Luis G. Giménez-Lirola

Polymicrobial respiratory disease in pigs

Abstract Respiratory disease in pigs is common in modern pork production worldwide and is often referred to as porcine respiratory disease complex (PRDC). PRDC is polymicrobial in nature, and results from infection with various combinations of primary and secondary respiratory pathogens. As a true multifactorial disease, environmental conditions, population size, management strategies and pig-specific factors such as age and genetics also play critical roles in the outcome of PRDC. While non-infectious factors are important in the initiation and outcome of cases of PRDC, the focus of this review is on infectious factors only. There are a variety of viral and bacterial pathogens commonly associated with PRDC including porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), porcine circovirus type 2 (PCV2), Mycoplasma hyopneumoniae (MHYO) and Pasteurella multocida (PMULT). The pathogenesis of viral respiratory disease is typically associated with destruction of the mucocilliary apparatus and with interference and decrease of the function of pulmonary alveolar and intravascular macrophages. Bacterial pathogens often contribute to PRDC by activation of inflammation via enhanced cytokine responses. With recent advancements in pathogen detection methods, the importance of polymicrobial disease has become more evident, and identification of interactions of pathogens and their mechanisms of disease potentiation has become a topic of great interest. For example, combined infection of pigs with typically low pathogenic organisms like PCV2 and MHYO results in severe respiratory disease. Although the body of knowledge has advanced substantially in the last 15 years, much more needs to be learned about the pathogenesis and best practices for control of swine respiratory disease outbreaks caused by concurrent infection of two or more pathogens. This review discusses the latest findings on polymicrobial respiratory disease in pigs.

A PCV2 vaccine based on genotype 2b is more effective than a 2a-based vaccine to protect against PCV2b or combined PCV2a/2b viremia in pigs with concurrent PCV2, PRRSV and PPV infection.

The predominant genotype of porcine circovirus (PCV) in the pig population today is PCV2b yet PCV2a-based commercial vaccines are considered effective in protecting against porcine circovirus associated disease. The objective of this study was to compare the ability of PCV2a- and PCV2b-based vaccines to control PCV2b viremia in a challenge model that mimics the U.S. field situation. Sixty-three pigs were randomly assigned to one of eight groups. Sixteen pigs were vaccinated with an experimental live-attenuated chimeric PCV1-2a vaccine based on genotype 2a and another 16 pigs with a chimeric PCV1-2b vaccine based on genotype 2b. Challenge was done 28 days post vaccination (dpv) using PCV2b (or a combination of PCV2a and PCV2b), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine parvovirus (PPV) to mimic what commonly occurs in the field. The experiment was terminated 21 days post challenge (dpc) or 49dpv. Pigs vaccinated with the chimeric PCV1-2b vaccine had significantly higher levels of PCV1-2b viremia and shedding of the PCV1-2b vaccine virus in feces and nasal secretions but also a more robust humoral immune response as evidenced by significantly higher ELISA S/P ratios compared to the PCV1-2a vaccination. Regardless of challenge, the PCV1-2b vaccination significantly reduced the prevalence and amount of PCV2 viremia compared to the PCV1-2a vaccination. Interestingly, in the non-vaccinated pigs concurrent PCV2a infection resulted in clinical disease and increased macroscopic lung lesions compared to pigs challenged with PCV2b alone, further supporting the idea that concurrent PCV2a/PCV2b infection is necessary for optimal PCV2 replication.

Effect of porcine epidemic diarrhea virus infectious doses on infection outcomes in naïve conventional neonatal and weaned pigs

Porcine epidemic diarrhea virus (PEDV) was identified in the United States (U.S.) swine population for the first time in April 2013 and rapidly spread nationwide. However, no information has been published regarding the minimum infectious dose (MID) of PEDV in different pig models. The main objective of this study was to determine the oral minimum infectious dose of PEDV in naïve conventional neonatal piglets and weaned pigs. A U.S. virulent PEDV prototype isolate (USA/IN19338/2013) with known infectious titer was serially ten-fold diluted in virus-negative cell culture medium. Dilutions with theoretical infectious titers from 560 to 0.0056 TCID50/ml together with a medium control were orogastrically inoculated (10ml/pig) into 7 groups of 5-day-old neonatal pigs (n = 4 per group) and 7 groups of 21-day-old weaned pigs (n = 6 per group). In 5-day-old pigs, 10ml of inoculum having titers 560–0.056 TCID50/ml, corresponding to polymerase chain reaction (PCR) cycle threshold (Ct) values 24.2–37.6, resulted in 100% infection in each group; 10ml of inoculum with titer 0.0056 TCID50/ml (Ct>45) caused infection in 25% of the inoculated pigs. In 21-day-old pigs, 10ml of inoculum with titers 560–5.6 TCID50/ml (Ct 24.2–31.4) resulted in 100% infection in each group while 10ml of inoculum with titers 0.56–0.0056 TCID50/ml (Ct values 35.3 –>45) did not establish infection in any pigs under study conditions as determined by clinical signs, PCR, histopathology, immunohistochemistry, and antibody response. These data reveal that PEDV infectious dose is age-dependent with a significantly lower MID for neonatal pigs compared to weaned pigs. This information should be taken into consideration when interpreting clinical relevance of PEDV PCR results and when designing a PEDV bioassay model. The observation of such a low MID in neonates also emphasizes the importance of strict biosecurity and thorough cleaning/disinfection on sow farms.

Identification and characterization of novel porcine astroviruses (PAstVs) with high prevalence and frequent co-infection of individual pigs with multiple PAstV types

Many astrovirus (AstV) species are associated with enteric disease, although extraintestinal manifestations in mammalian and avian hosts have also been described. In this study, the prevalence rates of porcine AstV types 1-5 (PAstV1-PAstV5) were investigated using faecal samples from 509 pigs of which 488 (95.9%) came from farms with a history of diarrhoea. All of the five known PAstV types were found to circulate in pigs in the USA, and co-infection of a single pig with two or more PAstV types was frequently observed. A high overall prevalence of 64.0% (326/509) of PAstV RNA-positive samples was detected, with 97.2% (317/326) of the PAstV RNA-positive pigs infected with PAstV4. Further genomic sequencing and characterization of the selected isolates revealed low sequence identities (49.2-89.0%) with known PAstV strains, indicating novel types or genotypes of PAstV2, PAstV4 and PAstV5. Some new features of the genomes of the PAstVs were also discovered. The first complete genome of a PAstV3 isolate was obtained and showed identities of 50.5-55.3% with mink AstV and the novel human AstVs compared with 38.4-42.7% with other PAstV types. Phylogenetic analysis revealed that PAstV1, PAstV2 and PAstV3 were more closely related to AstVs from humans and other animals than to each other, indicating past cross-species transmission and the zoonotic potential of these PAstVs.

The prevalence of Torque teno sus virus (TTSuV) is common and increases with the age of growing pigs in the United States

Infection with the Torque teno sus virus (TTSuV) is believed to be common yet limited information is available on the epidemiology of TTSuV. The objectives of this study were to develop novel and improve existing diagnostic methods for TTSuV infection and to investigate the prevalence of TTSuV species 1 (TTSuV1) and 2 (TTSuV2) in the USA. Three hundred and four blood or fetal thoracic fluid samples were collected from pigs on 40 US farms in 12 States. Samples were collected from fetuses and in pre-suckle neonates (n=73), suckling pigs (1-20 days of age; n=27), nursery pigs (21-55 days of age; n=60), finisher pigs (8-25 weeks of age; n=90) and adults (>25 weeks of age; n=54). Samples were tested by a new quantitative differential real-time PCR for TTSuV1 and TTSuV2 DNA and by ELISA for detection of anti-TTSuV2-antibodies. The prevalence of TTSuV1 DNA ranged from 8.2% (fetuses and neonates) to 81% (finisher pigs) and the prevalence of TTSuV2 DNA ranged from 3.7% (suckling pigs) to 67% (finisher pigs). Evidence of fetal TTSuV infection was minimal. Mixed infection of TTSuV1 and TTSuV2 was seen in 6.7% of the nursery pigs, 52.2% of the finisher pigs, and 22.2% of the mature pigs. The prevalence of TTSuV1 was higher than that of TTSuV2. Anti-TTSuV2 antibodies were not detected in the fetuses and neonates and the seroprevalence of TTSuV2 was between 3.8% and 100% in growing pigs. The results of this study indicate that vertical transmission may not be a main route of TTSuV transmission in pigs in the USA.

Characterization of porcine parvovirus type 2 (PPV2) which is highly prevalent in the USA

A novel porcine parvovirus designated as porcine parvovirus 2 (PPV2) was initially identified in Myanmar in 2001, in China during 2006-2007, and in Hungary in 2012. To investigate the presence and prevalence of PPV2 in the USA, a novel TaqMan(®) real-time PCR method was developed and used for a PPV2 survey using 483 lung samples, 185 fecal samples and 122 sera collected from pigs on 295 farms in 18 U.S. states. The overall prevalence of PPV2 was 20.7% (100/483) in lung samples and 7.6% (14/185) in fecal samples obtained from pigs of different age groups, and 1.6% (2/122) in sera or thoracic fluids obtained from neonatal pigs. Further genomic sequence comparison demonstrated that the 2011 U.S. PPV2 sequences have nucleotide identities of 95.4-97.7% with the 2006-2007 strains detected in China while the nucleotide identity was 94.7% with the 2001 strain detected in Myanmar, indicating persistent evolution of the virus. Phylogenetic and sequence homology analyses demonstrated a close relationship of PPV2 with members of the proposed genus PARV4-like virus, and the classification of PPV2 into this proposed genus is suggested.

Characterization of a Novel Porcine Parvovirus Tentatively Designated PPV5

A new porcine parvovirus (PPV), provisionally designated as PPV5, was identified in U.S. pigs. Cloning and sequencing from a circular or head-to-tail concatemeric array revealed that the PPV5 possesses the typical genomic organization of parvoviruses with two major predicted open reading frames (ORF1 and ORF2), and is most closely related to PPV4 with overall genomic identities of 64.1–67.3%. The amino acid identities between PPV5 and PPV4 were 84.6%–85.1% for ORF1 and 54.0%–54.3% for ORF2. Unlike PPV4, but similar to bovine parvovirus 2 (BPV2), PPV5 lacks the additional ORF3 and has a much longer ORF2. Moreover, the amino acid sequences of ORF1 and ORF2 of BPV2 showed higher homologies to PPV5 than to PPV4. The conserved motifs of the Ca2+ binding loop (YXGXG) and the catalytic center (HDXXY) of phospholipase A2 (PLA2) were identified in VP1 (ORF2) of PPV5, as well as in BPV2, but were not present in PPV4. Phylogenetic analyses revealed that PPV5, PPV4 and BPV2 form a separate clade different from the genera Parvovirus and Bocavirus. Further epidemiologic investigations of PPV4 and PPV5 in U.S. pigs of different ages indicated a slightly higher prevalence for PPV5 (6.6%; 32/483) compared to PPV4 (4.1%; 20/483), with detection of concurrent PPV4 and PPV5 in 15.6% (7/45) of lungs of infected pigs. Evidence for potential vertical transmission or association with reproductive failure was minimal for both PPV4 and PPV5. The high similarity to PPV4 and the lack of ORF3 may suggest PPV5 is an intermediate of PPV4 during the evolution of parvoviruses in pigs.

Kinetics of influenza A virus nucleoprotein antibody (IgM, IgA, and IgG) in serum and oral fluid specimens from pigs infected under experimental conditions.

Indirect influenza A virus (IAV) nucleoprotein (NP) antibody ELISAs were used to compare the kinetics of the NP IgM, IgA, and IgG responses in serum and pen-based oral fluid samples collected from 82 pigs followed for 42 days post inoculation (DPI). Treatment categories included vaccination (0, 1) and inoculation (0, 1) with contemporary H1N1 or H3N2 isolates. Antibody ontogeny was markedly affected by vaccination status, but no significant differences were detected between H1N1 and H3N2 inoculated groups of the same vaccination status (0, 1) in IgM, IgA, or IgG responses. Therefore, these data were combined in subsequent analyses. The correlation between serum and oral fluid responses was evaluated using the pen-based oral fluid sample-to-positive (S/P) ratios versus the mean serum S/P ratios of pigs within the pen. IgM responses in serum and oral fluid were highly correlated in unvaccinated groups (r=0.810), as were serum and oral fluid IgG responses in both unvaccinated (r=0.839) and vaccinated (r=0.856) groups. In contrast, IgM responses were not correlated in vaccinated groups and the correlation between serum and oral fluid IgA was weak (r∼0.3), regardless of vaccination status. In general, vaccinated animals exhibited a suppressed IgM response and accelerated IgG response. The results from this study demonstrated that NP-specific IgM, IgA, and IgG antibody were detectable in serum and oral fluid and their ontogeny was influenced by vaccination status, the time course of the infection, and specimen type.

Serological and Molecular Detection of Senecavirus A Associated with an Outbreak of Swine Idiopathic Vesicular Disease and Neonatal Mortality

ABSTRACT We performed a longitudinal field study in a swine breeding herd that presented with an outbreak of vesicular disease (VD) that was associated with an increase in neonatal mortality. Initially, a USDA Foreign Animal Disease (FAD) investigation confirmed the presence of Senecavirus A (SVA) and ruled out the presence of exotic agents that produce vesicular lesions, e.g., foot-and-mouth disease virus and others. Subsequently, serum samples, tonsil swabs, and feces were collected from sows (n = 22) and their piglets (n = 33) beginning 1 week after the onset of the clinical outbreak and weekly for 6 weeks. The presence of SVA RNA was evaluated in all specimens collected by reverse transcriptase quantitative PCR (RT-qPCR) targeting a conserved region of the 5′ untranslated region (5′-UTR). The serological response (IgG) to SVA was evaluated by the weekly testing of sow and piglet serum samples on a SVA VP1 recombinant protein (rVP1) indirect enzyme-linked immunosorbent assay (ELISA). The rVP1 ELISA detected seroconversion against SVA in clinically affected and non-clinically affected sows at early stages of the outbreak as well as maternal SVA antibodies in offspring. Overall, the absence of vesicles (gross lesions) in SVA-infected animals and the variability of RT-qPCR results among specimen type demonstrated that a diagnostic algorithm based on the combination of clinical observations, RT-qPCR in multiple diagnostic specimens, and serology are essential to ensure an accurate diagnosis of SVA.

Development of a novel fluorescent microbead-based immunoassay and comparison with three enzyme-linked immunoassays for detection of anti-Erysipelothrix spp. IgG antibodies in pigs with known and unknown exposure

A novel fluorescent microbead immunoassay (FMIA) using the recombinant polypeptide SpaA415 was developed for detection of anti-Erysipelothrix spp. IgG in pig sera. The diagnostic performance of the FMIA was evaluated on samples from pigs with known and unknown Erysipelothrix spp. exposure and compared to an in-house enzyme-linked immunosorbent assay (ELISA-1) based on the same capture antigen, and two commercially available ELISAs (ELISA-2 and ELISA-3). Sera from pigs experimentally infected with Erysipelothrix rhusiopathiae serotype 1a (n=60) or 19 (n=12), sera from pigs vaccinated with a commercial attenuated-live vaccine based on serotype 1a (n=12) or a commercial bacterin based on serotype 2 (n=12), and 90 field samples were utilized. The sensitivity on 22 true positive samples collected in the later stages of infection/post-vaccination was 100% for the FMIA and ELISA-1, 63.6% for ELISA-2 and 81.8% for ELISA-3. The earliest antibody response was detected 7days post inoculation with the FMIA (77.8%) and ELISA-1 (11.1%), and at 14days post-vaccination (dpv) with FMIA (50%) and ELISA-1 (50%). On field samples, a higher seroprevalence was found in pigs older than 21days with all four assays. Kappa analysis indicated that the FMIA and ELISA-1 had almost complete agreement whereas the agreement was slight with ELISA-2 and fair with ELISA-3. The sensitivity of both immunoassays based on the rSpaA415 antigen was higher compared to that of the two commercial ELISAs. The rSpaA415 FMIA has great potential as an inexpensive ELISA alternative for detection of antibodies against E. rhusiopathiae in the future.