective of this study was to evaluate the field efficacy of the ready-to-use combination vaccine Porcilis® PCV M Hyo (MSD Animal Health) by an observational historical study design using routinely generated herd productivity data. RESULTS: Data on mortality, average daily weight gain and feed conversion rate were collected as yearly averages for one year prior to and one year after implementation of Porcilis® PCV M Hyo vaccination from 20 nursery and 23 finishing herds. When comparing pre- and post-vaccination periods, the average improvements in productivity data amounted to - 0.4 percentage points for mortality (p = 0.014), + 5 g for average daily weight gain (p = 0.555) and - 0.06 feeding units(FU)/kg for feed conversion rate (p = 0.074) in nursery herds and - 0.5 percentage points for mortality (p = 0.012), + 34 g for average daily weight gain (p < 0.001) and - 0.04 FU/kg for feed conversion rate (p = 0.133) in finishing herds. Even though some nursery and finishing herds also previously vaccinated against PCV2 and/or Mycoplasma hyopneumoniae, this did not significantly affect the results. For finishers, these results were obtained when difference in arrival weights between the periods and shared ownership of the herds were additionally taken into account. CONCLUSION: In these 20 nursery and 23 finishing herds, previous findings from parallel-group vaccination studies concerning average daily weight gain for finishers were confirmed. Additionally, a significant effect on mortality for both nursery and finishing herds was demonstrated in this evaluation based on routinely generated herd productivity data.

Field evaluation of piglet vaccination with a Mycoplasma hyopneumoniae bacterin as compared to a ready-to-use product including porcine circovirus 2 and M. hyopneumoniae in a conventional French farrow-to-finish farm.

BACKGROUND: A controlled randomized trial was performed on a well-managed conventional French 180-sow farm. The trial compared the growth performances of piglets vaccinated at weaning (single shot) either with a commercial monovalent Mycoplasma hyopneumoniae bacterin vaccine or with a commercial bivalent vaccine (Porcilis® PCV M Hyo) against M. hyopneumoniae and porcine circovirus 2 (PCV2). The farm's porcine reproductive and respiratory syndrome status was stable, and most diseases (enzootic pneumonia, atrophic rhinitis, post-weaning multisystemic wasting syndrome) were controlled by routine vaccination. RESULTS: During the post-weaning phase, the growth performances of the piglets vaccinated with the bivalent vaccine were not significantly different from those vaccinated with the monovalent vaccine. However, during the fattening phase the group vaccinated with the bivalent vaccine had a significantly improved ADG (+34 g/d, p = 0. 047), resulting in a 5-day earlier shipment to slaughter. The group also had a shorter and lower PCV2 load in serum during the fattening period, and an improved lung lesions score. In both groups, three pigs died during the peak PCV2 viraemia (16-23 weeks of age). Immunohistochemistry of the lymph nodes showed that in the group vaccinated with the bivalent vaccine, none of these pigs had PCV2-like lesions, while 2 out of the 3 from the other group did. Results suggest that the added PCV2 valence in the vaccination protocol helps countering the negative impact of subclinical PCV2 infection on growth. The calculated return on investment of the added PCV2 vaccine valence was €1.7 extra revenue per slaughtered pig (€ 39 additional revenue per sow and per year), despite the fact that the cost of the bivalent vaccine was higher than the monovalent M. hyopneumoniae vaccine. CONCLUSION: In this healthy conventional sow farm, the combined M. hyopneumoniae and PCV2 vaccination was efficacious, convenient to administer and profitable.

A field efficacy and safety trial in the Netherlands in pigs vaccinated at 3 weeks of age with a ready-to-use porcine circovirus type 2 and Mycoplasma hyopneumoniae combined vaccine.

BACKGROUND: Respiratory diseases impair the health and welfare of growing pigs and impacts farmers' gains worldwide. Their control through a preventative medical approach has to be tailored according to the pathogens identified at farm level. In the Netherlands, several studies have emphasized the prominent role of Mycoplasma hyopneumoniae, Porcine Circovirus type 2 and Porcine Reproductive and Respiratory Syndrome Virus in such respiratory conditions. Further to the arrival on the Dutch market of the first commercially available bivalent vaccine against PCV2 and M. hyopneumoniae, Porcilis® PCV M. Hyo, a trial was designed to evaluate its safety and efficacy under local field conditions. MATERIAL AND METHODS: In a conventional farrow-to-finish 170-sow farm with a history of respiratory diseases and demonstrated circulation of both M. hyopneumoniae and PCV2, 812 piglets were randomised and included at weaning in either of the three following groups: PCVM (vaccinated with Porcilis® PCV M. Hyo), FLEX (vaccinated with CircoFLEX® and MycoFLEX®) or NC (negative control, injected with placebo). Piglets were vaccinated at 3 weeks of age (day 0) and a subset was bled and weighed at regular intervals up to slaughter. Lung slaughter checks were only performed on 64% of the pigs included on day 0. RESULTS AND IMPLICATION: No side effect of injection was observed in any of the three groups. Average daily weight gain was improved in both vaccinated groups as compared to the NC group, over the finishing period as well as from wean-to-finish. The PCVM group had a significantly lower PCV2 viremia area under the curve than the two other groups, and a significant reduction in the severity of the pneumonia-like lesions was observed at slaughter in the pigs of the PCVM group. A conservative estimate of the economic benefit of that vaccine was 2.84 € per finisher. This trial confirms that the vaccine is efficacious against the health and growth effects of PCV2 and M. hyopneumoniae, of practical advantage (single injection of a bivalent product) and well tolerated.

Field study on the safety and efficacy of intradermal versus intramuscular vaccination against Mycoplasma hyopneumoniae.

The present study compares the safety and efficacy of a needle-free, intradermal Mycoplasma hyopneumoniae vaccine to an intramuscular one. 420 piglets (21+3 days of age) were randomly assigned to two vaccination groups (intradermal vaccination V1 (n=138), intramuscular vaccination V2 (n=144)) and one unvaccinated control group (CG, n=138). As safety parameters clinical observations, local injection site reactions (ISR) and rectal temperatures were assessed. Average daily weight gain (ADWG) and pneumonic lung lesions (LL) were measured as efficacy parameters. ISRs were minor in V1. After both vaccinations, no adverse impact on appetite was observed and mean rectal temperatures remained within physiological range. ADWG during the fattening period was significantly higher in vaccinated groups (V1: 913.4 g, V2: 924.5 g) compared with CG (875.6 g). No differences in ADWG were observed between V1 and V2. Vaccinated pigs had a significantly reduced mean extent of LL compared with CG. V1 was superior in reducing the extent and prevalence of LL compared with V2. These results reveal that a needle-free intradermal vaccination is safe and efficacious in reducing both the prevalence and extent of lung lesions, as well as in improving performance parameters, in a farrow-to-finish farm with a late onset of M hyopneumoniae infection.

Field efficacy of Porcilis® PCV M Hyo versus a licensed commercially available vaccine and placebo in the prevention of PRDC in pigs on a French farm: a randomized controlled trial.

BACKGROUND: A controlled, randomised, and blinded trial performed on a conventional French farrow-to-finish farm compared the efficacy of a one-shot bivalent ready to use vaccine, Porcilis® PCV M. Hyo (group PCVM), to that of two commercial vaccines (Ingelvac® Circoflex® + Ingelvac® Mhyo, group ICIM), and to a placebo (group CTL), in preventing the health and economic impacts of Porcine Respiratory Disease Complex (PRDC). MATERIAL & METHODS: In this small-scale clinical study, all piglets in each group were administered the vaccine/placebo at weaning age (27 days old). Piglets from either of the three groups were bled at regular intervals from 3 weeks of age until slaughter, in order to assess the infection by the main PRDC infectious agents: Mycoplasma hyopneumoniae, PCV2 and PRRSV. Performance, lung checks and slaughter data were collected and analysed. RESULTS: PCV2 viremia was significantly reduced in both vaccinated groups as compared to the placebo group. Lung lesion score was significantly lower in group PCVM, as compared to groups CTL and ICIM. Average daily weight gain during the finishing period was not significantly different between both vaccinated groups and was significantly higher than in the placebo group (849 g/d in the latter). Carcass results provided a numerical advantage to PCVM group, through improved part of production eligible for premium payment, and superior farmer income; this was a trend and did not reach significance. CONCLUSION: The one-shot bivalent vaccine Porcilis® PCV M Hyo proved to be efficacious and convenient to use in a field context of active PCV2 and M. hyopneumoniae infections.

Protective immunity to Salmonella enterica is partially serogroup specific.

Pre-harvest reduction of Salmonella carriage by swine would benefit both animal health and food quality. While vaccination is an attractive pre-harvest intervention to reduce Salmonella levels in swine, the large number of potential Salmonella enterica serovars found in swine makes it critical that vaccines provide broad serotype efficacy. In order to directly compare the relative efficacy of Salmonella vaccines against serogroup-matched and serogroup-unmatched Salmonella, we vaccinated pigs with two commercially available Salmonella vaccines (either serogroup B or serogroup C1) and challenged with serovar-matched, serogroup-matched or serogroup-unmatched challenge strains. We found that while serogroup-matched vaccines provided relatively better efficacy than unmatched vaccines, serotype-unmatched vaccines also provided significant reduction of Salmonella carriage and shed. In addition, by measuring serogroup specific cell mediated (IFN-γ ELISPOT) and humoral (anti-LPS ELISA) immunity, we found that this serogroup specific efficacy correlates primarily with humoral immunity, while cell mediated immunity was mostly non-serogroup specific. While the practical relevance to pork quality of this serogroup-specific efficacy remains to be demonstrated, the large predominance of serogroup B Salmonella in swine suggests that a serogroup B Salmonella vaccine for swine would be of value to pre-harvest food safety interventions in swine.

Efficacy of vaccination of cattle with the Leptospira interrogans serovar hardjo type hardjoprajitno component of a pentavalent Leptospira bacterin against experimental challenge with Leptospira borgpetersenii serovar hardjo type hardjo-bovis.

OBJECTIVE: To evaluate the efficacy of vaccination with the Leptospira interrogans serovar hardjo type hardjoprajitno component of a pentavalent Leptospira bacterin against a virulent experimental challenge with Leptospira borgpetersenii serovar hardjo type hardjo-bovis strain 203 in cattle. ANIMALS: Fifty-five 6-month-old Holstein heifers. PROCEDURES: Heifers that were negative for persistent infection with bovine viral diarrhea virus determined via immunohistochemical testing and negative for Leptospira interrogans serovar pomona, Leptospira interrogans serovar hardjo, Leptospira interrogans serovar grippotyphosa, Leptospira interrogans serovar bratislava, Leptospira interrogans serovar canicola, and Leptospira interrogans serovar icterohaemorrhagiae determined via microscopic agglutination assay were enrolled in the study. Two heifers were separated and used for the challenge passage. The remaining heifers were vaccinated twice with a commercial pentavalent bacterin or a sham vaccine 21 days apart and subsequently challenged with L borgpetersenii serovar hardjo type hardjo-bovis strain 203. Urinary shedding, antibody titers, and clinical signs of leptospirosis infection were recorded for 8 weeks after challenge. RESULTS: Heifers that received the pentavalent bacterin did not shed the organism in urine after challenge and did not have renal colonization at necropsy. Heifers that were sham vaccinated shed the organism in urine and had renal colonization. CONCLUSIONS AND CLINICAL RELEVANCE: Results provided evidence that a pentavalent Leptospira vaccine containing L interrogans serovar hardjo type hardjoprajitno can provide protection against challenge with L borgpetersenii serovar hardjo type hardjo-bovis strain 203. It is important to demonstrate cross-protection that is vaccine specific against disease-causing strains of organisms that are prevalent under field conditions.

Insertion and deletion in a non-essential region of the nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome (PRRS) virus: effects on virulence and immunogenicity.

Developing a vaccine that can differentiate infected and vaccinated animals (DIVA) is a new challenge in the design of a vaccine for porcine reproductive and respiratory syndrome virus (PRRSV). Nonstructural protein 2 (nsp2) is the single largest viral product, and it has multiple roles in polypeptide processing and replication complex formation. Using reverse genetics and an infectious PRRSV cDNA clone, we constructed several deletion mutants in the non-essential region of nsp2. One mutant, which has a 131 amino acid deletion within a relatively conserved region of nsp2, was recovered and found to produce a viable virus. The deleted region was replaced with a peptide tag encoding eight amino acids. A recombinant virus containing the 131 amino acid deletion was found to produce normal virus yields in MARC-145 cells and porcine alveolar macrophages (PAM); however, gross and micro-histopathology showed that the virus was less virulent in pigs. The 131 amino acid peptide was expressed as a recombinant protein and used to coat enzyme-linked immunosorbent assay (ELISA) plates. This peptide was recognized by sera from pigs infected with wild-type virus, but not by sera from pigs infected with the deletion mutant. The results from this study show that nsp2 is an important target for the development of marker vaccines and for virus attenuation.

Functional mapping of the porcine reproductive and respiratory syndrome virus capsid protein nuclear localization signal and its pathogenic association.

PRRSV (porcine reproductive and respiratory syndrome virus) nucleocapsid (N) protein is the most abundant structural protein of the virus. During infection, the N protein is specifically localized to the nucleus and nucleolus in addition to its normal cytoplasmic distribution. Previously, a nuclear localization signal (NLS, 41-PGKK(N/S)KKKN)-null mutant virus (41-PGGGNKKKN) showed reduced viremia and increased production of neutralizing antibodies in infected pigs. However, the mutagenized NLS underwent strong selection pressure in the pig that resulted in partial or complete reversion and reacquisition of NLS function, and thus the biological effect of the NLS-null mutation needed further investigation. In the present study, a total of 9 "reversion resistant" mutants were generated by amino acid deletions and substitutions using an infectious cDNA clone. Two mutant clones (PG--SKKKS and PG--S-KKS) that produced progeny viruses were genetically stable for at least 20 passages in cell culture. Infection of pigs with those mutants induced neutralizing antibodies to higher titers than with wild-type virus. Both mutant viruses induced viremia of lower titer and of shorter duration than wild-type virus. RT-PCR from tonsils showed that both mutants persisted at a reduced level. Virus transmission to contact pigs was also lower in the mutant virus infected groups. No reversion to functional NLS was detected in either mutant from any pig. These data demonstrate that N protein nuclear localization is indeed associated with viral pathogenesis and host response to PRRS.

CD163 expression confers susceptibility to porcine reproductive and respiratory syndrome viruses.

Direct functional screening of a cDNA expression library derived from primary porcine alveolar macrophages (PAM) revealed that CD163 is capable of conferring a porcine reproductive and respiratory syndrome virus (PRRSV)-permissive phenotype when introduced into nonpermissive cells. Transient-transfection experiments showed that full-length CD163 cDNAs from PAM, human U937 cells (histiocytic lymphoma), African green monkey kidney cells (MARC-145 and Vero), primary mouse peritoneal macrophages, and canine DH82 (histocytosis) cells encode functional virus receptors. In contrast, CD163 splice variants without the C-terminal transmembrane anchor domain do not provide PRRSV receptor function. We established several stable cell lines expressing CD163 cDNAs from pig, human, and monkey, using porcine kidney (PK 032495), feline kidney (NLFK), or baby hamster kidney (BHK-21) as the parental cell lines. These stable cell lines were susceptible to PRRSV infection and yielded high titers of progeny virus. Cell lines were phenotypically stable over 80 cell passages, and PRRSV could be serially passed at least 60 times, yielding in excess of 10(5) 50% tissue culture infective doses/ml.

Mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus replicating in the cytoplasm, but the nucleocapsid (N) protein is specifically localized to the nucleus and nucleolus in virus-infected cells. A 'pat7' motif of 41-PGKK(N/S)KK has previously been identified in the N protein as the functional nuclear localization signal (NLS); however, the biological consequences of N protein nuclear localization are unknown. In the present study, the role of N protein nuclear localization during infection was investigated in pigs using an NLS-null mutant virus. When two lysines at 43 and 44 at the NLS locus were substituted to glycines, the modified NLS with 41-PGGGNKK restricted the N protein to the cytoplasm. This NLS-null mutation was introduced into a full-length infectious cDNA clone of PRRSV. Upon transfection of cells, the NLS-null full-length clone induced cytopathic effects and produced infectious progeny. The NLS-null virus grew to a titer 100-fold lower than that of wild-type virus. To examine the response to NLS-null PRRSV in the natural host, three groups of pigs, consisting of seven animals per group, were intranasally inoculated with wild-type, placebo, or NLS-null virus, and the animals were maintained for 4 weeks. The NLS-null-infected pigs had a significantly shorter mean duration of viremia than wild-type-infected pigs but developed significantly higher titers of neutralizing antibodies. Mutations occurred at the NLS locus in one pig during viremia, and four types of mutations were identified: 41-PGRGNKK, 41-PGGRNKK, and 41-PGRRNKK, and 41-PGKKSKK. Both wild-type and NLS-null viruses persisted in the tonsils for at least 4 weeks, and the NLS-null virus persisting in the tonsils was found to be mutated to either 41-PGRGNKK or 41-PGGRNKK in all pigs. No other mutation was found in the N gene. All types of reversions which occurred during viremia and persistence were able to translocate the mutated N proteins to the nucleus, indicating a strong selection pressure for reversion at the NLS locus of the N protein in vivo. Reversions from NLS-null to functional NLS in the tonsils suggest a possible correlation of viral persistence with N protein nuclear localization. These results show that N protein nuclear localization is non-essential for PRRSV multiplication but may play an important role in viral attenuation and in pathogenesis in vivo.

Construction and evaluation of genetically engineered replication-defective porcine reproductive and respiratory syndrome virus vaccine candidates.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an emerging pathogen causing significant economic losses in the swine industry worldwide. Two novel gene-deleted viruses were constructed and evaluated as vaccine candidates. Using the full-length infectious cDNA clone of North American PRRS isolate P129, the ORF2 and ORF4 genes (which encoded minor structural glycoproteins GP2a/2b and GP4, respectively) were individually deleted from the viral genome. Both deletion mutants were non-viable in MARC-145 cells and porcine alveolar macrophages, indicating that both genes are essential for virus replication. To rescue the replication-defective PRRSV, two complementing cell lines, MARC-2000 and MARC-400, were established to stably express the PRRSV GP2 and GP4 proteins, respectively. These cells were able to complement the deleted gene function of PRRSV in trans and supported production of the replication-defective DeltaORF2-PRRSV and DeltaORF4-PRRSV viruses. Both DeltaORF2-PRRSV and DeltaORF4-PRRSV viruses were propagated for 40-50 generations in the corresponding complementing cells and remained replication-defective in MARC-145 cells. To examine the immunogenic potential of the replication-defective PRRSV as vaccine candidates, four groups of pigs, 20 pigs per group, were immunized twice with DeltaORF2-PRRSV or DeltaORF4-PRRSV and challenged with the homologous virulent virus at 3 weeks post-immunization. In spite of the fact one group showed significant reduction in virus load, we could not demonstrate improvement from clinical diseases in this vaccination/challenge study. However, we did show that the cDNA clone of PRRSV can be a useful tool to genetically engineer PRRSV vaccine candidates and to study pathogenesis and viral gene functions.

Respiratory symptoms and airway obstruction in swine veterinarians: a persistent problem.

OBJECTIVE: There are many occupational hazards associated with the practice of swine veterinarians. To reassess the prevalence of respiratory complaints and pulmonary function abnormalities in this group. METHODS: This was a cross-sectional study conducted during the American Association of Swine Veterinarians annual meeting. Subjects completed a respiratory symptom/work history questionnaire and performed spirometry. RESULTS: Participants included 122 veterinarians (median age=42.5 years). Work-related symptoms included rhinitis symptoms (69%), cough and chest tightness (53%), wheezing (31%). Airway obstruction was seen in 24% of participants. Veterinarians with airway obstruction reported working more hours per week in hog barns than did practitioners with normal pulmonary function (P=0.009). CONCLUSIONS: Respiratory symptoms were common in the swine veterinarians tested as was airway obstruction. The association with these findings and hog barn exposure suggests that working in these facilities is still a risk factor for airway disease.

Comparison of embryonated chicken eggs with MDCK cell culture for the isolation of swine influenza virus.

Embryonated chicken eggs (ECE) and the Madin-Darby canine kidney (MDCK) cell line were compared for isolation of swine influenza virus (SIV) from nasal swabs and tissue samples. Samples originated from 30 pigs experimentally inoculated with 2 x 106 to 2 x 10(7) embryo infectious dose 50% (EID50)/mL of swine influenza strain A/Swine/Indiana/1726/88 (H1N1). The results were analyzed with McNemar's chi-squared test for symmetry. The results indicated that more samples were SIV-positive with ECE than with tissue culture (P < 0.001), suggesting that ECE remains the system of choice for isolation of SIV. It is recommend that routine use of both SIV isolation systems will increase the sensitivity of detection of virus shedding by considering the differences in growth and tropism of diverse SIV strains.