Coinfection with porcine epidemic diarrhea virus and Clostridium perfringens type A enhances disease severity in weaned pigs.

Clostridium perfringens is a constituent of the normal gut microbiome in pigs; however, it can potentially cause pre- and post-weaning diarrhea. Nevertheless, the importance of this bacterium as a primary pathogen of diarrhea in piglets needs to be better understood, and the epidemiology of C. perfringens in Korean pig populations is unknown. To study the prevalence and typing of C. perfringens, 203 fecal samples were collected from diarrheal piglets on 61 swine farms during 2021-2022 and examined for the presence of C. perfringens and enteric viruses, including porcine epidemic diarrhea virus (PEDV). We determined that the most frequently identified type of C. perfringens was C. perfringens type A (CPA; 64/203, 31.5%). Among the CPA infections, single infections with CPA (30/64, 46.9%) and coinfections with CPA and PEDV (29/64, 45.3%) were the most common in diarrheal samples. Furthermore, we conducted animal experiments to investigate the clinical outcome of single infections and coinfections with highly pathogenic (HP)-PEDV and CPA in weaned piglets. The pigs infected with HP-PEDV or CPA alone showed mild or no diarrhea, and none of them died. However, animals that were co-inoculated with HP-PEDV and CPA showed more-severe diarrheal signs than those of the singly infected pigs. Additionally, CPA promoted PEDV replication in coinfected piglets, with high viral titers in the feces. A histopathological examination revealed more-severe villous atrophy in the small intestine of coinfected pigs than in singly infected pigs. This indicates a synergistic effect of PEDV and CPA coinfection on clinical disease in weaned piglets.

Comparison of the pathogenicity of porcine reproductive and respiratory syndrome virus (PRRSV)-1 and PRRSV-2 in pregnant sows.

To date, few studies related to the evaluation of the pathogenicity of different PRRSV isolates using a reproductive model have been undertaken, and the main focus has remained on respiratory models using young pigs. This study aimed to evaluate the pathogenicity of two PRRSV-1 isolates (D40 and CBNU0495) and two PRRSV-2 isolates (K07-2273 and K08-1054) in a reproductive model. Pregnant sows were experimentally infected with PRRSV at gestational day 93 or used as an uninfected negative control. Sera were collected at 0, 3, 7, 14, and 19 days post-challenge (dpc) for virological and serological assays. At 19 dpc, all sows were euthanized, and their fetuses were recovered by performing cesarean section and immediately euthanized for sample collection. Here, compared to the other isolates, the CBNU0495 isolate replicated most efficiently in the pregnant sows, and K07-2273 produced the highest rate of reproductive failure even though it did not replicate as efficiently as the other isolates in sows and fetuses, indicating that vertical transmission and reproductive failure due to PRRSV infection do not have any significant correlation with the viral loads in samples from sows and fetuses. Similarly, the viral loads and the histopathological lesions did not show any correlation with each other, as the PRRSV-2-infected groups displayed more prominent and frequent histopathological lesions with lower viral loads than the PRRSV-1-infected groups. However, viral loads in the myometrium/endometrium might be related to the spreading of PRRSV in the fetuses, which affected the birth weight of live fetuses. This study contributes to a better understanding of the pathogenicity of the most prevalent Korean PRRSVs in a reproductive model.

Deposition of bioactive human epidermal growth factor in the egg white of transgenic hens using an oviduct-specific minisynthetic promoter.

Currently, transgenic animals have found a wide range of industrial applications and are invaluable in various fields of basic research. Notably, deposition of transgene-encoded proteins in the egg white (EW) of hens affords optimal production of genetically engineered biomaterials. In the present study, we developed a minisynthetic promoter modulating transgene transcription specifically in the hen's oviduct, and assayed the bioactivity of human epidermal growth factor (hEGF) driven by that promoter, after partial purification of epidermal growth factor (EGF) from transgenic hen eggs. Our minisynthetic promoter driving expression of chicken codon-optimized human epidermal growth factor (cEGF) features 2 consecutive estrogen response elements of the ovalbumin (OV) promoter, ligated with a 3.0 kb OV promoter region carrying OV regulatory elements, and a 5'-UTR. Subsequently, a 3'-UTR carrying the poly-A tail sequence of the OV gene was added after incorporation of the cEGF transgene. Finally, we partially purified cEGF from transgenic hen eggs and evaluated the biofunctional activities thereof in vitro and in vivo. In the in vitro assay, EW-derived hEGF exhibited a proliferative effect on HeLa cells similar to that of commercial hEGF. In the in vivo assay, compared to the nontreated control, transgenic hen egg-derived EGF afforded slightly higher levels of re-epithelialization (via fibroplasia) and neovascularization of wounded skin of miniature pigs than did the commercial material. In conclusion, transgenic hens may be used to produce genetically engineered bioactive biomaterials driven by an oviduct-specific minisynthetic promoter.

Performance of three multiplex real-time PCR assays for simultaneous detection of 12 infectious pathogens in mice affected with respiratory and digestive diseases.

Introduction: Research quality can be improved with reliable and reproducible experimental results when animal experiments are conducted using laboratory animals with guaranteed microbiological and genetic quality through health monitoring. Therefore, health monitoring requires the rapid and accurate diagnosis of infectious diseases in laboratory animals. Methods: This study presents a performance evaluation of a commercially available multiplex real-time PCR (mRT-PCR) assay for the rapid detection of 12 infectious pathogens (Set 1: Sendai virus [SeV, formally murine respirovirus], Mycoplasma spp., Rodentibacter pneumotropicus, and Rodentibacter heylii; Set 2: Helicobacter spp., Murine norovirus [MNV], Murine hepatitis virus [MHV], and Salmonella spp.; Set 3: Staphylococcus aureus, Streptobacillus moniliformis, Corynebacterium kutscheri, and Pseudomonas aeruginosa). To evaluate the efficacy of the mRT-PCR assay, 102 clinical samples encompassing fecal and cecal specimens were analyzed. The resulting data were then compared with the findings from sequence analysis for validation. Results: The assay's detection limit ranged from 1 to 100 copies per reaction. Specificity testing involving various viruses and bacteria indicated no cross-reactivity between strains. Additionally, the assay exhibited good reproducibility, with mean coefficients of variation for inter- and intra assay variation below 3%. The overall positive rate was 52.9% (n = 54), with the mRT-PCR assay findings matching sequence analysis results (κ = 1). MHV (n = 29, 28.4%) was the most prevalent pathogen, followed by Helicobacter spp. (n = 28, 27.5%), R. heylii (n = 18, 17.6%), Mycoplasma spp. (n = 14, 13.7%), MNV (n = 12, 11.8%), S. aureus (n = 9, 8.8%), P. aeruginosa (n = 4, 3.9%), and R. pneumotropicus (n = 1, 0.9%). Discussion: This assay offers a rapid turnaround time of 100 min, including 30 min for DNA preparation and 70 min for target DNA/RNA amplification. It ensures accuracy, minimizing false positives or negatives, making it a convenient tool for the simultaneous detection of infectious diseases in many samples. Overall, the propose­d assay holds promise for the effective detection of the most important pathogens in laboratory animal health monitoring.

Deletion of pentad residues in the N-terminal domain of spike protein attenuates porcine epidemic diarrhea virus in piglets.

Our previous study revealed that tissue culture-adapted porcine epidemic diarrhea virus (PEDV) strains, namely KNU-141112-S DEL2/ORF3 and -S DEL5/ORF3, were attenuated to different extents in vivo, suggesting that their independent deletion (DEL) signatures, including 2-amino acid (aa; residues 56-57) or 5-aa (residues 56-60) DEL in the N-terminal domain (NTD) of the spike (S) protein, may contribute to the reduced virulence of each strain. To investigate whether each DEL in the NTD of the S1 subunit is a determinant for the virulence of PEDV, we generated two mutant viruses, named icS DEL2 and icS DEL5, by introducing the identical double or quintuple aa DEL into S1 using reverse genetics with an infectious cDNA clone of KNU-141112 (icKNU-141112). We then orally inoculated conventional suckling piglets with icKNU-141112, icS DEL2, or icS DEL5 to compare their pathogenicities. The virulence of both DEL mutant viruses was significantly diminished compared to that of icKNU-141112, which causes severe clinical signs and 100 % mortality. Interestingly, the degree of attenuation differed between the two mutant viruses: icS DEL5 caused neither diarrhea nor mortality, whereas icS DEL2 caused mild to moderate diarrhea, higher viral titers in feces and intestinal tissues, and 25 % mortality. Furthermore, the icS DEL5-infected piglets displayed no remarkable macroscopic and microscopic intestinal lesions, while the icS DEL2-infected piglets showed histopathological changes in small intestine tissues, including moderate-to-severe villous atrophy. Our data indicate that the loss of the pentad (56GENQG60) residues in S alone can be sufficient to give rise to an attenuated phenotype of PEDV.

Evaluation of the Cross-Protective Efficacy of a Chimeric PRRSV Vaccine against Two Genetically Diverse PRRSV2 Field Strains in a Reproductive Model.

Despite the routine use of porcine reproductive and respiratory syndrome (PRRS)-modified live vaccines, serious concerns are currently being raised due to their quick reversion to virulence and limited cross-protection against divergent PRRS virus (PRRSV) strains circulating in the field. Therefore, a PRRS chimeric vaccine (JB1) was produced using a DNA-launched infectious clone by replacing open reading frames (ORFs) 3-6 with those from a mixture of two genetically different PRRSV2 strains (K07-2273 and K08-1054) and ORF1a with that from a mutation-resistant PRRSV strain (RVRp22) exhibiting an attenuated phenotype. To evaluate the safety and cross-protective efficacy of JB1 in a reproductive model, eight PRRS-negative pregnant sows were purchased and divided into four groups. Four sows in two of the groups were vaccinated with JB1, and the other 4 sows were untreated at gestational day 60. At gestational day 93, one vaccinated group and one nonvaccinated group each were challenged with either K07-2273 or K08-1054. All of the sows aborted or delivered until gestation day 115 (24 days post challenge), and the newborn piglets were observed up to the 28th day after birth, which was the end of the experiment. Overall, pregnant sows of the JB1-vaccinated groups showed no meaningful viremia after vaccination and significant reductions in viremia with K07-2273 and K08-1054, exhibiting significantly higher levels of serum virus-neutralizing antibodies than non-vaccinated sows. Moreover, the JB1-vaccinated groups did not exhibit any abortion due to vaccination and showed improved piglet viability and birth weight. The piglets from JB1-vaccinated sows displayed lower viral concentrations in serum and fewer lung lesions compared with those of the piglets from the nonvaccinated sows. Therefore, JB1 is a safe and effective vaccine candidate that confers simultaneous protection against two genetically different PRRSV strains.

Genomic characteristics and pathogenicity of natural recombinant porcine reproductive and respiratory syndrome virus 2 harboring genes of a Korean field strain and VR-2332-like strain.

Porcine reproductive and respiratory syndrome (PRRS), an economically-important disease caused by PRRS virus (PRRSV), has become endemic to most pig-producing countries. Point mutation and recombination are responsible for genetic heterogeneity, resulting in circulation of genetically-diverse strains. However, no natural recombinant PRRSV has yet been identified in Korea. Here, we successfully isolated natural recombinant PRRSV-2 (KU-N1202) using cell culture, investigated its genomic characteristics, and further evaluated its pathogenicity. KU-N1202 is a recombinant strain between Korean MN184-like and VR-2332-like strains. Specifically, ORF5 to partial ORF7 of the VR-2332-like strain was inserted into the backbone of a CP07-626-2-like strain. KU-N1202 induced mild-to-moderate clinical signs and mild histopathological changes with low viral loads in challenged pigs. Contact pigs showed minimal clinical signs and lower viral loads than those in the challenge group. This study demonstrates the genomic characteristics and pathogenicity of natural recombinant PRRSV-2, illustrating the potential importance of recombination in the field.

Detection of swine hepatitis E virus in the porcine hepatic lesion in Jeju Island.

Swine hepatitis E virus (HEV) is an emerging zoonotic pathogen due to its close genomic similarity to human HEV. The prevalence of swine HEV in the hepatic lesion of pigs from the Jeju Island was investigated by reverse transcriptase polymerase chain reaction (RT-PCR). In total, 40 pigs with hepatitis lesions were selected from 19 different farms, based on examination by microscopy. RTPCR findings revealed swine HEV in 22 cases (55%), including 18 suckling pigs and 4 growing pigs. Several histopathological lesions, including multifocal lymphoplasmacytic hepatitis, portal inflammation, and focal hepatocellular necrosis, were observed in liver sections of swine HEV PCR-positive pigs. The present study suggests that the prevalence of swine HEV is very high in the pig population in Jeju Island, and that pigs are infected at early stages of growth (under 2 months of age). The high prevalence of swine HEV in pigs in Jeju Island and the ability of this virus to infect across species puts people with swine-associated occupations at possible risk of zoonotic infection.

Recent vaccine technology in industrial animals.

Various new technologies have been applied for developing vaccines against various animal diseases. Virus-like particle (VLP) vaccine technology was used for manufacturing the porcine circovirus type 2 and RNA particle vaccines based on an alphavirus vector for porcine epidemic diarrhea (PED). Although VLP is classified as a killed-virus vaccine, because its structure is similar to the original virus, it can induce long-term and cell-mediated immunity. The RNA particle vaccine used a Venezuela equine encephalitis (VEE) virus gene as a vector. The VEE virus partial gene can be substituted with the PED virus spike gene. Recombinant vaccines can be produced by substitution of the target gene in the VEE vector. Both of these new vaccine technologies made it possible to control the infectious disease efficiently in a relatively short time.

Ectopic liver and gallbladder in a cloned dog: Possible nonheritable anomaly.

Ectopic liver and gallbladder are rare anomalies usually not accompanied by any symptoms and are found during surgical exploration or autopsy. We aimed to find a cause of this anomaly using somatic cell nuclear transfer (SCNT) technology, which can produce genetically identical organisms. A cloned beagle having ectopic organs was produced and died on the day of birth. Major and ectopic organs were fixed and underwent histologic analysis. SCNT was performed using cells derived from the dead puppy to produce reclones. Normality of internal organs in the original donor dog and recloned dogs was evaluated by computed tomography. While a liver without the gallbladder was located in the abdominal cavity of the cloned dog, a well-defined, reddish brown mass with a small sac was also positioned outside of the thoracic cavity. Histologically, they presented as normal liver and gallbladder. Five reclones were produced, and computed tomography results revealed that the original donor dog and reclones had normal liver and gallbladder structure and location. This is the first report of both ectopic liver and gallbladder in an organism and investigation on the etiology of these abnormalities. Normal organ structure and position in the original donor dog and reclones suggests that the ectopic liver and gallbladder is a possible nonheritable anomaly.

Proposed Motor Scoring System in a Porcine Model of Parkinson's Disease induced by Chronic Subcutaneous Injection of MPTP.

Destruction of dopaminergic neurons in the substantia nigra pars compacta (SNpc) is a common pathophysiology of Parkinson's disease (PD). Characteristics of PD patients include bradykinesia, muscle rigidity, tremor at rest and disturbances in balance. For about four decades, PD animal models have been produced by toxin-induced or gene-modified techniques. However, in mice, none of the gene-modified models showed all 4 major criteria of PD. Moreover, distinguishing between PD model pigs and normal pigs has not been well established. Therefore, we planned to produce a pig model for PD by chronic subcutaneous administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), neurotoxin. Changes in behavioral patterns of pigs were thoroughly evaluated and a new motor scoring system was established for this porcine model that was based on the Unified Parkinson's Disease Rating Scale (UPDRS) in human PD patients. In summary, this motor scoring system could be helpful to analyze the porcine PD model and to confirm the pathology prior to further examinations, such as positron emission tomography-computed tomography (PET-CT), which is expensive, and invasive immunohistochemistry (IHC) of the brain.

Epidemiological characteristics of pulmonary pneumocystosis and concurrent infections in pigs in Jeju Island, Korea.

Epidemiological characteristics of swine pulmonary Pneumocystis (P.) carinii and concurrent infections were surveyed on Jeju Island, Korea, within a designated period in 172 pigs submitted from 54 farms to the Department of Veterinary Medicine, Jeju National University. The submitted cases were evaluated by histopathology, immunohistochemistry, PCR/RT-PCR, and bacteriology. P. carinii infection was confirmed in 39 (22.7%) of the 172 pigs. Histopathologically, the lungs had moderate to severe lymphohistioctyic interstitial pneumonia with variable numbers of fungal organisms within lesions. Furthermore, porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV-2) co-infection was a common phenomenon (12.8%, 20.5%, and 48.7% were positive for PRRS, PCV-2, or both, respectively, as determined by PCR/RT-PCR). Infection was much more concentrated during winter (December to March) and 53.8% of the infected pigs were 7- to 8-weeks old. In addition, three pigs showed co-infection with bacteria such as Pasteurella multocida and Streptococcus suis. The results of the present study suggest that the secondary P. carinii infection is common following primary viral infection in swine in Korea. They further suggest that co-infection of P. carinii might be enhanced by the virulence of primary pathogens or might have synergistic effects in the pigs with chronic wasting diseases.