Histopathological findings of the nasopharynx-associated lymphoid tissue of pigs co-infected with porcine circovirus 2 and porcine reproductive and respiratory syndrome virus.

Porcine circovirus 2 (PCV2) causes porcine circovirus-associated disease, and co-infection with porcine reproductive and respiratory syndrome virus (PRRSV) severely affects the pig breeding industry. Both viruses target the macrophages in lymphoid tissues. Various porcine pathogens enter via the nasal cavity, and the nasopharynx-associated lymphoid tissue (NALT) acts as the mucosal immune system. However, the pathological analysis has not progressed. This study aimed to histologically examine the NALT of pigs with suspected PCV2 and PRRSV infections. Six pigs were subjected to necropsy, and their NALT, tonsils, and mesenteric lymph nodes were collected. Macrophages, lymphocytic depletion, multinucleated giant cells, intracytoplasmic inclusion bodies, and neutrophil infiltration increased in the NALT. In situ hybridization revealed positive signals for PCV2 in the NALT of all pigs and PRRSV in the NALT of three pigs. PCV2-positive macrophages were mainly identified in the follicles, whereas PRRSV-positive tissues were found primarily around the crypt and directly below the epithelium. Quantitative PCR revealed 108-1010 copies of PCV2 DNA/µL and 102-104 copies of PRRSV DNA/µL in the NALT. Therefore, both PCV2 and PRRSV were detected in the NALT of pigs. In conclusion, the infection and replication of both viruses in the NALT and tonsils may suppress host immunity and promote co-infection with other pathogens.

Effects of Probiotics on Growth and Immunity of Piglets.

Growth promoting antibiotics are used in modern animal husbandry to promote growth and avoid infections. Negative effects of these antibiotics on human health are a big concern and they need to be replaced. Probiotics are expected to be a good replacement for growth promoting antibiotics. In this study, we evaluated effects of probiotics on growth and immune status of liver and secondary lymphoid organs of piglets. Body weights of probiotic-fed piglets were heavier than those of control piglets (p < 0.05) at days 30 and 45 of the experiment. Relative populations of CD4+ and IgM+ cells isolated from the liver were significantly increased (p < 0.05 and p < 0.01, respectively) in probiotic-fed piglets compared to control piglets. CD4+CD8+ T cells were significantly decreased (p < 0.05) in jejunal Peyer's patches of treated piglets. Phagocytosis of MHC class II+ cells isolated from the liver of probiotic-fed piglets was significantly higher (p < 0.05) than that of control piglets. Phagocytosis of granulocytes isolated from the liver and peripheral blood of probiotic-fed piglets were also higher than those of control piglets. These results indicate excellent effects on growth and immune status of piglets. In conclusion, probiotics have beneficial effects on the growth and health of piglets and could be good replacement for growth promoting antibiotics.

Optimizing antimicrobial use by improving medication adherence among pig producers.

In Japan, many pig producers buy antimicrobials using veterinarians' prescriptions. Even if veterinarians prescribe antimicrobials for treatments and metaphylaxis, producers may be using them for prophylaxis on farms with a high incidence of chronic diseases. To reduce the frequency of antimicrobial use, we applied the concept of medication adherence and launched an intervention in eight farrow-to-finish farms. For the intervention farms, we attempted to implement measures for preventing the repeated onset of chronic infections that interfered with adherence, confirmed the need to continue medications, and discontinued medications if possible. For each farm, these interventions began at different time points after 2017 and continued until 2020. We evaluated the effectiveness of this intervention based on the amount of active ingredients/population correction unit and the total number of defined daily doses for the weight group/slaughter pig. The ratio of antimicrobial use for both indicators in the year 2020 compared with the start of the intervention decreased on all farms. Among the eight non-intervention farms, six showed an increase in both indicators. These results suggest that interventions for chronic diseases administered by producers and veterinarians lead to high adherence to prescriptions and responsible and prudent use of antimicrobials on pig farms.

Eimeria pragensis infection alters the gut microenvironment to favor extrinsic shiga toxin-producing Escherichia coli O157:H7 colonization in mice.

We examined the effects of Eimeria pragensis infection on intestinal peristalsis, goblet cell proliferation and intestinal flora in C57BL/6 mice. Intestinal peristalsis was evaluated by radiography using barium at 7 days post-infection (p.i.). The intestinal peristalsis of E. pragensis-infected mice was significantly suppressed compared with uninfected control mice. Twenty-three mice were divided into 5 groups of 4 or 5 mice each; 2 groups of mice were infected with E. pragensis and the others were kept uninfected. At 7 days p.i., E. pragensis-infected and -uninfected mice were sacrificed to examine goblet cell numbers in the intestines, and significant decreases were observed only in the infected mice. Shiga toxin-producing Escherichia coli (STEC) O157:H7 was inoculated orally in mice both infected and uninfected with E. pragensis at 7 days p.i., with the remaining mice used as uninoculated controls. When mice were sacrificed at 2 days after STEC inoculation, STEC was only detected in the intestines of E. pragensis-infected mice. Colonization of STEC was also confirmed by immunohistochemistry on the surface of epithelial cells in concurrently infected/inoculated mice. Also, an overgrowth of residential E. coli was observed only in E. pragensis-infected mice. These results suggest that E. pragensis induces the suppression of intestinal peristalsis and modifies the intestinal environment to facilitate artificially introduced STEC colonization and multiplication, in addition to residential E. coli overgrowth.

Prevalence of equine proliferative enteropathy in Hidaka district, Hokkaido, over five seasons.

Equine proliferative enteropathy (EPE) is an equine infectious disease that can lead to severe weight loss and hyperplasia of the intestinal mucosa due to infection with Lawsonia intracellularis. In this study, we investigated the prevalence of EPE in a major Thoroughbred breeding area: Hidaka district, Hokkaido, Japan. Of the 252 symptomatic horses that we tested, 192 EPE cases (76.2%), including 8 fatal cases, were confirmed from April 2015 to March 2020 by etiological and/or serological investigation. Most of the EPE cases were observed in foals (88.5%), with fewer cases in yearlings (7.3%) and adults (4.2%). Asymptomatic infection was observed in 62.9% of the horses kept with affected horses. These results suggest that EPE is an enzootic disease in Hidaka district.

The presence of adhesion factors NOX, α-enolase, TrmFO, P27, and VpmaX in Mycoplasma bovis wild isolates in Japan.

Background: Mycoplasma bovis causes various diseases such as bronchopneumonia, otitis media, arthritis, and mastitis in cattle. Mycoplasma bovis is often isolated from the deep pharynges of healthy cattle and is generally considered not to cause clinical symptoms while in the upper respiratory tract. In mycoplasma infections, adhesion to the host cells is a crucial step. In recent years, five new adhesins, NOX, α-enolase, TrmFO, P27, and VpmaX, have been reported in M. bovis strains from pneumonia cases. However, the presence of these adhesins in wild isolates has not been established. Aim: This study aimed to investigate the presence of these adhesin genes in wild isolates isolated from cattle nasal cavities and lesion sites (pneumonia, otitis media, arthritis, and mastitis) in various regions in Japan and clarify the relationship between adhesion and the symptoms caused by M. bovis infection. Methods: A total of 141 M. bovis wild isolates isolated from nasal cavities (healthy or sick cattle), lungs with pneumonia, ears with otitis media, joint fluids of arthritic animals, and milk of mastitic animals. Mycoplasma bovis type strain PG45 was also used. Specific polymerase chain reaction reactions were performed to detect nox, α-enolase, trmFO, P27, and vpmaX, which are adhesins of M. bovis. Results: This study reports 139 M. bovis wild isolates were positive for nox, α-enolase, trmFO, P27, and vpmaX, while two isolates each lacked α-enolase or P27 genes. Mycoplasma bovis PG45 also had all five adherens genes. Conclusion: Almost all M. bovis wild isolates possessed all nox, α-enolase, trmFO, P27, and vpmaX genes regardless of the lesion site or region of origin. This means no relationship was found between the presence of the five adhesins and lesion sites in M. bovis and M. bovis isolated from the nasal cavities of asymptomatic cattle have the same numbers and types of adhesins as isolates from symptomatic lesion sites (pneumonia, otitis media, arthritis, and mastitis). This suggests that not only M. bovis isolates from pulmonary lesions, but also M. bovis existing in the nasal cavity has the potential to causes symptoms in the host.

Analysis of the effect of feedback feeding on the farm-level occurrence of porcine epidemic diarrhea in Kagoshima and Miyazaki Prefectures, Japan.

When a large-scale epidemic of porcine epidemic diarrhea (PED) occurred in 2013 in Japan, feedback feeding (feeding feces and gut tissues of infected piglets) was attempted to impart immunity to sows and immunize nursing piglets via breastfeeding. This study evaluated the effect of feedback feeding on PED control at 172 farms in Kagoshima and Miyazaki Prefectures. Univariable and multivariable generalized linear models were used to analyze the associations between conduct of feedback feeding and damage from the outbreak (outbreak period and the number of piglet deaths) at the farm level. The within-farm outbreak period shortened over time after the regional outbreak began on Kyushu Island (P=0.009) and was longer on large-scale farms (mean 66.0 days, P=0.003) than small-scale farms (29.4 days) and on farms that used feedback feeding (145.2 days, P=0.059) than those that did not (66.0 days). The number of dead piglets decreased over time since the first regional case (P<0.001) and was higher at farrow-to-finish farms (3.8 piglets/sow, P<0.001) than reproduction farms (0.7 piglets/sow). The effect of feedback feeding on the number of dead piglets was not significant, but its interaction term with farm style had a significant effect (5.0 more piglet deaths at reproduction farms than fallow-to-finish farms, P=0.001). These results suggest that feedback feeding made the damage from PED worse, though it was well established at a later stage of the regional PED epidemic.

Indications and patterns of antimicrobial use in pig farms in the southern Kyushu, Japan: large amounts of tetracyclines used to treat respiratory disease in post-weaning and fattening pigs.

In Japan, reducing the use of antimicrobials in pig production is a significant issue. However, there are no published reports concerning porcine disease treatment, as related to the age of the pigs and the indications (e.g., organ system) in Japan. In this study, we analyzed the prescription records of 17 farrow-to-finish farms from 2014 to 2018 in southern Kyushu, Japan. The farms' antimicrobial usage was calculated as the active ingredient per population correction unit (PCU) or the number of treated pigs per PCU using the defined daily dose. All data were analyzed according to the indications and production stages (sows, suckling pigs, post-weaning pigs, and fattening pigs). In terms of active ingredients/PCU, tetracyclines were the most commonly used (43.2-59.3%), and the largest amounts of antimicrobials administered through feeds were for treating the respiratory organs of fattening pigs. In terms of the number of treated pigs/PCU, tetracyclines were most frequently used (16.3-31.1%), and a high frequency of antimicrobials administered through feeds was used for the treatment of respiratory organs in post-weaning pigs. In this study, it was confirmed that tetracyclines were used frequently as a herd treatment for respiratory diseases in post-weaning and fattening pigs in southern Kyushu, Japan. The findings suggest that it is necessary to improve the treatment and prevention of respiratory diseases in post-weaning and fattening pigs in order to reduce the frequencies of antimicrobial treatments.

Assessment of reproductive and growth performance of pigs on commercial swine farms in southern Kyushu, Japan.

The objective of the present study was to assess the productivity of pigs to investigate the time-dependent change in productivity and compare productivity according to breeding company group and porcine reproductive and respiratory syndrome (PRRS) status on commercial swine farms in southern Kyushu, Japan. Data used in the present study were 245 annual productivity records from 2014 to 2018 obtained from 72 farms. Average sow inventory was 481.2 sows. Mean numbers of pigs weaned per sow per year and market pigs sold per sow per year were 23.7 and 21.6, respectively. Pigs born alive per litter increased from 11.1 to 11.9 pigs from 2014 to 2018 (p < .05). Farms using domestic breeding companies had similar numbers in 2016 to those using international breeding companies, but fewer pigs in 2014, 2015, 2017, and 2018 (p < .05). Farms with an unknown or unstable PRRS status had fewer pigs born alive per litter and pigs weaned per sow per year than those with stable or negative PRRS statuses (11.2 ± 0.06 vs. 11.8 ± 0.08 pigs and 22.6 ± 0.38 vs. 25.0 ± 0.22 pigs, respectively; p < .05). These results can be used to establish feasible targets and standards of performance to identify problem areas and improve production.

Antimicrobial resistance profiles and phylogenetic groups of Escherichia coli isolated from healthy Thoroughbred racehorses in Japan.

In this study, we investigated the occurrence of antimicrobial resistance in commensal Escherichia coli isolated from healthy Thoroughbred (TB) racehorses in Japan. A total of 212 fecal samples were individually collected from TB racehorses from March 2017 to August 2018 at Japan Racing Association training centers. E. coli was isolated by using selective agar media, deoxycholate-hydrogen sulfide-lactose (DHL) and eosin methylene blue (EMB). A total of 417 E. coli isolates were examined against 10 antimicrobial agents by using the broth microdilution method. The 417 E. coli isolates were phylogenetically grouped using a multiplex polymerase chain reaction. The highest proportion of resistance was observed for streptomycin (30.9%, 129/417) followed by ampicillin (19.4%, 81/417), trimethoprim (15.8%, 66/417), tetracycline (8.4%, 35/417), chloramphenicol (2.6%, 11/417), kanamycin (1.2%, 5/417), nalidixic acid (0.5%, 2/417), cefazolin (0.2%, 1/417), colistin (0.2%, 1/417), and gentamycin (0%). Multidrug-resistant (MDR) E. coli was detected in 7.9% (33/417) of isolates. The proportions of resistance against ampicillin, streptomycin, kanamycin, and chloramphenicol and of multidrug-resistant phenotypes in E. coli belonging to phylogenetic group B2 were significantly higher than those of other groups. This study clarified the distribution of antimicrobial-resistant (AMR) E. coli in Japanese racehorses. A continuous monitoring program for antimicrobial resistance is required to control the spread of AMR bacteria in racehorses.

Antimicrobial-Resistant Enterococcus faecium and Enterococcus faecalis Isolated From Healthy Thoroughbred Racehorses in Japan.

In this study, the occurrence of antimicrobial-resistant (AMR) enterococci was evaluated in Thoroughbred (TB) racehorses in Japan. Fecal samples were collected from 212 healthy TB racehorses at the Miho and Ritto Training Centers of the Japan Racing Association from March 2017 to August 2018. Isolation and identification were performed by enterococcus selective medium and confirmed to the species using MALDI-TOF MS. Enterococcus faecium and E. faecalis isolates were subjected to antimicrobial susceptibility test against 11 antimicrobials by minimum inhibitory concentration based on recommendation from Clinical and Laboratory Standards Institute guidelines. Among 583 enterococcus isolates, E. faecium and E. faecalis were identified for 48.2% (281/583) and 7.4% (43/583), respectively. One isolate that was representing E. faecium (153 isolates) and E. faecalis (31 isolates) from each sample was selected for antimicrobial susceptibility test. The highest rate of resistance for E. faecium isolates was observed against enrofloxacin (57.5%; 88/153), followed by streptomycin (32.0%; 49/153), kanamycin (18.3%; 28/153), gentamycin (5.9%; 9/153), erythromycin (5.9%; 9/153), and oxytetracycline (4.6%; 7/153). For E. faecium isolates, the highest resistance was observed against streptomycin (90.3%; 28/31), followed by kanamycin (41.9%; 13/31), gentamycin (29.0%; 9/31), lincomycin (9.7%; 3/31), oxytetracycline (6.5%; 2/31), erythromycin (6.5%; 2/31), tylosin (6.5%; 2/31), enrofloxacin (6.5%; 2/31), and chloramphenicol (3.2%; 1/31). The results indicated that enrofloxacin and aminoglycosides were highly resistant among tested antimicrobials. Continuous monitoring studies are useful to increase the awareness of the potential for AMR bacteria to arise from imprudent use of antimicrobials in TB racehorses in Japan.

Assessment of abortion risk of sows on Japanese commercial farms infected with porcine epidemic diarrhea virus.

The objectives of this study were to assess abortion risk (AR) and the number of piglets that died during suckling periods per litter (DP) in farms infected with porcine epidemic diarrhea (PED) in relation to herd immunization procedures. Data were obtained from 91 farms in Japan that had PED infection during 2013 to 2014. The 91 PED-positive farms were asked the number of abortions that occurred and DP for 3 months (1 month before PED outbreak (previous month), 1 month after PED outbreak (the month of PED), and from 1 month after PED outbreak to 2 months after PED outbreak (following month)). AR in each month was calculated as the number of abortions divided by sow inventory. Both AR and DP in the month of PED were higher than those in the previous and following months (p < .05). Farms that performed a herd immunization procedure had higher AR and DP in the month of PED than those that did not perform the procedure (p < .05). In summary, PED occurrence increased AR and DP.

Multidrug-Resistant ESBL/AmpC-Producing Klebsiella pneumoniae Isolated from Healthy Thoroughbred Racehorses in Japan.

Extended-spectrum ß-lactamase (ESBL)- and AmpC ß-lactamase (AmpC)-producing Klebsiella spp. have become a major health problem, leading to treatment failure in humans and animals. This study aimed to evaluate the presence of ESBL/AmpC-producing Klebsiella spp. isolated from racehorses in Japan. Feces samples from 212 healthy Thoroughbred racehorses were collected from the Japan Racing Association Training Centers between March 2017 and August 2018. ESBL/AmpC-producing Klebsiella spp. were isolated using selective medium containing 1 µg/mL cefotaxime. All isolates were subjected to bacterial species identification (MALDI-TOF MS), antimicrobial susceptibility test (disk diffusion test), characterization of resistance genes (PCR), conjugation assay, and genetic relatedness (multilocus sequence typing/MLST). Twelve ESBL/AmpC-producing Klebsiella pneumoniae (ESBL/AmpC-KP) were isolated from 3.3% of horse samples. Antimicrobial resistance profiling for 17 antimicrobials showed all ESBL/AmpC-KP were multidrug-resistant (MDR). Only 1 isolate was confirmed as an ESBL producer (blaCTX-M-2-positive), whereas the other 11 isolates were plasmid-mediated AmpC (pAmpC) producers (blaCMY positive). On the basis of MLST analysis, the ESBL-KP isolate was identified as sequence type (ST)-133 and four different STs among AmpC-KP isolates, ST-145, ST-4830, ST-4831, and ST-4832, were found to share six of the seven loci constituting a single-locus variant. This is the first study to show K. pneumoniae carrying MDR pAmpC isolated from a racehorse.

IncI1 Plasmid Associated with blaCTX-M-2 Transmission in ESBL-Producing Escherichia coli Isolated from Healthy Thoroughbred Racehorse, Japan.

In our previous study, extended spectrum ß-lactamase (ESBL)-producing Escherichia coli (ESBLEC) were isolated from healthy Thoroughbred racehorse feces samples in Japan. Some ESBL genes were predicted to be located on the conjugative plasmid. PCR-based replicon typing (PBRT) is a useful method to monitor and detect the association of replicons with specific plasmid-borne resistant genes. This study aimed to evaluate the plasmid replicon associated with ESBLEC isolated from healthy Thoroughbred racehorses at Japan Racing Association Training Centers in Japan. A total of 24 ESBLECs isolated from 23 (10.8%) individual Thoroughbred racehorse feces samples were used in this study. ESBL gene transfer was performed using a conjugation assay. Then, replicon types of ESBLEC isolates and their transconjugants were determined using PBRT. Pulsed-field gel electrophoresis (PFGE) was performed to look at the clonality of the ESBLECs isolates. ESBLECs were detected from 10.8% of healthy Thoroughbred racehorses. The blaCTX-M-2 was identified as the dominant type of ESBL gene, followed by blaCTX-M-1 and blaTEM-116. In this study, only the blaCTX-M-2 and the IncI1 plasmid were transferred to transconjugants. The PFGE results showed that ESBL genes were distributed in diversity of ESBLECs. This finding suggested that the IncI1 plasmid was associated with the dissemination of blaCTX-M-2 in Thoroughbred racehorses in Japan.

Extended-spectrum ß-lactamase-producing Escherichia coli isolated from healthy Thoroughbred racehorses in Japan.

Extended-spectrum ß-lactamase-producing Escherichia coli (ESBLEC) have become a major health concern in both human and veterinary medicine. These bacteria could become a critical problem in equine medicine due to the limited number of antimicrobial drugs available. However, there are no previous reports of ESBLEC isolated from horses in Japan. The objectives of this study were to investigate the occurrence of ESBLEC isolated from feces in healthy Thoroughbred racehorses in Japan. Feces samples were collected from 147 healthy Thoroughbred racehorses by equine veterinarians at the Japan Racing Association (103 from Miho Training Center and 44 from Ritto Training Center) between March 2017 and April 2018. Samples were screened for ESBLECs using MacConkey agar supplemented with 1 µg/ml cefotaxime. Detection of ESBL genes was performed by PCR and confirmed by DNA sequencing. Horizontal transmission was demonstrated by conjugation assay. In this study, 24 ESBLECs were isolated from twelve horse feces samples (8.2%). All ESBLECs harbored blaCTX-M-2, and both blaTEM-1 and blaCTX-M-2 were detected in nine isolates (37.5%). ESBLECs showed resistance to all ß-lactam antibiotics (100%) tested, followed by trimethoprim (66.7%), streptomycin (62.5%), tetracycline (25.0%), and oxytetracycline (25.0%). Horizontal transmission was successfully demonstrated by conjugation assay in eight of 13 isolates, and blaCTX-M-2 was detected by PCR in all transconjugants. This study showed that racehorses in Japan are potential reservoirs of ESBLECs.

Total serum protein reference value as a clinical diagnostic index of equine proliferative enteropathy.

Equine proliferative enteropathy (EPE) caused by Lawsonia intracellularis is characterized by hypoproteinemia. There are currently no reliable reports that provide a reference value for the total serum protein (TP) concentration to clinically diagnose EPE. The objective of this study was to statistically determine the reference value. Feces and sera of 99 foals with EPE-like clinical signs and of 35 healthy foals were obtained. The samples were used for specific-gene detection of L. intracellularis, TP measurement, and specific-antibody detection against L. intracellularis. Based on these results, the optimal reference value for the TP concentration as a clinical diagnostic index of EPE was found to be ≤ 4.8 g/dl. This clinical diagnostic index will provide an effective approach for diagnosing EPE.

Effect of intervention practices to control the porcine epidemic diarrhea (PED) outbreak during the first epidemic year (2013-2014) on time to absence of clinical signs and the number of dead piglets per sow in Japan.

Porcine epidemic diarrhea (PED) is an emerging and/or re-emerging disease of pigs in several countries, with high morbidity and mortality in suckling piglets. Farms affected with PED perform various intervention practices to control and/or eliminate the PED virus. The objectives of the present study were to assess the effect of biosecurity measures and intervention practices to control PED on time to absence of clinical signs (TAC) and number of dead suckling piglets during TAC. A questionnaire was administered to 120-PED affected farms located across Japan between 2013, when the first case was reported in Japan, and 2014. Farms were asked to provide information on farm characteristics and internal or external biosecurity measures during PED outbreak, as well as on intervention practices to control PED. The TAC was defined as the number of days from the date that clinical PED signs appeared to the date that clinical PED signs disappeared. The number of dead piglets per sow (DP/S) was calculated as the number of dead suckling piglets during TAC divided by the sow inventory. Regarding the effect of biosecurity measures during PED outbreak on TAC and DP/S, longer TAC was observed in Actinobacillus pleuropneumoniae-positive farms and farms outsourcing pig transport to the slaughterhouse (p <  0.05). In addition, farms with divided truck entrances had lower DP/S than those without divided entrances (p <  0.05).Regarding the effect of intervention practices to control PED on TAC and DP/S, farms that performed feedback at 2 weeks or later after PED outbreak had longer TAC and higher DP/S than other farms (p <  0.05). Farms that fixed the hours staff worked in farrowing barn had lower DP/S than the other farms (p <  0.05). In conclusion, variables associated with long TAC were Actinobacillus pleuropneumoniae -positive farms, farms outsourcing pig transport to the slaughterhouse, and farms performing feedback at 2 week or later after PED outbreak. Additionally, those associated with high DP/S were farms without divided entrances, farms without a fixed hours worked in the barn, and farms that performed feedback at 2 week or later after PED outbreak.

Assessment of reproductive performance in F1 sows exposed to the porcine epidemic diarrhea virus at different periods of production stage on farms with different hygienic environments.

Porcine epidemic diarrhea (PED) occurred in Japan in 2013 after an interval of 7 years. The present study assessed individual productivity of sows exposed to PED virus at different periods of the production stage. The present study was performed at three commercial farms that had PED outbreak during the month of December 2013. Herd immunization was conducted for all gilts and sows. The production records were obtained for sows that were alive during the PED outbreak at each farm. The sows were categorized into six groups based on the period in which they were exposed to PED virus between days 0-30 (G1), 31-60 (G2), 61-90 (G3), or after 91 days of pregnancy (G4), during lactation (L), and after weaning (W). The control group was assigned based on the records before the period of PED outbreak (uninfected group). The number of sow's records obtained from farms A, B, and C were 1056, 1137, and 1035, respectively. Compared with the uninfected group, there was no reduction in the number of pigs born alive in the G1-G4 groups. Sows of the G4 and L groups, however, had 4-9 pigs fewer pigs weaned, and a 36%-77% greater pre-weaning mortality than the uninfected group (P < 0.05). There was no difference in farrowing rate and number of pigs born alive at subsequent parities among the sow groups. There were no interactions between sow groups and parity for sow productivity.

Molecular characterization of US-like and Asian non-S INDEL strains of porcine epidemic diarrhea virus (PEDV) that circulated in Japan during 2013-2016 and PEDVs collected from recurrent outbreaks.

BACKGROUND: Since late 2013, porcine epidemic diarrhea virus (PEDV) has reemerged in Japan and caused severe economic losses to the swine industry. Although PEDV vaccines have been used widely, the disease has swept rapidly across the county, and is commonly observed in PED-vaccinated farms, and has recurred in domestic herds. To better understand PEDVs responsible for the reemerging outbreaks in Japan, full-length spike (S), membrane (M), and nucleocapsid (N) genes of 45 PEDVs collected in Japan during 2013-2016, were sequenced and analyzed. RESULTS: Phylogenetic analysis based on S gene sequences revealed that all the recent field PEDVs were genetically distinct from the classical Japanese strains, and were classified into three genotypes: North American (NA), S INDEL, and Asian non-S INDEL. Our data suggested a possibility that multiple parental PEDV strains were introduced into Japan from abroad at the same time or similar times. The newly identified Japanese strains showed the closest relationship to the US strains. Two sublineages of Japanese strains circulating in Japan were similar to two sublineages identified in the US, suggesting common ancestors for these strains. In comparison with two vaccine strains used in Japan, the field strains had various changes in epitope regions, glycosylation sites, and phosphorylation sites. These substitutions, particularly observed in epitope regions of the S (521, 553, 568, and 570), M (5), and N (123, 252, and 255) proteins, may have affected antigenicity and vaccine efficacy, resulting in an unsuccessful PEDV control. Sequence comparisons between PEDVs collected from primary and secondary outbreaks in three herds revealed that the disease has developed to an endemic stage in which PEDV could persist for nearly two years in the herds or local regions, causing subsequent epidemics. CONCLUSIONS: These results elucidate the genetic characteristics, origin, and molecular epidemiology of PEDVs circulating in Japan, as well as the PEDV strains causing recurrent outbreaks. This study provides a better insight into the PEDVs responsible for recent outbreaks in Japan, and could potentially help to develop measures for controlling and preventing the disease.